The presented results are an attempt to identify the changes taking place during a punch test experiment and the development of fungal impurities of powdered food materials over long-term storage at 75% RH. The potato starch and wheat flour market has a large share of the global production of bulk materials. The growing interest in powdered food materials requires additional production expenditure. This is associated with an increase in storage time of the discussed product and providing it with the appropriate conditions. The samples of potato starch and wheat flour were stored in perforated containers in a climatic chamber at 75% humidity and 21 °C for five months and then samples were measured by a punch test in a Lloyd LRX materials testing machine. The graphs obtained in the potato starch punch test differed significantly from wheat flour. The thickening of potato starch was observed in the form of layers, while potato starch was uniformly thickened throughout the experiment. The conditions of 75% humidity and 21 °C can be described as the beginning of the caking process. In potato starch, linear sections were observed, which changed the length of their storage time and, additionally, was correlated with the appearance of fungal contamination. These results may suggest the influence of fungi on the phenomenon of bulk material caking. Statistics show that the consumption of wheat flour in 2018 reached 385 million tons, while the potato starch world market reached 3.7 million tons. Both of these food powders play an important role not only in the food industry, but also in other industries such as pharmacy, the textile and paper processing as well as the chemical industry. Moreover flour and starch are increasingly being proposed as carriers for microorganisms in production of biopreparation for agricultural use . TherefoAdditional problems associated with improper storage conditions that result in the deterioration of product quality are biological agents. One of them is microorganisms, especially molds, whose spores can be present at any stage of the production process. When temperature and humidity exceed to the minimum limit for the development of microorganisms, they start to grow. For mold, the lower limit of relative humidity can start from 60–70% . TherefoThe most recent research described in the scientific literature is focused on the issues concerning the influence of humidity on physical parameters of powders and changes in their composition, as well as their influence on powder flowability and caking degree. Methods used to assess caking may include rheometric tests , blowingThere are also studies on water activity and microbiological impurities in powders. Abdullah et al.  studied In this study, we are focused on the relationship between the degree of caking of food powders correlated with the development of fungal impurities during long-term storage in the highest humidity limit accepted as safe in typical warehouses (RH 75%). The determination of the caking was examined by the punch test. The aim of this study was to evaluate the changes in potato starch and wheat flour during the punch test after storage them under controlled conditions. Additionally, related occurred changes to fungi contamination of the tested material, as well as to assess and identify the depth of caking in material samples. Powders, which are the most commonly used materials in the loose materials industry, have been selected for testing based on their different properties. Potato starch is a more homogeneous system, while wheat flour has other ingredients besides starch. In addition, these materials have a different morphological structure of molecules, so both of them, although they belong to powders, represent different properties.Potato starch  and wheat flour type 500  were used in the study. The materials were dried in a SUP-30 laboratory dryer  to a maximum moisture content of 3% ± 0.5. The process of drying the powders to achieve the aforementioned humidity level was aimed at unifying the water content of the samples, so that in each sample the initial water content was at the same low level. Humidity measurements were performed using Mettler Toledo HG63 moisture analyzer . Cylindrical perforated vessels with a diameter of 24 mm, a height of 30 mm and mesh diameter 1.5 mm were used in the study, the purpose of which was to maximize the exposition of the material to humidity. The perforated cylinders had previously been sterilized in an autoclave  to eliminate microorganisms originating from the vessel. The prepared materials were sieved through a sieve into measuring vessels to create more homogeneous deposit. Each vessel was completely filled with powder, whose net weight was about 6 g. Next, prepared samples were stored in a climatic chamber  at 75% humidity and 21 °C for five months. These conditions were intended to resemble the upper limit of the conditions prevailing in warehouses ,10. The –1. The measurement started when the indenter touched the surface of the material in the vessel and was carried out until a depth of 14 mm.The procedure of measurement by means of a punch test is based on the method presented by Wajs et al. (2020) . The sam−1 dilution was chosen for the analysis, from which the colonies were counted after five days as recommended by the standard. The measurements were made in triplicate. After counting, the grown colonies were converted to colony forming units (CFU) per gram of dry matter (CFU g−1) of tested material.Measurement of the degree of fungal contamination was assessed based on the determination of the number of yeasts and molds by counting colonies according to the protocol described in the ISO 21527:2009 . Samplesp-value is the calculated probability . A moving average was used to assess the development of fungal contamination. The use of a moving average was to mitigate the impact of random fluctuations in the results. The obtained CFU results were analyzed every six periods. Additionally, a matching line was inserted with the 95% confidence intervals (dotted lines).The obtained results were calculated with averages together with standard deviation, and then analysis of variance was carried out using software Statistica . Information on the statistical significance was given as 0.95 confidence intervals shown as dotted lines on the graphs. In the first stage, the runs of the punch test were analyzed to estimate changes of the strength value that characterized the studied materials. Among the obtained graphs representing the discussed features of both powders, we could distinguish groups characterized by similarity. Three types of graphs describing potato starch were obtained. The model groups of the graphs presented in The potato starch graphs are characterized by specific points that can be observed in each of them. Peak loads are the maximum values of periodic increases in strength, which are related to the gradual compaction of the material. The diagrams also show the first increase and the second increase, which describe the approximately linear sections of the increase between the peaks. Type A was characterized by one peak and one linear increase. This type of graph can be observed from days 1–7. From day 9, type B diagrams have been selected. They appeared until day 84 and were characterized by the presence of two peaks and two linear increases in force values. For the period over 84 days, type C graphs appeared. At the beginning, they were characterized by three peaks, while from day 114 of storage, another additional peak started to appear. Additionally, during the whole experiment in potato starch, a linear increase was observed after the first peak. The second growth developed over time and resembled an exponential function. The wheat flour treatments are markedly different from those observed for potato starch . DiffereAlong with the time of storage in elevated humidity, the potato starch is compacting and strengthening, resulting in an increasing number of peaks. The first peak load appears throughout the experiment and takes very low values up to 1 N. It was observed at a similar depth not exceeding 1.5 mm. It is, therefore, bound to the surface of the powder, and its values suggest the moment of penetration through the potato starch surface. The penetration tests of lumped skimmed milk powder  also shoFrom the ninth day of storage, a second peak was observed in measurements of potato starch. Until the 78th day, it appeared irregularly at 4–13 mm depths, after the 78th day the peak depth decreased and it appeared more regularly at 5–7 mm. During the whole experiment, it took values from 1 N to 5 N. In addition, in the first and second peak, we can observe an increase in strength up to 18 days, which may be associated with an increase in material density. No further significant changes in these peaks may suggest that after this time a stabilized material layer was formed. After day 84, thickening of potato starch and the formation of the next layers were observed, which can be plotted as the third peak and from day 114 onwards as the fourth peak. The third peak load is the only peak that showed a downward trend in force values. This decrease may be related to the formation of the next layer of material (the fourth peak). In the case of peaks 1 and 2, the values increased slightly, until the moment of stabilization of the material’s core and the appearance of three peaks. When the third layer of material forms, the situation is similar. In the initial days of its appearance, the values will be at a similar level, but over time they do not stabilize and their value decreases due to giving way to the next peak and moving closer to the surface. In addition to the above-mentioned downward trend of mean values of the third peak, among the remaining peaks no statistically significant differences in the matching lines were observed and no correlation between the variables was shown. The resulting layers of material and increasing force values suggest that under the influence of storage with increased humidity, the material became more stable and gained strength. The layering and the thickening of potato starch can be the beginning of the caking process. This means that conditions of 75% humidity and 21 °C can be the beginning of the caking process. A similar conclusion was drawn by Billings et al. . They teA similar comparison of conditions of 100% RH and 76% at 20 °C was carried out by Fitzpatrick et al.  on skim An additional element that was observed in potato starch during the punch test were linear increases in strength. The analysis was based on the differences between the end of the growth and the start point. The calculated results are shown in Both the first and second increase decreased during the time of storage of the tested potato starch. A linear matching function is also inserted into the chart, which also indicates that this distance is shortening. The shortening of the linear sections suggests that the material strengthening has become more homogenous.p = 0.00) correlation with the storage time. This correlation may result from the appearance of the next peaks in the following days as well as the second linear growth, which results in the shortening of the first linear segment. The situation is similar for the second linear growth. Additionally, here we can observed a strong (r = −0.801) negative linear and a significant (p = 0.002) correlation with the time of storage. However, this increase did not systematically appear during the measurements. This decrease can also be caused by the formation of the next layer of material, which is illustrated in the graphs as the fourth peak load. Another explanation for the shortening of the second increase may be the fact that fungal contamination appears at the same time as this decrease.p = 0.000) and a high (r = 0.710) linear correlation was observed between the linear section and storage time. The matching function indicates that the values of the linear growth section increased with the storage time and this is reflected in the graphs, as there were more disorders in the initial days of measurement in the end of the graphs. As the storage time passed, the test line was more "smooth", which may be related to the settling of wheat flour and its stabilization.An increase in the strength of the second peak has been observed since the date of appearance of the fungus. It is known that moisture can affecting the density of the material. It may suggest that the development of microorganisms could also affect the density of the potato starch layer and, thus, increase the strength value. A similar effect was obtained in the work Wajs et al. , where tThe wheat flour charts are mainly characterized by linear growth. Only in the initial phase of measurement, a force peak was observed, which was related to the sensor breaking through the powder surface. However, it can be observed that the linear section in wheat flour was increasing, which differs from potato starch samples values. In the first days of measurements, where the material was not yet sufficiently thickened, the sensor pierced the powder surface rapidly. This resulting in a long time for the indenter to adapt to the material and, thus, the length of linear growth was shorter. With the storage time, the material has consolidated and less susceptible to the sensor hitting the surface. Teunou et al.  studied The different behavior of potato starch and wheat flour during the penetration test, resulting in distinct shapes of flow charts, a number of peaks, strength values, and linear growths may be due to the separate botanical origin of the materials and, consequently, other properties of the powders. Internal properties, specific for a particular powder, include, among others, parameters describing the morphology of particles, which can have a significant impact on the flow properties of powders. Fitzpatrick et al.  analyzedMoreover, the analyzed materials differ in their chemical composition. In wheat flour, apart from starch, there are additional components such as vitamins, proteins and others  which maThe ratio of amylose to amylopectin, as well as the previously mentioned particle size and other physical and chemical properties, can also influence the behavior of starch in increased moisture . This isThe degree of fungal contamination was determined as additional analysis of materials. The contamination of wheat flour with fungi was similar to that of potato starch. In wheat flour, the first appearance of fungal contamination at a very low level occurred on the 9th day, slightly earlier than in starch (14th day). This may be due to slightly better conditions for development than in potato starch, due to the compactness of additional ingredients such as proteins, vitamins, etc. . It was The longer the potato starch was exposed to moisture, the higher the depth of the compacted material, which can be observed in the form of shortened linear growth segments and an increasing number of peaks.The conditions of 75% humidity and 21 °C can be regarded as the beginning of the caking process. Adsorption of water from the environment contributes to an increase in the concentration of starch and the formation of material layers. As the moisture content of the material increases, the risk of caking in the material and settling on the walls of storage tanks increases.Different punch test graphs in wheat flour and potato starch suggest that the caking process is different in both powders. The shortening of the linear growths and the zoom of the peaks in potato starch may show a gradual clumping at several points in the sample and then the joining of the resulting thickened layers, while the wheat flour is homogeneous in the entire volume of the sample.The low strength values of wheat flour suggest that this material has a low affinity for caking under the given conditions.The correlation of the shortening of the first linear growth section in potato starch, with the development of fungal contamination, may suggest that mold affects caking. However, the amount of mold in both wheat flour and potato starch is very low. To completely exclude or confirm the effect of fungal contamination on the caking phenomenon, an experiment with the above-mentioned powders should be carried out.The increase of fungal impurities in the potato starch under the conditions of 21 °C 75% RH begins from day 42; therefore, it can be assumed that until this day the material is not microbiologically contaminated. However, this concerns mold and yeast only. After that time, to use the starch, the fungal contamination level of the material should be performed, and other groups of microorganisms should be tested.


