Virus-like particles (VLPs) are empty, nanoscale structures morphologically resembling viruses. Internal cavity, noninfectious, and particulate nature with a high density of repeating epitopes, make them an ideal platform for vaccine development and drug delivery. Commercial use of Gardasil-9 and Cervarix showed the usefulness of VLPs in vaccine formulation. Further, chimeric VLPs allow the raising of an immune response against different immunogens and thereby can help reduce the generation of medical or clinical waste. The economically viable production of VLPs significantly impacts their usage, application, and availability. To this end, several hosts have been used and tested. The present review will discuss VLPs produced using different yeasts as fermentation hosts. We also compile a list of studies highlighting the expression and purification of VLPs using a yeast-based platform. We also discuss the advantages of using yeast to generate VLPs over other available systems. Further, the issues or limitations of yeasts for producing VLPs are also summarized. The review also compiles a list of yeast-derived VLP-based vaccines that are presently in public use or in different phases of clinical trials. Mycobacterium leprae and species of Plasmodium) [The term vaccine originated from the Latin word “Vacca”, meaning cow. Vaccines are a biological formulation of weakened or killed pathogens (sometimes closely related species of the target pathogen), which, upon administration, help protect an individual against the target disease(s) by imitating an infection. Generally, vaccines are prophylactic except for the vaccine against rabies, which is curative . Primarismodium) ,4.The arrival of subunit vaccines, which involved only a few components of the pathogen, overcame the loophole associated with the traditional vaccine development regime. Apart from the surface proteins , subunit vaccines may also include pathogen-specific toxin(s) and polysaccharides . The moshuman papillomavirus, or HPV), is expressed and purified from yeast against HPV, whose infection may lead to cervical cancer. Thus, in the contemporary world, vaccines are not just a tool to fight against infectious diseases but are also used to prevent certain types of cancer associated with viral infections [Initially, vaccines were used only against infectious diseases , but now vaccines are considered the most promising means to combat certain types of cancer. For example, Gardasil-9  . AdditioAfter briefly introducing vaccines, the different development platforms used, and their shortcomings, it is now time to focus on one of the emerging platforms in the field of vaccine development. The intended purpose of this review is to discuss the suitability of VLPs as a means of vaccine development and drug delivery, with a particular focus on VLPs derived from yeast. We will also discuss how VLPs are a better platform than purified proteins for triggering immune responses in the host. The present review also highlights the reasons that make yeast the most acceptable model for the expression and purification of VLPs; the review also includes associated concerns and success stories about this concept. We also discuss the major yeast species used to express and purify VLPs. All of this is discussed under separate headings.VLPs, ghost viruses, or dummy viruses lacking genetic material are nanostructures first observed in sera samples from hepatitis patients in 1968 . VLPs caThe feature that makes VLPs important in vaccine development is their high density of epitopes, particulate nature, and lack of genetic material that restricts their replication and makes them safe for the host ,36,37,38The high epitope density and particulate nature of VLPs make them ideal systems for mounting immune responses. It is essential to mention that VLPs can mount both humoral and cellular immune responses ,42,43,44Escherichia coli) [S. cerevisiae) [Due to their importance in vaccine development and drug delivery, efforts were made to express and purify VLPs from different biological systems and identify the most suitable hosts for producing VLPs commercially. To this end, VLPs are successfully expressed and purified from both the prokaryotic system   as well evisiae) , insect evisiae) ,49, mammevisiae) ,50, and evisiae)  , fall under GARS , which is another significant advantage of using yeast-based systems for the development of VLPs [K. phaffii, can be grown to a high cell density on a commercial scale, which is not feasible with animal cells. The rapid growth of yeast cells  is another advantage [S. cerevisiae . Yeasts such as nt cells . Under cnt cells . The yeant cells . Althougnt cells . Apart fLPs. See .In the previous section, we discussed some salient features that make yeast a suitable host for the commercial production of VLPs. Indeed, many commercially available vaccines are produced using yeast (discussed in detail in the next section). Despite this, several issues limit the use of the yeast-based system for commercial expression and purification of VLPs. In this section, we will point out the reasons that hamper the full utilization of yeast-based platforms for the commercial production of VLPs. One of the critical issues with using yeast to produce proteins (which will be used as vaccines) is the inability of yeast cells to perform protein glycosylation like humans .K. phaffii from Biogrammatics showed a significant reduction in glycosylation of expressed proteins (https://www.biogrammatics.com (accessed on 17 December 2022).Further, proteins in yeast cells are, in general, more heavily glycosylated compared to animal cells, including human cells . AdditioAnother issue limiting the use of yeast for producing VLPs is that many viruses are enveloped. To date, only one study has shown the possibility of producing enveloped VLPs (eVLPs) using yeast as a host . The sucS. cerevisiae and K. phaffii are known to grow on cheap media and can grow well in big industrial vessels ; however, the secretion of fully assembled VLPs into culture media is possible in a few cases.  