Gold nanoparticles (AuNPs) are the most thoroughly studied nanoparticles because of their remarkable optical properties. Color changes in assays that use AuNPs can be easily observed with the naked eye, resulting in sensitive colorimetric methods, useful for detecting a variety of biological molecules. However, while AuNPs represent an excellent nano-platform for developing analytical methods for biosensing, there are still challenges that must be overcome before colloidal AuNPs formulation can be successfully translated into practical applications. One of those challenges is the ability to immobilize AuNPs in a solid support. There are many difficulties with controlling both the cluster size and the adhesion of the coatings formed. In addition, many of the techniques employed are expensive and time-consuming, or require special equipment. Thus, a simple and inexpensive method that only requires common lab equipment for immobilizing AuNPs on a surface using Starch Hydrogels has been developed. Starch hydrogels confer a 400% increase in stability to the nanoparticles when exposed to changes in the environment while also allowing for macromolecules to interact with the AuNPs surface. Several starch derivatives were tested, including, dextrin, beta-cyclodextrin and maltodextrin, being dextrin the one that conferred the highest stability. As a proof-of-concept, a SlipChip microfluidic sensor scheme was developed to measure the concentration of DNA in a sample. The detection limit of our biosensor was found to be 25 ng/mL and 75 ng/mL for instrument and naked eye detection, respectively. Gold nanoparticles (AuNPs) are the most thoroughly studied nanoparticles because of their remarkable optical properties used in a variety of applications such as sensing, detection, and imaging, making them an excellent material for diagnostic purposes ,2,3,4. TNanocomposite hydrogels can be generally synthesized through three different mechanisms that involve a combination of nanoparticles and hydrogels . Among tCAS 741965), Sigmacote siliconizing reagent (CAS SL2), Potato starch , Maltodextrin , NaCl and MgCl2 were purchased at Sigma-Aldrich. Dextrin  and beta-cyclodextrin  were purchased from Acros Organics. Sheared Salmon Sperm DNA (CAS AM9680) and Invitrogen DEPC-Treated Water (CAS AM9906) were obtained from ThermoFisher Scientific. Gold nanoparticles (AuNPs) with a diameter of 20 nm  of AuNPs suspension was placed in a 2-mL Eppendorf tube (Eppendorf 4515D) and centrifuged at 14,000 rpm for 20 min. The supernatant (mostly citrate buffer) was removed until 40 μL of the AuNPs suspension have left. A second aliquot of AuNPs suspension was added and centrifuged again under the same conditions. The supernatant was removed until 50 μL of the AuNPs suspension have left. A stock solution of 0.1 g/mL Potato Starch was prepared by mixing 1.0 g of starch with 10.0 mL of deionized water. AuNPs-Starch suspensions were prepared by adding the appropriate amount of the starch stock solution to the concentrated AuNPs until the 1-mL mark of the tube. Tubes were heated in boiling water for 3 min while gently rocking side to side. Then, the tubes were let to cool down at room temperature for 1 h, at which time the solution had gelled. AuNPs-Starch suspensions were prepared at five different starch concentrations . For each, drops of 10 μL were placed on a Hammerhill hydrophilic paper  and allowed to dry overnight at 20 °C. Images of each starch concentration on paper were acquired using an HP DeskJet Plus scanner  and processed using ImageJ by converting images into gray scale and calculating the intensity with the Analyze tool. Results were then graphed in Microsoft Excel (Microsoft Office 365). All results were expressed as the mean ± SD.For the UV/Vis absorption assays, Corning 96-Well Plates  were used. In each well, 10 μL of the AuNPs suspension were added followed by 10 μL of a salt solution. The absorption spectra were recorded on a Synergy HTX spectrophotometer  from the Hauptman Woodward Institute, Buffalo, NY, USA. The wavelength was measured from 300 nm to 700 nm. Data processing analysis was done on Microsoft Excel (Microsoft Office 365). All results were expressed as the mean ± SD.2 60 W laser cutter. Each layer is made individually from cast acrylic. The inlet and outlet ports in the top layer and the microfluidic channel in the middle layer are made by cutting all the way through their respective acrylic layers using the following settings . The bottom layer has an engraved channel made by using the following settings .A microfluidic device is developed following the Slip Chip approach . The devDNA stock solutions were prepared by heating an aliquot of Sheared Salmon Sperm DNA in boiling water for 5 min and quickly placing it on ice for 15 min. Serial dilutions from this aliquot were done to prepare eight DNA solutions with concentrations ranging from 10 ng/μL to 10 μg/μL. For biosensor assays, the bottom channel of the microfluidic device was filled with 20 μL of AuNP-Starch suspension at a starch final concentration of 10 mg/mL. The middle channel was filled with 4 M NaCl solution. The DNA solution was injected through the inlet into the bottom channel. The Slip Chip was incubated at room temperature for 3 min at which time the middle layer would be switched so the NaCl solution interacted with the DNA-AuNPs mixture. A change in color on the solution was detected by the naked eye as well as UV/Vis characterization.To test the ability of Starch hydrogels to fix AuNPs to a surface, five solutions of Starch-AuNPs were prepared at different concentrations. Drops of these solutions were placed on paper and allowed to air dry. After the drops were fully dry, the color difference between the adsorbed samples was discerned visually and it was noticeable that those samples with the highest concentration of Starch allowed AuNPs to remain stable under dry conditions, this is known because of the red color of the sample indicating that the AuNP is not aggregated, while those samples with low or no Starch caused AuNPs to aggregate upon drying A. PapersGold nanoparticles and Starch-suspended gold nanoparticle samples were analyzed using a UV-Vis spectrophotometer. 630/Abs525 ratio level of aggregation [A quantitative study using UV-Vis absorption spectroscopy was performed to analyze the effect of depletion stabilization. The peak ratio at 630 nm and 525 nm was employed to quantitatively scale the aggregation effect on the particles since there is a linear dependence of the Absregation . Therefo2 to test the limit of stability that Starch provides , since cfDNA in plasma is associated with cancer and other pathologies. Circulating DNA is present in the blood of all individuals, but it has been found that patients with cancer have far greater levels of circulating DNA in their blood than do healthy subjects . Howeverβ-cyclodextrin. Dextrin is a mixture of polymers of D-glucose units linked by α-(1→4) and α-(1→6) glycosidic bonds produced by the hydrolysis of Starch or Glycogen [α (1→4) glycosidic bonds [β-cyclodextrin is a cyclic derivative of starch, prepared from partially hydrolyzed starch  by an enzymatic process. All three polysaccharides are able to form hydrogels [In addition to studying the effect that Starch hydrogels may have on the stability of gold nanoparticles, other starch derivatives were also studied including dextrin, maltodextrin and Glycogen . Maltodeic bonds  and β-cyydrogels .β-Cyclodextrin, these samples began to aggregate at 50 mM of NaCl and reached full aggregation at 200 mM, while the control samples reached full aggregation at 1 M. Meanwhile with Maltodextrin an opposite effect was observed because the AuNPs got aggregated even faster, at 25 mM (versus the 50 mM of the control sample) and, in the case of the 10 mg/mL suspension, samples became fully aggregated at 100 mM of NaCl. It is also important to note that the 25 mg/mL and the 50 mg/mL solutions of Maltodextrin/AuNPs suspension could not be prepared because the solutions aggregated upon contact with the AuNPs. When comparing the structures of these four polysaccharides, it can be seen that Starch and Dextrin have in common the α-1,6-glycosidic bond, allowing to form a branched and more complex structure. It is possible that these branches are involved in providing stability to the AuNP, but more research is needed to understand the exact mechanism of action.To study the stability effect of more hydrogels on the gold nanoparticles, other AuNPs-Starch samples were titrated with increasing amounts of NaCl solution . In the A method was developed by which AuNPs can be bound to a surface. The Starch hydrogel suspension has shown that it can stabilize the AuNPs by depletion stabilization, where at a certain polymer concentration, the repulsive energy barrier becomes high enough to allow particles to be kinetically stabilized. A depletion stabilization instead of steric stabilization was considered herein because the Starch hydrogel should block the particle surface from absorbing other molecules. The experiment with DNA solutions also suggests that other molecules are still able to interact with these AuNPs. Additionally, a proof-of-concept of a label-free, colorimetric biosensor that utilizes immobilized AuNPs on an optically transparent substrate was developed. This biosensor provides a straightforward and efficient system that can be easily implemented in most laboratories. While the findings herein are promising, a lot still remains to be done to optimize this device for clinical trial research. These include: (1) further characterization of the biosensor’s sensitivity and dynamic range as a function of the size of the DNA fragment and its binding affinity in absolute terms, i.e., molecules per unit area, (2) direct kinetics and sensitivity in steady-state reactions, and (3) characterization in terms of the effect of colloid size and shape on the biosensors respon


The visible absorption spectra (500-800 nm) for each starch sample were used as a unique fingerprint, and then elaborated by cluster analysis. The cluster analysis managed to distinguish data of two clusters, a cereal type cluster and a potato/tapioca/rice starch cluster. The cereal subclusters extensively distinguished wheat/barley/rye starches from corn starches. Data for cultivars were mostly in good agreement within the same subclaster. The proposed method that combines cluster analysis and visible absorbance data for starch-triiodide complex was able to distinguish starch of different botanical origins and cultivars within the same species. This method is simpler and more convenient than standard time-consuming methods.The botanical origin of starch is of importance in industrial applications and food processing because it may influence the properties of the final product. Current microscopic methods are time-consuming. Starch consists of an origin-dependent amylose/amylopectin ratio. Triiodide ions bind characteristically to the amylose and amylopectin depending on the botanical origin of the starch. The absorbance of the starch-triiodide complex was measured for: wheat, potato, corn, rye, barley, rice, tapioca and unknown origin starch; and within the different cultivars. Each starch sample had specific parameters: starch-triiodide complex peak wavelength maximum ( Waxy corn, wheat and potato commercial starch samples were purchased from Sigma-Aldrich, Merck . Commercial starch of unknown (US) origin was purchased from Kemika . Potassium triiodide solution was prepared using iodine and potassium iodide, both purchased from Sigma–Aldrich, Merck. Glacial acetic acid was purchased from Panreac  and sodium acetate trihydrate was purchased from J.T. Baker .The sample seed coats were peeled off, and an alkaline steeping method  in a previously prepared acetate buffer solution (pH=6.0) in a closed 100-mL volumetric flask. After the solution was heated and stirred for 10 min in a sonic bath , it was allowed to cool to room temperature and was then diluted to 100 mL with deionized water in a volumetric flask. For each starch type, five independent solutions were prepared each day to avoid microbial degradation.The potassium triiodide solution was prepared by dissolving solid iodine (600 µM) in a 0.03 M potassium iodide solution. Iodine is very toxic and easily sublimates, which makes it difficult to weigh; the weighing procedure should be fast but accurate. Iodine has low water solubility; thus, it was dissolved in the following order. First, potassium iodide was dissolved in a small volume of water, making it possible to dissolve slowly the iodine. After the iodide solution was vigorously stirred, the iodine was completely dissolved, and the volumetric flask was filled to the mark.d=400 µm optical fibre, measuring cuvette holder and AvaLight-DH-S light source, with AvaSoft v. 7.0. software  =0, 0.25, 0.50, 0.75, 1.0 and 1.25 mL. The previously prepared potassium triiodide solution was then added to the starch-filled volumetric flasks in opposite incremental volume order V(I3-)=1.25, 1.0, 0.75, 0.5, 0.25 and 0 mL. The volumetric flasks were filled to the mark with deionized water, stirred in a sonic bath for 5 min and then were ready for further investigation. Subsequently, the spectrophotometric measurement data were collected and analysed using licensed Statistica v. 12 software . Rye (Danovski cv.) showed the highest positive shift (+71 nm), waxy corn and corn starch the highest negative (-44 and -46) and potato starch (+7) the lowest shift towards the commercial starch of unknown origin. Rye, barley and wheat varieties exhibited λmax in very narrow λ region. The absorbance increase (∆A) at each λmax was calculated. Potato and rice starch showed the highest absorbance increase, 0.474 and 0.422, respectively. This indicates that the highest amount of triiodide is complexed with starch.The commercial starch of unknown origin was used as a zero for In this paper we used the usually negative influence of amylopectin (that immobilizes some triiodide ions) on amylose determination  as an advantage to get the whole absorbance spectra and actually get the individual fingerprint of each starch sample. These fingerprint datasets were then analysed for the first time and compared with the cluster analysis.Clustering aims at discovering natural groupings of items, thus revealing interesting data patterns in their relations  sample was placed within cluster 1, but it was separated from clusters 1.1 and 1.2, and also distinguished from cluster 1.3. The reason for this could be explained through the origin of the unknown origin starch. At our request, the producer provided the information that this starch (US) was of wheat origin but slightly debranched by the enzymes. When observing the position of the US in cluster 1, this makes sense, it is close enough to cereal-type starch samples in clusters 1.1 and 1.2 and still quite distinguished from corn-type starch in cluster 1.3.On the contrary, ,,The observed distances in different starch spectra separated in clusters see  were in Table 2 ,,. It shouThe presented research successfully combined spectrophotometric data obtained by measuring the starch-triiodide complex (as a unique fingerprint of each starch sample) and statistical cluster analysis to distinguish the starch samples based on different origin, including among different cultivars. The proposed method uses simple instrumentation, is more convenient and easier to use than the standard microscopic methods, and provides an alternative to our recently developed electrochemical methods. The future work will include more comprehensive research on testing the performance of the proposed method. The method will be tested by supervised machine learning method of automatic classification of samples data and compared to other methods for recognition of starch source. The presented method offers a potential application in artificial food determination and also in food processing industry for starch origin control and increase in quality of final product, but potentially, also in botanic determinations and origin relations of different species.