mentioned, another vaccine, Mosquirix against Plasmodium falciparum, responsible for malaria, is commercially approved [The first VLP-based vaccine to hit the market was Gardasil, followed by Gardasil9; both products were branded under Merck and were against HPV-induced cancer. GlaxoSmithKline introduced another VLP-based vaccine (Cervarix) against HPV. It is important to note that Cervarix was effective against both the HPV16 and HPV18 viral strains. Cervarix from GlaxoSmithKline was taken off the market (USA) in 2016, while Gardasil and Gardasil9 are still available for public use. Notably, Gardasil and Gardasil9 are from yeast, while Cervarix is from the Baculovirus expression system, which uses High Five Rix4446 cells derived from the insect approved .Above, we mentioned the VLP-based vaccines currently in public use. Apart from this, several VLP-based vaccines are under clinical trials. An example vaccine against HIV is under clinical trial . Apart fhttps://www.advimmuno.com).Based on the points discussed above, during the last few years, VLPs have gained significant importance and acceptance as a means of vaccine and drug delivery. Apart from their use in a vaccine, VLPs are also noted for their potential use in drug delivery  as well as for the delivery of nucleic acids (DNA/RNA) ,232,233.S. cerevisiae and K. phaffii, other yeast species, including H. polymorpha, species of Kluyveromyces, Y. lipolytica, X. dendrorhous, and S. pombe, have also been used for VLP expression and purification. Additionally, over the years, more and more studies have appeared in which K. phaffii was used to produce VLPs. Although eVLPs have yet to be produced using yeast , this remains one of the concerns with using yeast for VLP production. Since considerable effort and resources are used during the purification of VLPs, the secretion of fully assembled VLPs into culture media is ideal, making the purification of VLPs economical. The secretion of VLPs is common in the case of animal cells, but in the case of yeast, only a few studies are available where VLPs have been successfully secreted into media [S. cerevisiae and K. phaffii are also used for producing VLPs, whether using those species on an industrial scale is feasible remains to be determined. VLPs purified from S. cerevisiae are already approved by the FDA and are currently in public use. It will be interesting to see when we will have VLPs purified from K. phaffii, which is FDA-approved and available for public use. It is anticipated that we may soon have a VLP-based vaccine where K. phaffii is used as a host. Preclinical evaluation of HBc VLPs (purified from K. phaffii) against hepatocellular carcinoma is underway [K. phaffii. Since K. phaffii can grow to high cell densities on cheap media, the use of VLPs purified from K. phaffii may also reduce the cost of VLP-based vaccines.Interestingly, more and more VLPs are now expressed in yeast. Apart from to media ,145,189.underway ,235. ThiK. phaffii, the authors used the GS115 strain. However, using protease-deficient strains  to minimize the degradation of an expressed protein is recommended [On a commercial scale, the purification of VLPs uses a chromatography column (such as POROS HS50). These columns get clogged by yeast lipids (due to the irreversible binding of lipids to resin) and lipid droplets. Therefore, to prevent or minimize any such issue, it is essential to use yeast strains that do not form lipid droplets . In mostommended . IntraceIn the end, several hosts are available for the production of VLPs, each offering some advantages over the others. Certainly, yeast has some significant benefits as a host for the commercial production of VLPs compared to other available hosts. However, the inability of yeast to secrete VLPs of important viruses remains a critical bottleneck. Therefore, more work in strain engineering will be needed to fully tap the potential of yeast for the production of VLPs. It may involve host engineering, antigen engineering, modification of culture media, and buffers, thus representing several channelings and opportunities.


The ubiquity of pedigrees in many scientific areas calls for versatile and user-friendly software. Previously published online pedigree tools have limited support for complex pedigrees and do not provide analysis of relatedness between pedigree members.We introduce QuickPed, a web application for interactive pedigree creation and analysis. It supports complex inbreeding and comes with a rich built-in library of common and interesting pedigrees. The program calculates all standard coefficients of relatedness, including inbreeding, kinship and identity coefficients, and offers specialised plots for visualising relatedness. It also implements a novel algorithm for describing pairwise relationships in words.QuickPed is a user-friendly pedigree tool aimed at researchers, case workers and teachers. It contains a number of features not found in other similar tools, and represents a significant addition to the body of pedigree software by making advanced relatedness analyses available for non-bioinformaticians.The online version contains supplementary material available at 10.1186/s12859-022-04759-y. The inbreeding coefficient of King Charles II of Spain (1661–1700) has been estimated to approximately 0.25 , HaploForge [2], pedigreejs [3], and Progeny [4], respectively. For ped_draw and pedigreejs, the pedigree was loaded from the ped file, while HaploForge and Progeny required manual creation. In all cases, the result is inadequate. C The pedigree as shown in QuickPed.


In this study, after APL cells were treated with different concentrations of realgar, the cell survival rate, apoptotic assay, morphological changes, ATP levels and cell cycle arrest were assessed. The expression of Bcl-2, Bax, Cytochrome C (Cyt-C) and apoptosis-inducing factor (AIF) at the mRNA and protein levels were also measured by immunofluorescence, quantitative PCR (qPCR) and Western blotting. We found that realgar could significantly inhibit APL cell proliferation and cell death in a time- and dose-dependent manner. Realgar effectively decreased the ATP levels in APL cells. Realgar also induced APL cell cycle arrest at the S and G2/M phases. Following realgar treatment, the mRNA and protein levels of Bcl-2 were significantly downregulated, whereas the levels of Bax, Cyt-C, and AIF were significantly upregulated. In summary, realgar can induce APL cell death via the Bcl-2/Bax/Cyt-C/AIF signaling pathway, suggesting that realgar may be an effective therapeutic for APL.Acute promyelocytic leukemia (APL) is a specific subtype of acute myelogenous leukemia (AML) characterized by the proliferation of abnormal promyelocytes. Realgar, a Chinese medicine containing arsenic, can be taken orally. Traditional Chinese medicine physicians have employed realgar to treat APL for over a thousand years. Therefore, realgar may be a promising candidate for the treatment of APL. Nevertheless, the underlying mechanism behind realgar therapy is largely unclear. The present study aimed to investigate the effect of realgar on cell death in the APL cell line (NB4)  Acute promyelocytic leukemia (APL), which is caused by a balanced translocation t15;17)q22;q12) that results in the fusion transcript PML-RARA, is a rare type of acute myeloid leukemia (AML) that accounts for 10–15% of newly diagnosed cases [5;17, has been applied for more than 1800 years by traditional Chinese doctors . Similarthan ATO . Since 1than ATO , 21. In than ATO –27. Simithan ATO . In Chinthan ATO . Chen etthan ATO . In a clthan ATO . In addiDespite the fact that realgar has been used to treat APL in China for many years, its molecular mechanism remains unclear. The purpose of this study was to investigate the effect of realgar on the death of the APL cell line NB4 and to clarify the underlying mechanism. In this study, APL cells were treated with different concentrations of realgar, and cell viability, apoptosis, morphological changes, ATP levels and cell cycle arrest were measured, and we evaluated, for the first time, the expression of Bcl-2, Bax, Cyt-C and AIF at the mRNA and protein levels following realgar treatment. Our study is helpful for understanding the potential mechanism of realgar in the treatment of APL.4S4) with a purity of 93.4% was obtained from Hubei Pharmaceutical Co., Ltd. . In the current investigation, 3 g of realgar was sieved through a 500 mesh sieve, added to distilled water to a volume of 300 mL, and mixed for 30 minutes using a magnetic stirrer. It was then put into an M-110EH high-pressure microjet granulator  for 10 cycles at 30,000 rpm, followed by 30 minutes of ultrasonic vibration. After grinding, the realgar nanoparticles were spherical and uniform in size, and the average diameter measured by a Malvern Zetasizer Nano-ZS  was approximately 72.63 nm. The details of realgar nanoparticles Dynamic Light Scattering (DLS) and particle size are shown in Realgar containing arsenic sulfide . All experiments were performed on an ultraclean bench.The APL cell line  was cultured in RPMI-1640 media  supplemented with 10% fetal bovine serum , 100 μg/mL streptomycin  and 100 U/mL penicillin . APL cells were cultured at 37°C in an incubator with 5% CO4/mL) were added to each well of the 96-well plate. Next, realgar solutions at concentrations of 0, 12.5, 25.0, 50.0 and 75.0 μg/mL  were added to each well. Then, the cells were cultivated for 24, 48, and 72 hours in an incubator at 37°C with 5% CO2 and water saturation humidity. Finally, each well’s optical density (OD) at 450 nm was determined using a microplate reader . The experiment was carried out three times independently. Furthermore, 50% inhibitory concentration (IC50) was the concentration of realgar required to inhibit the growth of APL cells by 50%.APL cells in the logarithmic growth phase were cultured in 96-well plates . In brief, 100 μL of the cell solutions  of realgar  for 24 h. At the indicated times, morphological alterations were observed using a light field microscope .NB4 cells were seeded in 6-well plates at a density of 1.2 × 105 cells/well in 6-well plates, and then the cells were exposed to various concentrations  of realgar  for 24 h. Next, the cells were collected at the specified time, gently resuspended by adding 195 μL Annexin V-FITC Binding Buffer , incubated with 5 μL Annexin V-FITC and 10 μL PI staining solution for 20 minutes at room temperature (20−25°C) and protected from light, and placed on ice for detection by flow cytometry . Annexin V-positive and PI-negative cells indicated the early stages of apoptosis, whereas both Annexin V- and PI-positive cells indicated the late stages of apoptosis.NB4 cells were plated at a density of 1.2 × 105 cells were seeded in a six-well tissue culture plate, and various concentrations  of realgar were added and incubated for an additional 24 h. Next, the cells were collected, washed with 1× PBS, and centrifuged. Then, the obtained cells were rewashed in PBS, fixed in 70% ice-cold ethanol and kept at −20°C. Finally, the cells were rewashed with PBS and stained with ethidium bromide, followed by analysis by flow cytometry .Cell cycle arrest analysis was carried out by flow cytometry after APL cells were exposed to different concentrations  of realgar and stained with the DNA-specific dye ethidium bromide. In short, 1.2 × 10To investigate the effect of realgar on the production of ATP, the ATP content was evaluated using an ATP assay kit . Briefly, APL cells were cultured with various concentrations of realgar for 24 hours , and then ATP-releasing reagent was added and ultrasonicated