Majority of cancer-related deaths worldwide is attributed to non-small cell lung cancer (NSCLC). G protein-coupled receptor 56 (GPR56) is overexpressed and associated in the progression of NSCLC. The aim of this study is to use immunoinformatics approach in designing a multi-epitope vaccine to target overexpressed GPR56 which can potentially activate antibody-mediated cell death mechanisms and inhibit pathways involved in the proliferation, migration and survival of NSCLC. Herein, the reported overexpression of GPR56 was further investigated by conducting a differential gene expression analysis of NSCLC samples from GEO DataSets (GSE29249). Results confirmed significant overexpression of GPR56 in NSCLC compared to adjacent normal samples. A multi-epitope vaccine (Fvax) was constructed in silico by adjoining B lymphocytes (BL) and helper T lymphocytes (HTL) epitopes from the extracellular sequence of GPR56. Population coverage (PC) of HTL epitopes was also estimated. To enhance its immunogenicity, sequences of flagellin domains were fused as adjuvant. Fvax was evaluated in silico for antigenicity, allergenicity, peptide toxicity, physicochemical properties and cross-reactivity. Its tertiary structure was predicted, refined, and validated followed by structural epitope prediction. Lastly, Fvax DNA was optimized and cloned in silico. This is the first work to design a potential vaccine against GPR56-overexpressing NSCLC. Fvax has 3 BL and 7 HTL immunogenic epitopes on GPR56. In silico evaluations suggest that Fvax is antigenic, non-toxic, non-allergenic, stable, and has accessible BL epitopes with high PC worldwide for HTL epitopes. Overall, results showed that Fvax is a potential vaccine against NSCLC. The approach of this study efficiently minimized the number of tests, cost and time required to select the best epitopes and to design a vaccine for the treatment of NSCLC. Lung cancer is the major cause of cancer-related deaths globally . Approximately 87% of lung cancer cases are non-small cell lung cancer (NSCLC) . Patients with NSCLC often have different therapeutic needs depending on the tumor presentation. Combinations of chemotherapeutic agents, radiation therapy, immunotherapy, and surgery are currently used; however, these are often accompanied by tumor regression, metastasis and adverse events. To date, there is no single remedy available to completely treat NSCLC due to its complications. Advancements in the understanding of the immune system lead to the development of immunotherapy against cancer. Studies on cancer vaccines targeting tumor-associated antigens (TAA) have recently entered clinical trials with encouraging results . TAA includes overexpressed signalling receptors on the surface of cancer cells. G protein-coupled receptors (GPRs) are signalling receptors playing important roles in cell signal transductions, cell adhesion and cell-cell interactions. GPRs have been implicated in the development of diseases like cancer, one of these is the G Protein-Coupled Receptor 56 (GPR56) with 693 amino acid (aa) residues, encoded by Adhesion G-Protein Coupled Receptor G1 (ADGRG1) gene. Immunohistochemistry and overexpression studies showed that GPR56 is overexpressed in NSCLC and is involved in promoting its proliferation and invasion . Moreover, Song and colleagues (2016) found that the expression of GPR56 is correlated with the TNM stage of NSCLC. Overexpression of GPR56 has been also associated in the progression of other cancer types . Taking advantage of the overexpression and roles of GPR56 in NSCLC, designing a vaccine containing BL and HTL epitopes to induce immune responses against GPR56-overexpressing NSCLC is a potent strategy.One of the requirements to activate TAA-specific helper T lymphocytes (HTL) which has escaped clonal deletion is to break the natural immune tolerance towards that TAA. A signal must be triggered by pathogen-derived molecules containing pathogen-associated molecular patterns (PAMPs) which are recognized by pathogen-recognition receptors (PRR) expressed on the surface of immune and non-immune cells. Among these PRRs are toll-like receptors (TLR). TLR5 is a member of TLR family known to bind and recognize flagellin . Flagellin is a pathogen-associated protein found in flagellated bacteria, and has been successfully employed as safe and strong adjuvant to vaccines in clinical trials . Thus, this work incorporated flagellin domains from Salmonella typhimurium (fliC) as adjuvant.The main purpose of this work is to design a novel multi-epitope vaccine against GPR56-overexpressing NSCLC, which can also potentially augment available therapies. Peptide-based vaccines can elicit immune responses against cancer cells . The development of peptide-based vaccines has been more efficient and cost-effective through the use of in silico techniques and bioinformatics tools. Confirmation of the overexpression of GPR56 in NSCLCPrior to this study, the overexpression of GPR56 in NSCLC and various cancer types have been reported. As a confirmatory step, gene expression profile of ADGRG1 (encoding GRP56) from Gene Expression Omnibus (GEO) database (GSE29249) was analyzed with GEO2R. Tissue profiles from NSCLC samples (n=6) were compared to normal adjacent samples (n=6). Data log transformation was applied in the analysis.Prediction of candidate linear B lymphocyte epitopesThe extracellular sequence of GPR56 (Q9Y653) with 377 aa (26-402) was retrieved from UniProt database to predict linear BL epitopes using multiple tools. Default thresholds in Emini Surface Accessibility (ESA), BepiPred Linear Epitope (BLE), and Kolaskar and Tongaonkar Antigenicity (KTA) tools at Immune Epitope Database (IEDB) and ABCPred Server were used to generate linear BL epitopes. Consensus BL epitopes with 20 residues from at least 2 servers were further assessed for antigenicity, allergenicity, and toxicity. Prediction of helper T lymphocyte epitopes50 <500 nM which classifies epitopes as good binders . Consensus sequences binding to at least 4 of the most frequent MHC II alleles with 15 residues were further evaluated for antigenicity, allergenicity and toxicity.The binding affinity of HTL epitopes were predicted from the extracellular sequence of GPR56 using SMM-align method in NetMHCII 1.1 of IEDB Server. This method was validated to be superior to other MHC II epitope prediction methods . HTL epitopes were retrieved using the most frequent MHC II alleles , and ICEvaluation of antigenicity, allergenicity and toxicity of epitopesAntigenic epitopes were identified in Vaxijen 2.0 server using tumor as target with threshold ≥ 0.5. Vaxijen is an alignment-independent method which predicts antigen with 70%-89% accuracy . Potential allergenic sites were assessed using AllergenFP v 1.0. The highest Tanimoto similarity index of an epitope matching an allergen from the database is used to identify whether the epitope is a probable allergen . Lastly, to avoid potential toxicity, epitopes were scanned in ToxinPred server using default SVM parameters. Estimation of population coverageThe use of multi-epitopes could have larger population coverage (PC). In this study, the set of candidate HTL epitopes with their corresponding MHC II alleles was queried to estimate the worldwide PC using the Population Coverage tool in IEDB. In addition, PC was also calculated in areas with higher incidence rate of lung cancer including USA, Japan, China and countries that are in Europe . Construction of Fvax Candidate BL and HTL epitopes were adjoined together using GPGPG linkers. Amino acid sequences of fliC (P06179) were linked as an adjuvant. FliC N-terminal region from D0-D1 (2-175) was adjoined with the N-terminus of multi-epitope construct via flexible EAAAK linker. Then, the C-terminus of the construct was fused with the C-terminal region of fliC D1-D0 (398-495) via EAAAK linker. Valine was added at the N-terminus of the construct to increase its half-life. To aid in efficient purification, 6-histidine tag was added at the C-terminus.Evaluation of cross-reactivity, antigenicity, allergenicity, and toxicity Possible cross-reactivity of Fvax against proteins expressed in Homo sapiens was evaluated using protein-protein BLAST (BLASTp). Default parameters were used to search for the possible hits against UniProtKB and SwissProt databases. Allergenicity, antigenicity and toxicity of the whole construct were further assessed in AllergenFP v.1.0, Vaxijen 2.0, and ToxinPred server, respectively. Estimation of physicochemical properties of FvaxMolecular weight, amino acid composition, isoelectric point (pI), half-life, instability index, thermostability  and the grand average hydropathicity (GRAVY) of Fvax were calculated using ProtParam tool in ExPASy (https://web.expasy.org/protparam/).Secondary and tertiary structure prediction, refinement and validation of FvaxPercentage composition of the secondary structures in Fvax was estimated using GOR4 tool (https://npsa-prabi.ibcp.fr/NPSA/npsa_gor4.html). RaptorX was used to identify solvent accessible and disordered regions (http://raptorx.uchicago.edu/StructurePropertyPred/predict/) in the vaccine. The tertiary structure of Fvax was predicted using GalaxyTBM tool  which employs multiple-template approach and was evaluated to be one of the top TBM servers . The initial structures were refined using GalaxyRefine tool . To check the validity and to compare the initial and refined structures, ERRAT, ProSA-web, and PROCHECK tools were employed. ERRAT (https://servicesn.mbi.ucla.edu/ERRAT/) analyzes nonbonded atom-to-atom interactions in the input structure as compared to x-ray crystallography structures . ProSa-web server (https://prosa.services.came.sbg.ac.at/prosa.php) calculates the z-score which estimates the deviation of the structure from the validated x-ray crystallography and NMR structures of native proteins . PROCHECK (https://servicesn.mbi.ucla.edu/PROCHECK/) gives Ramachandran plots and shows the percentage of residues lying within the most favoured, and disallowed regions. A model with good quality is expected to have at least 90% of residues within the most favoured region . The best tertiary structure model for Fvax was viewed using PyMol and was further assessed for structural BL epitopes.Prediction of structure-based epitopes on Fvax The sequence of epitopes must be accessible and protruded enough so BCRs can bind to it. Ellipro (http://tools.iedb.org/ellipro/) predicts discontinuous and linear BL epitopes based from the protrusion index (PI) of a residue, and provides PI score for each protruded sequence. This tool was used to identify structural epitopes in Fvax.In silico cloning optimization of FvaxFor the efficient cloning of Fvax in E. coli K12 strain, in silico codon optimization was conducted using Java Codon Adaptation Tool (JCAT) (http://www.jcat.de/). It calculates the GC-content and the Codon Adaptation Index (CAI) used to approximate the level of gene expression. The closer the CAI-value to 1, the more it is expressed in a host . Finally, the optimized sequence of Fvax was inserted into pET-30(+) as a vector using SnapGene tool.Overexpression of GPR56 in NSCLC tissue samples Results showed that ADGRG1 is overexpressed approximately twice higher in NSCLC than the normal adjacent tissues. Probe ILMN_2384122 shows significant differential gene expression between NSCLC group and normal group with p-value 0.040, and 1.1 log2-fold change NSCLC/normal or 2.144–fold increase . While pCandidate linear BL epitopesESA, BLE, KTA, and ABCPred prediction tools resulted to initial linear BL epitopes with 1-76 residues. From these epitopes, 3 epitopes (20aa) consensus from at least 2 servers, are classified to be antigenic, non-allergenic and non-toxic. Candidate HTL epitopesResulting epitopes from NetMHCII 1.1 were trimmed down to 7 HTL epitopes (15aa). Each has a good binding affinity to at least 4 of the most common human MHC II alleles and are classified as antigenic, non-allergenic, and non-toxic. Population coverage of HTL epitopes The estimated worldwide PC for the set of HTL epitopes in Fvax is 83.81%. Moreover, the PC for the epitopes in areas with high incidence rates of lung cancer are 87.47% in Europe, 60.8 % in China, 75.04% in Japan, and 90.2% in USA.Fvax constructCross-reactivity, antigenicity and toxicity of FvaxFvax has no significant sequence similarity with other known human proteins except for GPR56 which implies specificity towards the target. The percentage identity of Fvax with GPR56 is 54.13%  indicating significant sequence similarity. Fvax is classified as antigenic (0.6306) in tumor target, and is also non-allergenic with highest Tanimoto similarity index (0.87) to Q9Y4F5. Furthermore, no toxic peptide sequences were identified within the whole construct.Physicochemical properties of FvaxFvax is consist of 499 residues with molecular weight of 52,707.25 Da, and pI of 6.37 which can be used for its purification using isoelectric focusing. The estimated half-life of Fvax is 100 hours in mammalian reticulocytes in vitro, >20 hours in yeast in vivo, and > 10 hours in E. coli in vivo. The instability index is 38.64 which classifies Fvax as stable (<40). In addition, aliphatic index of 78.08 indicates thermal stability while the negative value of its GRAVY (-0.463) classifies it to be hydrophilic. Secondary structure of FvaxPrediction, refinement, and validation of the tertiary structure of FvaxPredicted tertiary structure models were validated and further refined for improvement. The quality of the initial structure has notably improved in the refined version. ERRAT quality factor increased from 85.13 to 89.43 . RamachaStructural BL epitopesTo further assess if the 3 linear BL epitopes included in Fvax are still accessible enough to bind with BCRs though they are adjoined with other sequences forming a tertiary structure, the protruded regions on the tertiary structure of Fvax, known here as structural BL epitopes, were identified. Ellipro predicted 7 linear  and 3 diIn silico cloning optimization of FvaxThe CAI-value for the optimized codons of Fvax is 0.92 which is very close to 1. While GC-content is 55.64% which is within the optimal range (30%-70%). These results convey that the optimized codons of Fvax can be highly expressed in E. coli K12 strain as host. The reported overexpression of GPR56 in NSCLC, which was further confirmed in this study, and its association with the mechanisms involved in the progression of NSCLC, motivated the researcher to find novel BL and HTL epitopes from the extracellular domain of GPR56. Evaluated antigenic and immunogenic sequences of GPR56 using various immunoinformatics tools can potentially activate immune responses against GPR56-overexpressing NSCLC. Studies suggest that anti-tumor mechanisms against overexpressed receptors involve signalling blockade which affects cancer-associated pathways, complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) . Most of these mechanisms require cross-linking of antibodies on the surface of tumor cells or certain receptors on effector cells, making these mechanisms more effective towards cancer cells due to the overexpression of TAAs on their surface . On the The activation of BL using peptide-based vaccines requires the presentation of MHCII-peptide complex to an activated HTL. One of the major challenges in the development of peptide vaccines is the MHC haplotype-restricted antigen recognition. Thus, this study included HTL epitopes with good binding affinity towards the most frequent MHC II alleles. Candidate HTL epitopes have high PC worldwide (83.81%), and more importantly, have large PC (60.8% to 90.2%) in several areas with high incidence rate of lung cancer (majority is NSCLC) emphasizing the immunogenicity of Fvax as a potential immunotherapeutic agent.Due to the intrinsic nature of peptides from GPR56, the presence of PAMPs in biomolecules known to bind to PRRs, is required to elicit immune response. In this study, the type of PRR activated by a specific adjuvant was carefully considered. Activation of some TLRs was reported to have tumor-stimulating activities . On the other hand, TLR5 signalling was found to have anti-tumor effects in NSCLC cells . A well studied PAMP known to activate TLR5 is flagellin. Flagellin has four domains, namely, D0, D1, D2 and D3. Studies showed that highly conserved D0 and D1 play critical roles in binding and activating TLR5 . The binding of flagellin with TLR5 was also demonstrated to enhance antitumor immune responses . Due to reported efficacy, safety and anti-tumor benefits, flagellin was incorporated as an adjuvant in this work. And to avoid other sequences that may result to adverse reactions, only D1 and D0 were fused with the epitopes of Fvax.The form and stability of vaccines should be considered to ensure efficient delivery of targeted actions. This work focused on developing multi-epitope vaccine which has several advantages over single peptides. Superiorities include simultaneous induction of immune response due to the adjoined antigenic and promiscuous epitopes, linked adjuvant which increases immunogenicity of the epitopes, and exclusion of sequences that can result to adverse effects. Fvax construct consists of 3 linear BL and 7 HTL epitopes which were assessed to be antigenic, non-allergenic and non-toxic. Epitopes were adjoined using GPGPG linkers proven to present sufficient epitopes in vivo . While flexible EAAAK linkers were used to preserve the bioactivity  of flagellin D0-D1 in Fvax. The addition of valine residue at the N-terminus of Fvax confers