on ice for 10 minutes, followed by centrifugation at 12,000 g at 4°C for 10 minutes to collect the supernatants. Finally, a MultiMode Microplate Reader  was used to determine the results.NB4 cells were treated with realgar under optimal conditions  as determined by the CCK-8 assay. Following treatment, the cells were spread onto slides, fixed with 4% paraformaldehyde for 20 minutes at 4°C, and permeabilized in 0.1% Triton for 10 minutes at room temperature. Then, the cells were incubated with 10% goat serum (Beyotime Institute of Biotechnology) for 30 minutes at room temperature before the primary antibody reaction. After that, the slides were treated for 8 hours at 4°C with rabbit anti-human polyclonal antibody against RARa , followed by FITC-conjugated goat anti-rabbit IgG  at room temperature for 1 h. Next, microscopy was used to observe the cells after counterstaining with DAPI . A fluorescence microscope  and a confocal laser scanning microscope  were used to observe the cells.5/well) were seeded in six-well plates and treated for 24, 48 and 72 h with various concentrations  of realgar solution. Then, the cells were collected, and total RNA was extracted by an RNA Tissue/Cell Rapid Extraction Kit . Total RNA was quantified by a microspectrophotometer, followed by generation of single-stranded cDNA from 600 ng total RNA. Furthermore, qPCR was performed in order to measure the gene level using cDNA synthesized by reverse transcription. −ΔΔCt technique.We used qPCR to investigate the expression of Bcl-2, Bax, Cytochrome C (Cyt-C), and apoptosis-inducing factor (AIF) mRNA levels in cells treated with different concentrations of realgar. In brief, APL cells . The extracted proteins were denatured with reducing sample buffer at 100°C for 7 min. Then, 40 μg of protein was loaded onto a 10% polyacrylamide gel. Proteins were separated  with Tris-HCl running buffer in an electrophoresis apparatus . Then, the proteins were transferred  from the acrylamide gel to a polyvinylidene fluoride (PVDF) membrane. Blots were blocked with 5% bovine serum albumin (BSA) for 1 h, followed by incubation with monoclonal anti-Bax , Bcl-2 , AIF , and Cyt-C  antibodies at 4°C overnight. Subsequently, the membranes were washed frequently with Tris-buffered saline Tween , followed by incubation with goat anti-rabbit IgG antibody  at room temperature for 1 h. After repeated washings with TBS-T, the blots were treated with a chemiluminescent detection kit . The internal reference was represented by an antibody against actin. The quality of the protein was assessed using a Typhoon PhosphorImager .P < 0.05 was considered to be significant. SPSS 25.0 was used to analyze the data.All experiments were performed at least three times independently, and the data are expressed as the mean ± the standard deviation. For the significance test, one-way analyses of variance and post hoc methods depending on Newman–Keuls tests were applied. The dose- and time-dependent responses of NB4 cells to realgar are shown in As illustrated in –2E, we fAnnexin-V-FITC and PI double staining was used to distinguish between live cells, early apoptotic cells, and late apoptotic/necrotic cells after realgar treatment of NB4 cells . We obseTo verify whether realgar suppressed the proliferation of NB4 cells by triggering cell cycle arrest, we evaluated the change in the cell cycle distribution after NB4 cells were treated with realgar. After 24 hours of treatment with different concentrations of realgar for 24 hours, the ATP levels in NB4 cells declined from 100% to 76.409%, 54.008%, and 48.825% . The resIn this study, the impact of different concentrations of realgar on the levels of Bcl-2, Bax, Cyt-C, and AIF gene expression in NB4 cells was investigated using qPCR. As shown in –6D, the Typically, deregulated cell proliferation and cell apoptosis are intimately coupled. Therefore, we evaluated the effect of realgar on cell apoptosis by evaluating the expression of apoptosis-associated proteins in NB4 cells upon realgar stimulation. Based on the cell proliferation data, the optimal concentration and stimulation time  were used in this experiment. The results of the immunoblotting analysis showed that realgar treatment clearly downregulated the expression of the antiapoptotic protein Bcl-2 and promoted the expression of the proapoptotic protein Bax. In addition, the levels of Cyt-C and AIF were also elevated  and 7B.To further verify this outcome, we performed western blotting analysis . The resFor more than 1500 years, traditional Chinese doctors have employed realgar, a traditional Chinese medicine, to treat hematological malignancies by oral administration , 32. EmpA CCK8 assay was used to analyze the inhibitory impact of realgar on cell proliferation. We observed that by increasing the concentration of realgar cultured with the cells and lengthening the culture period, APL cell viability gradually decreased . Chen etAt present, apoptosis is generally thought to be the most common mechanism of cell death . We founCell cycle arrest has become one of the most successful cancer therapies after years of study into the cell cycle and its role in cancer development . Flow cyMitochondria are vital to cell survival. They not only produce ROS but also generate ATP through the oxidative phosphorylation system. In pathological situations, the malfunctioning of mitochondria results in ATP exhaustion . We inveApoptosis, also called programmed cell death, is a kind of cell death that is vital for maintaining homeostasis . It has To determine the underlying mechanism of realgar-induced APL cell death, we evaluated the effect of realgar on mitochondrial function. The expression of Bcl-2, Bax, Cyt-C, and AIF at the mRNA and protein levels was evaluated before and after exposure to various concentrations of realgar using immunofluorescence, western blotting, and qPCR experiments. Our results showed that Bcl-2 was decreased and Bax, Cyt-C, and AIF were increased at the mRNA level in a concentration-dependent manner after APL cells were