stability as implied by the drastic increase in its half-life from 4.4 hours  to 100 hours in mammalian reticulocytes. Moreover, the instability index classifies Fvax to be stable which implies longer protein half-life in vivo based from its dipeptide composition . In addition, its GRAVY value indicates that Fvax is hydrophilic; thus, it can be easily dissolved in polar solvents like water.Data from this study showed that the whole Fvax construct is antigenic using tumor as target. The antigenicity of epitopes is also dependent to their exposure in the vaccine conformation where BCRs can easily access. The secondary structures of the candidate BL epitopes (181-250) are random coils and extended strands, indicating exposure of these sequences. These epitopes are also found within the disordered regions of Fvax. Disordered regions have less folding structures; thus, sequences are less likely buried. The linear BL epitopes are also located within the solvent accessible regions which further emphasize their exposure. The tertiary structure of Fvax was predicted, refined, and validated. Results from the analysis justified the need for refinement and validated the tertiary structure model of Fvax. Prediction of structural epitopes also showed that the 3 linear BL epitopes included in the vaccine are highly protruded and accessible in the tertiary structure of Fvax. This work further evaluated the safety of Fvax as an immunotherapeutic agent. Vaccines that generate immune responses against very similar protein sequences in humans may result to adverse reactions. Favourably, Fvax was not found to have significant sequence similarity with other proteins expressed in humans except for GPR56, indicating its specificity towards the target. The construct was also classified as non-allergen, and non-toxic indicating its safety as a potential immunotherapeutic agent.In conclusion, this is the first work to design a vaccine against GPR56-overexpressing NSCLC. In silico evaluations showed that Fvax confers stability, safety, and contains epitopes that can potentially stimulate BL and HTL immune responses. Nonetheless, the application of Fvax is anticipated to be authenticated both in vitro and in vivo.


ApoE−/−Svep1+/−) compared to Svep1 wild-type mice (ApoE−/−Svep1+/+) and ApoE−/−Svep1+/− mice displayed elevated plaque neutrophil, Ly6Chigh monocyte, and macrophage numbers. We assessed how leukocytes accumulated more inside plaques in ApoE−/−Svep1+/− mice and found enhanced leukocyte recruitment from blood into plaques. In vitro, we examined how SVEP1 deficiency promotes leukocyte recruitment and found elevated expression of the leukocyte attractant chemokine (C-X-C motif) ligand 1 (CXCL1) in EC after incubation with missense compared to wild-type SVEP1. Increasing wild-type SVEP1 levels silenced endothelial CXCL1 release. In line, plasma Cxcl1 levels were elevated in ApoE−/−Svep1+/− mice. Our studies reveal an atheroprotective role of SVEP1. Deficiency of wild-type Svep1 increased endothelial CXCL1 expression leading to enhanced recruitment of proinflammatory leukocytes from blood to plaque. Consequently, elevated vascular inflammation resulted in enhanced plaque progression in Svep1 deficiency.A missense variant of the sushi, von Willebrand factor type A, EGF and pentraxin domain containing protein 1 (SVEP1) is genome-wide significantly associated with coronary artery disease. The mechanisms how SVEP1 impacts atherosclerosis are not known. We found endothelial cells (EC) and vascular smooth muscle cells to represent the major cellular source of SVEP1 in plaques. Plaques were larger in atherosclerosis-prone Svep1 haploinsufficient (The online version of this article (10.1007/s00395-020-00828-6) contains supplementary material, which is available to authorized users. SVEP1  is genome-wide significantly associated with CAD/MI   and aortae   for 12 weeks followed by histological analysis of the aortic root. Total plasma cholesterol levels rose similarly in both genotypes  Fig. a. To invApoE−/−Svep1+/+ and ApoE−/−Svep1+/– mice using flow cytometry. Plaque macrophage , Ly6Chigh monocyte , and neutrophil  numbers increased in ApoE−/−Svep1+/– over ApoE−/−Svep1+/+ mice  monocytes/macrophages in plaques of ApoE−/−Svep1+/– mice . Whether this population derives from recruited Ly6Clow/high blood monocytes or from Ly6Clow lesional macrophages is currently poorly understood , blood neutrophils , blood Ly6Chigh monocytes , and blood Ly6Clow monocytes  were comparable between ApoE−/−Svep1+/– and ApoE−/−Svep1+/+ mice  monocytes and neutrophils from naïve transgenic mice (Ubc-GFP) and adoptively transferred these cells into either ApoE−/−Svep1+/– or ApoE−/−Svep1+/+ mice which were fed a WD for 6 weeks . Using flow cytometry 24 h after the transfer, we detected more GFPhigh myeloid cells inside plaques in ApoE−/−Svep1+/– in comparison to ApoE−/−Svep1+/+ mice  indicating that SVEP1 affects leukocyte recruitment , were incubated with either wild-type SVEP1 or missense SVEP1 (SVEP1_p.D2702G). While investigating a panel of inflammatory transcripts, we found that incubation with SVEP1_p.D2702G, i.e. mutant SVEP1 without wild-type SVEP1, led to an increase in chemokine (C-X-C motif) ligand 1 (CXCL1) mRNA levels [+ 49.2 ± 0.15 (%), p = 0.03; Fig. CXCL1 expression and Svep1-deficient mice showed enlarged plaque sizes, we wondered whether SVEP1 directly impacts endothelial CXCL1 expression. After incubation with concentrated conditioned media containing wild-type SVEP1, CXCL1 protein levels in EC supernatant decreased when exposed to SVEP1 compared to control , p < 0.01; Fig. Leukocyte recruitment is a process in which EC and circulating leukocytes act in concert to mediate rolling, adhesion, and transmigration . Hence, SELE expression in EC when exposed to wild-type SVEP1 compared to control . SELE encodes E-selectin, a cell adhesion molecule that together with chemokines is pivotal in mediating leukocyte recruitment [In addition to the chemokine CXCL1, we also detected lower ruitment . Our datSVEP1 phenome-wide association study (PheWAS) in 445,504 individuals included in UK Biobank [padj = 0.02) and inguinal hernia  but a reduced risk of uterine fibroids   leads to decreased SVEP1 function, we analyzed EC phenotypes after incubation with either SVEP1 or SVEP1_p.D2702G. Adding SVEP1_p.D2702G was associated with increased expression of the chemokine CXCL1 and the adhesion molecule SELE suggesting wild-type SVEP1 plays a rather atheroprotective role that was altered in the presence of the SVEP1_p.D2702G variant. In CAD cases, we further found reduced SVEP1 mRNA levels compared to controls. In addition, our data show that SVEP1 exerts regulating effects on EC resulting in decreased CXCL1 and SELE expression in the presence of higher SVEP1 levels in vitro. Both CXCL1 and E-selectin mediate leukocyte recruitment from blood to plaques and hence play a crucial role in the early stages of atherosclerosis when dysfunctional EC secrete chemokines/upregulate adhesion molecules and thereby induce monocyte and neutrophil adhesion [Svep1 also displayed elevated Cxcl1 plasma levels under proatherogenic conditions. Also, the observed increased atherosclerotic plaque formation and more numerous neutrophils, Ly6Chigh monocytes, and macrophages inside plaques of ApoE−/−Svep1+/− compared to ApoE−/−Svep1+/+ mice accord with the downstream effects of CXCL1 and E-selectin in atherosclerosis [ADAMTS7 gene has been linked to atherosclerotic plaque formation in humans [Knowledge of the molecular and cellular functions of SVEP1 is limited. Besides its role in lymphatic vessel formation , 23, SVEs lethal , 23 suggnditions , 35. Thiadhesion , 37. In clerosis , 37. Of clerosis . The ADAn humans , 30, 33 Drug targets identified by genetic analyses reportedly have higher success rates in the drug development pipeline . Our finIn summary, we hypothesize that SVEP1 which is produced by vascular cell types and secreted to the ECM of the vascular wall acts as a silencing factor on EC activity by reducing the expression of chemokines and cell adhesion molecules, e.g., CXCL1 and E-selectin. If wild-type SVEP1 protein levels are reduced, e.g. via degradation by MMPs, or in the presence of mutant SVEP1 (SVEP1_p.D2702G missense variant), CXCL1 and E-selectin expression rises leading to enhanced inflammatory cell influx. Figure SVEP1, other cell types might also produce SVEP1 and thus influence plaque monocyte/macrophage phenotypes.Whereas in this study, we postulate an atheroprotective effect of SVEP1 on EC, we cannot exclude further functions of SVEP1, particularly in other cell types. Specifically, the CAD risk variant has been shown to be associated with slight effects on blood pressure . As SVEPSvep1 which might not fully replicate the effects of the human risk variant since loss of intact wild-type SVEP1 may vary. However, we detected reduced SVEP1 mRNA levels in vascular tissues from patients with CAD, as compared to controls, an event reflected by the mouse model we used. Third, and most importantly, findings from in vitro experiments do not necessarily reflect in vivo situations. In most in vitro approaches, the environment that naturally surrounds cells—in our case the ECM—is missing. Consequently, in vitro assays are somewhat artificial. We found that loss of SVEP1 leads to increased CXCL1 levels, both in vitro and in vivo. This indicates that at least with respect CXCL1 release, our in vitro data resemble our in vivo findings. It is of course possible that soluble SVEP1 exerts further in vivo effects that are not addressed in this study.Our study has several limitations. First, we do not know how SVEP1 mediates its effects on EC on a molecular level. ECM proteins have diverse effects on adjacent cell types that may surpass the known interaction with integrin α9β1. Second, we investigated a heterozygous knockout of Supplementary file1 (DOCX 18387 KB)Below is the link to the electronic supplementary material.