treated with realgar . In addiQi et al. studied the differential proteome after realgar was used to interfere with the APL retinoic acid-resistant cell line NB4-R1 , 50. A tAs shown in in vitro in a dose- and time-dependent manner. Realgar treatment of APL cells can reduce ATP production, damage the nucleus, and induce APL cell cycle arrest at the S and G2/M phases. In addition, it can also disrupt mitochondrial homeostasis by decreasing Bcl-2 expression and increasing Bax, Cyt-C, and AIF expression at the mRNA and protein levels. Therefore, Bcl-2, Bax, Cyt-C, and AIF may play essential roles in APL cell death. Our findings provide new insight into realgar-induced APL cell death involved in mitochondria-mediated apoptosis, and indicate that realgar may be a potential therapeutic for APL in clinical practice. Animal studies should be performed next to confirm the therapeutic effects of ZnO NPs on MM.Overall, our results demonstrate that realgar can effectively induce APL (NB4) cell death Supplementary Figure 1


Microfluidic paper-based analytical devices (µPADs) are leading the field of low-cost, quantitative in-situ assays. However, understanding the flow behavior in cellulose-based membranes to achieve an accurate and rapid response has remained a challenge. Previous studies focused on commercial filter papers, and one of their problems was the time required to perform the test. This work studies the effect of different cellulose substrates on diffusion-based sensor performance. A diffusion-based sensor was laser cut on different cellulose fibers  with different structure characteristics, such as basis weight, density, pore size, fiber diameter, and length. Better sensitivity and faster response are found in papers with bigger pore sizes and lower basis weights. The designed sensor has been successfully used to quantify the ionic concentration of commercial wines with a 13.6 mM limit of detection in 30 s. The developed µPAD can be used in quantitative assays for agri-food applications without the need for any external equipment or trained personnel. The advantages of miniaturizing fluid handling are appreciated in different sectors, such as in food, agri-food industry, or in biochemical, biomedical, and pharmaceutical applications ,2,3,4. T+ or troponin diazinyl}benzene-1-sulfonate) which is used as pH indicator, was prepared by dissolving 40 mg of it in 40 mL of water. Due to its pH transition rate (turns to red at pH < 3.0 and yellow at pH > 4.0), it is suitable for the expected pH value of wines and tartaric acid solutions (at or lower than pH 3.7) . CommercMehrdel et al.  used comThe equation for convection–diffusion is defined as:2/s [Equation (3) couples the convection–diffusion transport phenomena under the laminar regime and it is introduced in the model as user-defined scalar. In this study, the specie that diffuses is the proton 0−9 m2/s . Since t [(Deff) : (4)DeffThe porous substrate is modeled through the following parameters: the porosity The pore shape factor for 0.4 to φ=140 . Table 43 and 0.001003 kg/m.s, respectively. At the inlets of the lateral channels, the initial species concentration is set to 0.05 M.The working fluid is assumed to be liquid water under standard room conditions (RT = 25 °C and HR = 50%) with a density and viscosity of 998.2 kg/mThe velocity is defined at the inlets. The initial flow velocity at the inlets is estimated from previous capillary flow characterization done with the different paper substrates, measuring the time required to fill the inlet branches, see The convergence residuals are set to 1 eability . The difBased on the measured fluid velocities and calculated viscous resistance in all the paper substrates, 2) is used for the numerical analysis.Owning to the fact that the investigated geometry is identical to the conducted study by Mehrdel et al. , therefoThe 30-degree model with a 3-inlet substrate was laser cut (using a NEJE7000mW laser) in the different paper substrates. The 3D-printed support described in  was usedSince the diffusion width varies along the length of the sensor (see Equation (3)), it is necessary to establish a benchmark time in order to obtain repeatable and accurate results.A Dino-Lite MS325B microscope is used to take pictures of the defined measurement line  is set as “Time zero”. The pictures for analysis are taken at 30 s, 60 s and 120 s after the “Time zero”. The ambient and the projected light are the same in all the assays.The errors in this work might have occurred during numerical simulations and experimental sections. To ensure that the mathematical method is as close as possible to the real flow, careful efforts have been made, such as considering the effect of discretization and choosing the cells’ size accurately to not affect the final results, as well as choosing the residuals of the results between consecutive iterations correctly to avoid producing a false convergence.In the experimental section, the errors might have been rooted in mistakes done by staff or devices. To minimize these errors, every assay was repeated at least 5 times for every configuration of solutions on each paper substrate.Furthermore, careful efforts were made to make sure that all the assays were carried out at similar temperatures, ambient light, and magnification rates.Y-axis, the limit of detection (LOD) can be found through the following formula:Finally, by considering the generated standard deviation from the measured diffusion width and regression line, which can be calculated as, Based on the characteristics of the cellulose fibers, a computational fluid dynamic model is evaluated for each paper substrate, and the results obtained from the model are then validated experimentally.The flow velocity and the diffusion width in the porous medium for all the paper substrates based on their cellulose fibers’ characteristics are numerically obtained from the steady-state results of the simulations. There are various parameters that influence the motion of fluid in porous media. For instance, Whatman papers, because of the higher density and lower length of their cellulose fibers, showed more fluidic resistance in their matrix compared to Sisal papers. Therefore, the species in Whatman’s paper can diffuse more, see On the other hand, Numerical results obtained by steady-state simulations revealed that Whatman papers showed a wider diffusion width than Sisal papers, while the fluid velocity over the X-direction (measurement line) in Whatman papers was less than in the Sisals. Therefore, to have a comparison between these papers in terms of the time needed to have a measurable diffusion width at the measurement line, the time transient simulation is studied.Several paper substrates are prepared and tested (at least 5 tests for every concentration of solutions on each paper substrate). One millimeter diffusion width can be easily measured in the portable experiment setup shown in According to the results plotted in Due to the small pore size and low permeability of Whatman grade 5, Mehrdel et al.  studied Hence, to have a comparison between two Whatman filter papers (5 and 40), the interaction between the pH indicator and the reagents is analyzed at 120 s after the “Time zero” for Whatman 40, as well. X-axis is the concentration based on (g/L) unit, which was converted from the molar (M) unit (concerning the molar mass of tartaric acid) and the Y-axis is measured diffusion width based on the millimeter (mm) unit, which was converted from the micrometer (µm) unit.Moreover, according to According to One of the primary benefits of µPADs is the ability to provide results in a short period. In a diffusion-based sensor, there is a balance between the diffusion of molecules in the transversal direction of the flow and the flow of fluids along the sensor. If there is no flow along the sensor, the diffusion is extremely localized and hard to measure. Heretofore, different lab-made Sisal paper substrates, which had different characteristics see , are comWhile due to high viscous resistance see , WhatmanMoreover, the shift of the pH indicator color is quite visible to the naked eye even after 30 s. In lab-made Sisal papers, based on their cellulose fiber characteristics (bigger pore size and length of the cellulose fiber), and higher permeability, the fluid flows faster and decreases the time to generate a co-flow and measuring diffusion. The more intense the diffusion phenomenon is happening, the better mixing with the pH indicator and the sedimentation of methyl orange salts occurs, which establishes a limit on the performance of the sensor. Between these two papers , even though the permeability is quite similar, the thickness is different (143 µm and 301 µm), respectively. When the thickness is low, the sedimentation increases, and as time passes it can decrease the diffusion velocity of molecules.From the measured diffusion widths over the measurement line at 30 s after the “Time zero”, a calibration plot for each proposed sensor made out of lab-made Sisal papers is obtained, see 2) show wider diffusion widths compared to the ones with 50 g/m2 of basis weight. The reason for the greater difference in diffusion widths between paper substrates with 50 g/m2 of basis weight (S0_50 and S1000_50) is due to the sedimentation of the methyl orange salts. The bigger average pore size of S0_50 causes an increase in the fluid velocity through its porous medium and after a certain time, the salts form fibers and prevent further interaction between the reagents. Meanwhile, in S1000_50 , the species have more time and space to diffuse. Apart from the pore size and basis weight, another parameter that was not considered in the simulation model, but has an important role in the experimental results, is the thickness. Based on the obtained results, paper substrates with a higher thickness (S0_100 and S1000_100) show wider and more clear diffusion width compared to the papers with less thickness  showed wider diffusion width for wine and the measured diffusion width of wine for S0_100 was 1726 ± 48.1 µm and S1000_100 showed a diffusion width of 1711 ± 53.5 µm. Moreover, based on the calculated regression line, the S0_50 paper substrate displayed a 6.1 g/L of total acid concentration for the wine sample, whereas the same values for S0_100, S1000_50 and S1000_100 were 6.6, 7.5 and 7.8 g/L, respectively. According to HPLC measurements, the total acid concentration of white wines varies from 5.64 to 10.7 g/L [The diffusion width of wine Is also measured, being 1024 ± 25.6 µm and 1323 ± 38.7 µm for S0_50 and S1000_50, respectively. As expected, sisal papers with a higher basis weight  diagnostics that enable researchers to perform statistical tests, analyze differences between groups, and determine the effectiveness of treatments, is studied. Prior works have documented the influence of different characteristics of papers by taking advantage of lab-made papers (such as hardwood and softwood fibers) compared to commercial papers. Results pointed out that papers with lower basis weights and higher porosities improved the performance of paper-based devices ,46. CasaApart from the commercial papers (Whatman filter papers), which bring some limitations to having control over different parameters, here we used lab-made Sisal papers to investigate the effect of different characterizations of paper on the performance of the diffusion-based sensors. Lab-made paper with nonwood cellulose fiber  has low basis weight and high porosity . These parameters (particularly bigger pore size which has an important effect on permeability) allow the easy diffusion of molecules through the porous media. Sensors made with such paper deliver high-clarity assay results. Furthermore, since less time is required for the cellulose to be extracted from the sisal plant, as in our lab-made papers, this type of sensor can be more sustainable .In this work, the effect of paper substrate characteristics  on the performance of diffusion paper-based sensors by quantitatively measuring an analyte rather than only detecting its presence, is