The hepatocellular carcinoma (HCC) represents a serious malignancy worldwide especially in China. Our transcriptome analysis identifies a novel long non‐coding RNA (lncRNA) termed HLNC1. However, the function of HLNC1 in HCC remains to be determined.USP49 as HLNC1 binding partner.Novel lncRNAs were screened using lncRNA profiling. Relative expression was quantified by qRT‐PCR. In vitro experiments such as migration and viability assays were performed. In vivo implantation experiments were conducted to investigate tumorigenic functions. RNA‐RNA interaction assay was performed to determine USP49 transcript as the HLNC1 binding partner. HLNC1‐USP49 interaction dramatically destabilized USP49. Heat‐shock factor 1 (HSF1) was shown to directly induce HLNC1 expression. The therapeutic potential of targeting HLNC1 was investigated using antisense oligonucleotides (ASOs). The ASO construct which significantly depleted HLNC1 expression could strongly attenuate xenograft tumor growth.We found that HLNC1 was markedly upregulated in HCC samples and cell lines. HLNC1 could promote viability and migration of HCC cells. Meanwhile, we could also observe an oncogenic effect of HLNC1 in vivo. By RNA‐RNA interaction assay, we unraveled Our data suggested that HLNC1 may advance HCC progression and act as a potential target for intervention. We identified a novel lncRNA termed HLNC1 and HLNC1 was significantly upregulated in HCC samples. The ASO construct which significantly depleted HLNC1 expression could strongly attenuate xenograft tumor growth. HLNC1 may advance HCC progression and act as a potential target for intervention. Fraction of these transcripts with >200 nucleotides in length has been defined as long non‐coding RNAs (lncRNAs).lncRNA on chromosome 1 (HLNC1) by lncRNA profiling. We found HLNC1 is significantly upregulated in HCC tissues and human specimens. HLNC1 primarily localizes in the cytoplasm and promotes viability and migration of HCC cells. Meanwhile, HLNC1 also plays an oncogenic role to advance in vivo tumor growth. Mechanistic studies showed that HLNC1 interacts with USP49 mRNA via its N‐terminal region. HLNC1‐USP49 interaction dramatically destabilizes USP49 transcripts. Interestingly, HSF1 is responsible for HLNC1 induction and HLNC1 promoter displays enhanced HSF1 occupancy. The therapeutic effect of HLNC1 is further evaluated using antisense oligonucleotides (ASOs) and ASO‐mediated HLNC1 silence profoundly inhibits HCC growth and metastasis. Collectively, we have shown an oncogenic role for the novel lncRNA HLNC1 in HCC and HLNC1 may serve as a potential therapeutic target for pharmaceutical intervention.In current work, we identified a novel lncRNA termed hepatocellular carcinoma‐associated 22.1293T and HCC cell lines were obtained from Shanghai Institute of Cell Biology. HCC cells were maintained in DMEM with high glucose, 8% FBS, 150 µg/mL penicillin, and 100 U/mL streptomycin. Throughout all the experiments, passage number varied between 3rd and 10th for all cells. For every experiment, it was ensured that cells had been passaged at least thrice after thawing. Cell lines were verified by PCR without contamination by mycoplasma.2.2The HCC samples and paired normal adjacent tissues were collected from the primary sites in patients as previously described from February 2015 to Jun 2018.2.3Total RNAs were extracted with Trizol reagent, according to the manufacturer's instructions. For lncRNA expression, the first‐strand cDNA was synthesized using Superscript I (Invitrogen) with random primers. qPCR was performed by a miScript SYBR Green PCR Kit (Qiagen). The primers are shown in Table 2.4The lentivirus plasmid for stable RNA interference and the short hairpins targeting HLNC1 (shHLNC1) were designed and purchased from BGI (Beijing). A non‐targeting silencing RNA was used as negative control (shCtrl). Virus packaging was performed in 293T cell after co‐transfection of pLVX lentivirus plasmids using Lipofectamine 2000. To established stable HLNC1 overexpression clones, sub‐confluent cells were transfected with an empty pCMV expression plasmid (control) or pCMV vector containing HLNC1 (HLNC1) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After 48‐h transfection, G418 (600 mg/L) was added for selecting stable clones.2.5post hoc. At least three replicates were shown. P < .05 was statistically significant.Statistics were calculated using SPSS (version 16). Data were represented as mean ± SD. Mann‐Whitney test was selected for comparison between two groups and ANOVA was used for detecting significance among more than three groups followed by LSD 33.1To explore HCC‐associated lncRNAs, we performed RNA sequencing (RNA‐seq) in normal and cancerous tissues Figure . Results3.2Since HLNC1 was significantly upregulated in HCC cell lines and tissues, we then explored the effect of HLNC1 in vitro. Stably transfected HepG2 and KYN‐2 cell lines were generated to significantly overexpress HLNC1 Figure  or knock3.3Since HLNC1 has an oncogenic role in vitro, we anticipated that HLNC1 might also exhibit an oncogenic effect in vivo. We found that HLNC1 overexpression indeed resulted in an expansion of tumor volumes whereas decreasing HLNC1 played an inhibitory effect Figure . Meanwhi3.4ubiquitin carboxyl‐terminal hydrolase 49 (USP49) mRNA was a putative target for HLNC1 binding . The secondary structure was predicted via online tool RNAfold . Six ASOs were designed and qRT‐PCR data showed that all ASOs significantly decreased HLNC1 expression in HepG2 cells Figure . Since A4USP49 mRNA to increase USP49 turnover. HSF1 was shown to directly activate HLNC1 transcription. Therefore, we identified a novel HSF1‐HLNC1‐USP49 axis during hepatocellular carcinoma progression. Targeting the HSF1‐HLNC1‐USP49 axis by specific antisense oligonucleotides for HLNC1 has presented a promising effect to eradicate HCC cells.In this work, we identified a novel lncRNA named HLNC1 and evaluated its role in hepatocellular carcinoma. HLNC1 transcription was dramatically elevated in hepatocellular carcinoma tissues and cell lines. HLNC1 displayed significantly oncogenic effects in vitro and in vivo. Mechanistically, HLNC1 interacted with HSF1 is a conserved helix‐turn‐helix transcription factor which is activated when cells encounter heat‐shock, chemical agents, or other forms of environmental stresses.USP49 mRNA stability. The direct USP49 and HLNC1 binding strongly raised the degrading rate of USP49 transcripts whereas silencing HLNC1 had an opposite effect. The upregulation of HLNC1 in HCC through HSF1 may partially enhance cancer progression via decreasing the level of USP49 mRNA and USP49 proteins were consistently downregulated. Therefore, the novel HSF1‐HLNC1‐USP49 axis may add a novel layer of complexity in HCC.The USP49 has been identified as a tumor suppressor protein which participates in DNA damage response via p53‐positive feedback loop.Rx has already been shown to alter apolipoprotein expression to treat calcific aortic valve stenosis (CAVS) in a clinical trial.Given the oncogenic role of HLNC1, the therapeutic effect of HLNC1 targeting is potentially beneficial for HCC inhibition. Recently, ASOs have been demonstrated to be an effective strategy to target RNA in vivo.USP49 transcripts leading to HCC progression. The novel HSF1‐HLNC1‐USP49 axis may serve as a target for pharmacological intervention to eradicate HCC cells.In conclusion, we have identified a novel lncRNA HLNC1 with potential oncogenic function. HSF1 contributes to HLNC1 induction which then destabilizes Supplementary MaterialClick here for additional data file.