investigated. The use of a CFD model capable of taking into account certain paper properties  can help in the substrate selection for the co-flow type of sensors. The experimental tests were in agreement with the numerical results and validated that Whatman filter papers  required a longer time to produce results than lab-made Sisal papers (bigger pore size). However, certain characteristics not considered in the model such as basis weight and paper thickness are also important.2, and S1000_50 with an average pore size and basis weight of 31 ± 5 µm and 47.2 ± 0.9 g/m2, respectively, showed a better limit of detection than lab-made papers with a higher basis weight (S0_100 and S1000_100) and commercial Whatman papers . In the case of Sisal 0_50 and 1000_50, due to their characteristics and flow behavior through their porous media, it was easier to capture an acceptable diffusion width in a shorter time, which facilitated its measurement and accordingly, enhanced the limit of detection. The sensor made of non-refined lab-made Sisal paper with 50 g/m2 of basis weight (S0_50) exhibited an LOD of 13.6 mM when evaluating the ionic strength of wines by comparison to different tartaric acid solutions in 30 s, which is approximately 3 times lower than when using Whatman grade 5 (41.3 mM in 120 s).The experimental results revealed that lab-made paper substrates with bigger pore sizes and lower basis weights, particularly S0_50 with an average pore size of 39 ± 3 µm and basis weight of 50.9 ± 1 g/mHowever, there are other parameters of interest as well, such as paper additives, paper manufacturing and refining processes, which in turn may affect the fiber length which impacts the capillary tortuosity and uniformity of the paper substrate; these can be considered in order to further optimize the quantitative measurement of the results and tune the generated capillary flow.


Can the male citation advantage  be explained by gender homophily bias, i.e., the preference of scientists to cite other scientists of the same gender category? Previous studies report much evidence that this is the case. However, the observed gender homophily bias may be overestimated by overlooking structural aspects such as the gender composition of research topics in which scientists specialize. When controlling for research topics at a high level of granularity, there is only little evidence for a gender homophily bias in citation decisions. Our study points out the importance of controlling structural aspects such as gendered specialization in research topics when investigating gender bias in science. Gender bias is an ongoing topic in science studies. There is evidence for various forms of gender differences, as a recent review in Science suggests: “Women have fewer publications … and collaborators … and less funding … and they are penalized in hiring decisions when compared with equally qualified men. The causes of these gaps are still unclear” . At the only because they belong to the same gender category. We look at authors’ gender expression through names, but cannot distinguish this from authors’ gender identity which might differ. We also applied a binary concept of gender that only distinguishes between women and men. Thus, our analyses rely on a simplified concept of gender. Nevertheless, our analyses should provide a first insightful analysis of the extent to which preferences versus structural aspects lead to citation inequalities.To date, literature shows mixed evidence of a possible gender citation gap. Some studies find no gender differences in citations or that female authors receive more citations than male authors –12, whilOur notion of homophily captures preferences that go beyond structural reasons for gendered citation patterns. Such structural aspects exist for example with the gendered specialization on research topics, which can result in male scientists citing more male-authored papers than female scientists (and vice versa). However, we conceptualize homophily as the “bias” that leads same gender peers to cite each other more often than what a baseline model of gender-blind selection of relevant literature would predict . EvidencIn our study, we tested the hypothesis that gendered citation patterns can emerge on the macro level due to structural aspects alone, with no gender homophily being at play. With gendered citation patterns, we mean the fact male scientists cite male-authored papers more often than female scientists (and vice versa) when looking at the pool of all scientists, regardless of their research area. Scientists’ gender is strongly related to the topic they are working on . It follTo identify homophily bias, it is therefore important to control for structural aspects that make some papers to more adequate sources to be cited than other papers. Besides research quality, the most important structural aspect to define such risk pools of papers is certainly topic similarity . Previous homophily studies have already tried to control for this topic similarity by considering the journals authors have published in , 19–28. https://facultyopinions.com/; previously F1000Prime) that contains this information for papers published in 2002–2020. The keywords assigned to these papers were curated by an editorial team at Faculty Opinions in cooperation with leading scientists and clinicians in the corresponding biological and medical research fields. For each of the papers, the data also include information from reviews of experts in these fields [Our goal was therefore to test more rigorously whether gendered citation patterns are caused by a gender homophily bias. To precisely control for the risk pool of papers to be cited, we measured the topic similarity between papers based on (the combination of) keywords that were assigned manually by experts. We drew on data provided by Faculty Opinions  shows that for male-authored focal papers, the share of male-authored citing papers is about 12.6 percentage points higher than for female-authored focal papers. However, no further