Subcapsular renal hematoma (SRH) is a challenging condition, which may jeopardize kidney function or constitute a life-threatening event. This is particularly true in single-kidney patients, such as kidney-transplant recipients. SRH may exert an excessive pressure on the surrounding parenchyma, thus resulting in hypoperfusion and ischemia, with high risk of acute kidney failure and graft loss. Moreover, SRH may precede an overt renal rupture with subsequent hemorrhage and hemodynamic instability. The indication to an interventional management for this condition is still a matter of debate, with some authors advocating the high possibilities of spontaneous resolution and others advocating the high-risk of graft loss and even internal bleeding in case of overt renal rupture. Herein, we report the case of a 51-year-old simultaneous pancreas-kidney transplantation recipient who presented a SRH following a mild trauma. The therapeutic choices were carefully balanced on the specific case, and the conservative management proved successful. Subcapsular renal hematoma (SRH) is a rare although challenging condition that may constitute a life-threatening event. It is defined as a localized collection of blood underneath the renal capsule. SHR may exert excessive pressure on the surrounding parenchyma, causing renal hypoperfusion and refractory hypertension  or sometimes ischemia . Moreove2.The patient was a 51-year-old man who received diagnosis of type 1 diabetes mellitus at the age of 13, and underwent successful SPKT for the presence of brittle type 1 diabetes mellitus with severe hypoglycemic episodes and stage-4 chronic kidney disease at our University Hospital when he was 36 years old. The pancreatic-duodenal graft was placed in right iliac fossa, the exocrine drainage was made through a direct anastomosis between donor duodenum and recipient small bowel, and the endocrine drainage was made through a venous anastomosis in the right iliac vein. The renal graft was placed in the left iliac fossa. Maintenance immunosuppressive therapy included tacrolimus, mycophenolic acid, and prednisone. In the postoperative period, both the pancreatic and renal graft maintained normal function, and insulin therapy was dismissed. He underwent regular follow-up as an outpatient at our Department, where there is an active kidney and pancreas transplantation follow-up program , 8, for He went to the Emergency Department of a peripheral Hospital for persistent pain in the left iliac fossa. The pain presented following a traumatic episode during a sexual intercourse and underwent a progressive increase in intensity. The patient was referred to our University Hospital for the ultrasound evidence of a subcapsular hematoma on the convex surface of the renal graft.2, glucose 98 mg/dL, HbA1c 38 mmol/mol, hemoglobin 14.7 g/dL, white blood cells 11.72 × 103/μL. Contrast-enhanced computerized tomography showed a subcapsular hematoma of 6.5 × 3 × 12 cm with regular urinary outflow, without signs of contrast leakage outside the vascular bed   or wait-To the best of our knowledge, this is the first report of SRH in a SPKT recipient. In this case, a conservative approach proved successful, leading to the resolution of the hematoma without invasive procedures. This strategy required a careful noninvasive monitoring in the presence of surgical expertise readily available in case of need. Pros and cons of conservative and interventional approaches should be carefully weighted, and the decision-process should be tailored on the specific case. In this specific situation, the additional presence of a pancreatic graft did not influence significantly our therapeutic choices, being the immunosuppressive regimen carefully followed and the glycemic control stable. However, the presence of two different grafts, both of which should possibly be preserved, should be considered when taking decisions about the management of SHR.


This work presents a bio-inspired grasp stiffness control for robotic hands based on the concepts of Common Mode Stiffness (CMS) and Configuration Dependent Stiffness (CDS). Using an ellipsoid representation of the desired grasp stiffness, the algorithm focuses on achieving its geometrical features. Based on preliminary knowledge of the fingers workspace, the method starts by exploring the possible hand poses that maintain the grasp contacts on the object. This outputs a first selection of feasible grasp configurations providing the base for the CDS control. Then, an optimization is performed to find the minimum joint stiffness (CMS control) that would stabilize these grasps. This joint stiffness can be increased afterwards depending on the task requirements. The algorithm finally chooses among all the found stable configurations the one that results in a better approximation of the desired grasp stiffness geometry (CDS). The proposed method results in a reduction of the control complexity, needing to independently regulate the joint positions, but requiring only one input to produce the desired joint stiffness. Moreover, the usage of the fingers pose to attain the desired grasp stiffness results in a more energy-efficient configuration than only relying on the joint stiffness  modifications. The control strategy is evaluated using the fully actuated Allegro Hand while grasping a wide variety of objects. Different desired grasp stiffness profiles are selected to exemplify several stiffness geometries. The human hand shows a high level of dexterity that allows to perform numerous complex tasks. Therefore, it is often used as inspiration for designing robots to operate in human environments.TMThe five-fingered Shadow Dexterous HandWith the aim to introduce principled simplification strategies in the design or control of robotic hands, recent works use underactuated and compliant mechanisms. These are endowed with compliant elements and fewer actuators than degrees of freedom, proving to be an effective way to reduce the design complexity of robotic hands and prostheses, while maintaining their bio-inspired shape  the Configuration Dependent Stiffness (CDS), and (ii) the Common Mode Stiffness (CMS). CDS and CMS mechanisms were firstly observed in the human arm stiffness control. Patterns of stiffness variation depending on the arm posture (CDS) were observed for the human arm in Mussa-Ivaldi et al. , while tSimilarly, regarding the stiffness control of the human hand, Milner and Franklin found that humans can modify the endpoint stiffness geometry by varying the fingers posture (CDS), being this process more energy-efficient than using muscle co-contraction . Furthermore, the method targets to use the minimum required sensory information, taking as input only the joint positions.The approach detailed in this paper proposes to exploit the advantages given by the CDS and CMS concepts in robotic hands. The aim is to obtain bio-inspired and energy-effective grasps with a simplified control strategy where This work completes and extends the preliminary study presented in Ruiz Garate et al. . Though On the other hand, the approach presented in this paper differs from the one in Ruiz Garate et al.  from a fTherefore, this paper presents the first attempt to provide a complete generalized method for the regulation of the grasp stiffness, based on the exploration of the grasp workspace. After a thorough explanation of our control approach (section 2), we show its performance using the fully actuated Allegro Hand, a robotic hand with 4 fingers and 16 DoFsThe bio-inspired control strategy proposed in this paper exploits the introduced concepts of CDS and CMS to regulate the grasp stiffness of a robotic hand.K∈ℝ6 × 6 is a symmetric matrix that relates the wrench Δw∈ℝ6 applied to an object to its displacement Δu∈ℝ6:Before detailing the control policy, some mathematical background regarding grasp stiffness is needed. Grasp stiffness is a fundamental tool for modeling and controlling compliant robotic grasps. Its main characteristics are discussed in Kao and Ngo , and morG is the grasp matrix relating the contact forces and moments transmitted through the contact points, to the set of wrenches that the hand applies on the object. In our case, nc the number of contact points on the object. where nq is the total number of finger joints in the handwith Kc as well as the resulting grasp stiffness K can be represented by translational and rotational stiffness ellipsoids. Each of these are characterized by a particular geometry (defined by the ellipsoid axes) and volume (magnitude).The endpoint stiffness of the fingers K toward a desired Kd while preserving the grasp contact points. We focus on the translational part of the desired grasp stiffness, i.e., Kd, t, influencing the translational interaction force profiles that play an important role in manipulation of daily objects. For example, doing a precise object placement in a hole, the grasp should be most stiff in the hole direction, whereas it should be compliant in the other ones.The objective of the proposed method is to manipulate the grasp stiffness q (CDS) and the joint stiffness Kq (CMS). The CDS control is implemented by manipulating the vector q that contains the joint angular positions q = . The changes in the hand pose reflect into geometrical variations of the grasp stiffness ellipsoid. On the other hand, the CMS control regulates the synergistic finger stiffness Kq, mainly providing modifications of the stiffness ellipsoid volume. For every finger f, Kq = αΓf, where α is the CMS parameter (Nm/rad) and Γf is a constant normalized vector implementing the coordinated stiffening of the hand fingers  index .Trajectory generation.These processes are described in more detail in the following subsections. For every new grasp, the first two steps need to be executed. Then, the desired stiffness can be changed only requiring to perform the two last ones.q, and (ii) the virtual hand from which the reference joint positions qref are defined such that the actual hand slightly squeezes the object and stabilizes the grasp. This last pose drives the fingers of the virtual hand inside the object  the actual hand based on the measurement of the joint positions The encoder readings of the joints positions are the only sensory information the method relies on. This means that no information needs to be acquired from the real hand in terms of force pressure, orientation, object position, etc. The fact that no extra force or visual information is required makes the method generalizable to almost any hand and object. However, to do so the following assumptions are made:We focus on fingertip grasps, as with power grasps the possibility of having in-hand manipulation is very limited. For the sake of simplicity all contacts are modeled as hard finger contacts where only forces (and no moments) are transmitted , it has to be attached to one of the fingers, similarly to Sundaralingam and Hermans . The thuDue to possible model inaccuracies, for the main stiffness computations the contact locations are approximated to be exactly at the fingertips, whose positions can be retrieved from direct kinematics.qref should drive the fingers toward the inside of the object to stabilize the grasp  corresponding to that force can be found as dq = JT(JJT)−1dx is then applied to the virtual hand model, while keeping the object in place. Figure Similarly to Ruiz Garate et al. , at thisλopt = Eyopt. If the grasp proves to be stable, this configuration is finally commanded to the real hand, assuring the stability of the initial grasp  and Qc in Figure From the fingers workspace obtained preliminary, configurations keeping similar fingertip locations as the initial ones are stored  is defined as:As outlined in Figure fminbnd  is used, optimizing α by first computing the grasp stiffness and then trying to find the stabilized grasp.To obtain PCR from each configuration, depending on α, new reference finger joint positions are found that stabilize the grasp. This is done following the same procedure as in section 2.2.1. In order to run this optimization, the function Ke is defined as a weighted function on the difference between the desired and actual stiffness parameters described previously (Equations (3)–(5)):The stiffness error This step provides the CMS control for each of the previously found configurations adapting mainly the volume of the grasp stiffness depending on the attainable stability. If the found α is smaller than the one defined for the initial grasp, the latter is kept. Nonetheless, this value can be afterwards manipulated by the user depending on task specifications or external inputs.Ke is kept as the optimal. In this way, the achievement of the desired stiffness is prioritized in the process (CDS control).The optimization stops if incompatible contact points are found during the stabilization and previous values are kept. From all the feasible configurations that result in a stable grasp, finally the one with the lowest The previous steps provide a final stable configuration that best matches the desired stiffness characteristics in terms of orientation and shape. To move from the original configuration to the final one, a trajectory is needed Figure .t = 0.05 s. At each step, the following function is optimized:Based on the work of Sundaralingam and Hermans , we compfmincon function  is used with a maximum of 50 iterations.The joint stiffness obtained from the grasp optimization is applied from the beginning of the trajectory. This value is always equal or higher than the initial stiffness to provide better stability.To evaluate the proposed method, we carried out experiments with the Allegro Hand , a 4-fingered robotic hand with 16 independent torque-controlled joints, 4 joints per fingercf as τg is the gravity torque vector compensating for the weight of the hand. This value was provided with the hand software. In this way, when the joint stiffness is set to very low values, the hand is controlled in gravity compensation mode.where Γf =  for the index, middle, and little gingers, and Γf =  for the thumb. These values are chosen based on the mechanical characteristics of the hand and the selected vertical set-up .To compute the grasp stiffness, the joint stiffness synergy is defined as For the stability optimization, a minimum and maximum contact force of 2 and 5 N respectively are specified. This avoids getting a reference position that goes too much inside the object which, due to the hand configuration, would provide a big push from the upper fingers and prevent the lower finger (the thumb in this case) from moving. Based on the technical specifications from the hand, during the optimization α is allowed to vary between 2 and 6 Nm/rad. The initial value of α is set to 4 Nm/rad.To test the different aspects of the algorithm, we decided to use several objects from the YCB set .1 and θ2) are also visually depicted in Figure Table 1 decreases for all tested cases. This decrease ranges from 50.14% in the worst case to 99.24% in the best. Similar decreases are observed for the secondary axis, where the reduction spans from 66.06 to 91.94%.Difference between desired and initial main axis orientation θFor the cases where a manual translational stiffness is defined (lemon and pudding box), the error in the length ratio β is decreased  or remains almost the same . In the case when the objective stiffness is defined as a rotation of the initial one, the initial error in length ratio is zero. Therefore any change will increase it. This error is in any case kept quite low (below 0.9) except for the apple and the metal mug, where it reaches a value of 4. However, as it can be observed in Figures The minimum required synergistic joint stiffness parameter α Table  is generPCR varies from an initial range of [0.00241–0.5352] to a final of [0.0036–1.8541]. If the minimum found stiffness is considered, PCR decreases for all cases except the apple and gelatin box. However, if the lower bound on the initial stiffness is considered, PCR only decreases significantly in the case of the banana.Finally, Figure From the results shown in Table Regarding the error in the length ratio, as we prioritize the axes orientation, β is only improved if it does not cause major impact in these orientations. Therefore, while in the case of the lemon Table  the erroWith respect to the synergistic joint stiffness parameter α, Table In spite of that, though the joint stiffness magnitude might not be the most important factor to determine stability, it does play a significant role. Actually, as it can be seen from the PCR values in Table During the experiments, the three-finger grasp is more widely tested because it clearly shows the changes on the hand configuration while being able to grasp a big diversity of objects. Only one four-finger grasp is tested. This is due to the constraint put by the stability criterion. We consider that the grasp is stable if all contact points are within the limits of the friction cone. However, this condition might be too strict for the cases with many contacts, as some might not necessarily be stable, whereas the overall grasp is. Other stability criteria could be studied to deal with numerous contact cases, for example by starting from the one presented in Pozzi et al. . Here, tAs stated in the introduction, whereas in Ruiz Garate et al.  a grasp Moreover, differently from Ruiz Garate et al.  and follAs we start from the premise that no sensory information is available apart from the encoder position, there is no visual information regarding the object shape. Therefore, we generalize the objects as spheres, cuboids, or cylinders. Experiments show that this generalization is sufficient for most daily objects , based on the proposed assumptions on the object shapes and contact points.Future work will envisage the study of alternative and less conservative criteria to analyze the grasp stability. Also, the application of the control paradigms to other robotic hands would be interesting. At a higher level, the human hand−arm system could be studied during grasp stiffness regulation in manipulation tasks, for a possible development of a CDS/CMS control of the equivalent artificial hand−arm composite.VRG and MP significantly contributed to the development of algorithm, the execution of experiments, the analysis of results, and the writing of the manuscript. AA significantly contributed to the development of the algorithm, the analysis of results, and the writing of the manuscript. DP significantly contributed to the analysis of results and the writing of the manuscript. All the authors approved the submitted version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


According to the 2020 Journal Citation Report by Clarivate Analytics, the impact factor of Balkan Medical Journal arose by 1.53 in 2019, which reflects 27% increase compared to last year. It has become one of the most prestigious journals in the Balkans and Turkey because of this new score. Based on this development, I would like to talk about what has been done in the last 4 years on behalf of the editorial team.After taking over as editor-in-chief in autumn 2016, for the first time in the history of the journal, an editorial independence agreement was signed between the editor and the owner of the journal. However, in the early days of this new appointment, the editorial team faced a publisher change. The publishing house change which is one of the most difficult processes and changes of a journal was successfully realized with the support of the Dean and the intensive work of the editorial board. From 2017, we decided to continue with the Galenos publishing house. In November 2016, the news that the journal was accepted to be indexed in the PubMed/MedLine increased our motivation. Therefore, as stated in the history of the journal  of the Balkan Medical Journal . We haveOne of the most challenging changes in 2018 was the switch to the online submission system. In the second half of 2018, as a result of the depreciation of the Turkish lira, we had to reduce the cost of the journal. After a long-term search by the editorial team and the publisher, we decided that the Manuscript Manager peer review system would meet all our needs . With thAnother job waiting to be done for us was the journal’s legal regulation. The editorial independence agreement between the owner of the journal and the editor alone did not guarantee the institutional independence of the journal therefore, a legal regulation was needed. We thought that the implementation of the journal legal regulation would guarantee the independent and responsible management of the journal, protect the freedom of publication, and ensure the transparency of the team at work. In addition, the effort to maintain the editorial freedom had posed distinct challenges arising from regional dynamics in the Balkan region. After working closely with the Dean’s Office and rectorate’s legal commission, the Balkan Medical Journal Legal Regulation came into force in January 2019 .As a member of IJMCE, in January 2019, Balkan Medical Journal declared compliance with the ICMJE data sharing statement . As a joIn the second quarter of 2020, the whole world was shocked by the new coronavirus pandemic. The COVID-19 outbreak pressured journals and authors to quickly publish many academic articles about the new coronavirus. This edition led to some editorial practices that ignore ethical principles. During this period, we witnessed many qualified journals withdrawing or retracting COVID-19 articles. This period has shown how important the competence of editorial board members is . In addi