variables are included in this model. Model M2 (blue) shows that this effect reduces to about 7.1 percentage points when controlling for keywords . This means that a gender-specific selection of scientists into different topics is–at least partly–responsible for the observed gendered citation patterns. Controlling for further factors  in Model M3 scarcely changes the effect of the gender of the focal papers’ authors. We controlled for publication year and number of authors, since empirical analyses suggest that the share of female authors increased over time , and thaIn line with some previous studies , 27, 29,X shared keywords. With increasing X, the pairs are increasingly similar in terms of keywords .However, the inclusion of keywords in the form of binary variables in a regression model only allows controlling for each keyword independently of other keywords. Since research is usually reflected by more than one keyword , topics may be better represented by certain (dependent) combinations of keywords. To consider this, we generated pairs of focal papers such that one paper is authored only by male scientists and the other paper is authored only by female scientists see . For eacX , the difference approaches the shape of a normal distribution. The shape of a normal distribution could be expected if there is no gender homophily in citations: with no homophily bias, on average, the difference in the share of male-authored citing papers would be zero, and the differences would be distributed symmetrically around this average . These results suggest that after controlling for the topic on a sufficiently high level of granularity , gender homophily can be scarcely observed.We deem the keywords in the Faculty Opinions database a reliable approach for measuring the topic similarity between papers, since these keywords are based on expert knowledge and provide a more consistent measurement compared to keywords idiosyncratically chosen by authors . AlthougWe therefore tested whether our results also hold for alternative similarity measurements  and a seFor identifying comparable pairs of papers, the approach based on titles and abstract is the most efficient alternative to using Faculty Opinions keywords . Therefore, we used this approach to expand the analyses to all papers from the WoS published in 2012 (the mean publication year for the papers in the Faculty Opinions dataset). For these data, the observed degree of gender homophily is generally smaller than for the Faculty Opinions data. Since these results are based on the less precise approach to measure topic similarity based on titles and abstracts, we can be even more sure that there is no marked gender homophily that goes beyond gender compositions of research fields.We were able to replicate our results in various further robustness checks  that there is a strong homophily bias. The very small evidence for homophily bias that remains in our study after controlling for topic similarity suggests that other mediators are not very meaningful. Since previous studies have shown that more productive, senior authors collect more citations [Just as in previous studies, we were unable to conduct a randomized experiment and instead had to rely on large-scale bibliometric data. The main takeaway from our study is the necessity of using adequate measures for controlling mediating factors when studying gender bias: only when all relevant mediators are controlled with exact measurements can the itations , senioriSimilar to previous studies on gender homophily in citations, our approach to identify the authors’ gender based on their first names implies an imprecise concept of gender. It is unclear to what extent this approach measures only a person’s gender expression and not their possibly different gender identity. Thus, our results do not allow to differentiate between these notions of gender. Our approach to infer the scientists’ gender is also limited to a binary concept of gender, which means that we cannot draw any conclusion about scientists with non-binary gender. Future research could address these issues by applying a more differentiated concept of gender.Gender (homophily) bias is also suspected in many other realms of science, including reviews of publications, grant assignments or decisions to select co-authors or peers for acknowledgments , 37, 38.Our study also points out, in accordance with many other empirical studies e.g., –44, thatThe Faculty Opinions data that we used in this study includes expert ratings of the papers’ scientific quality, which are given in the form of "good," "very good," and "excellent." Thus, only papers at a high quality level were selected for inclusion in the database. Information about the topic of the papers is given in the form of keywords assigned by experts . There are 334 different keywords occurring in the database, and an expert may have assigned multiple of these keywords to a paper. Since keywords and quality ratings have been assigned by experts in the field (and in many cases by more than one expert per paper), we can assume a high accuracy of the data.We matched the papers in the Faculty Opinions database  with metadata on authors and topics from the WoS. From these data, we used the author names (to infer the authors’ gender) and the publication year for both the focal papers and all of their citing papers. For this purpose, we used an open source application for assigning a gender category  to first names . At the S1 File(PDF)Click here for additional data file.


With anti-PD-1 antibodies serving as a representative drug, immune checkpoint inhibitors (ICIs) have become the main drugs used to treat many advanced malignant tumors. However, immune-related adverse events (irAEs), which might involve multiple organ disorders, should not be ignored. ICI-induced myocarditis is an uncommon but life-threatening irAE. Glucocorticoids are the first choice of treatment for patients with ICI-induced myocarditis, but high proportions of steroid-refractory and steroid-resistant cases persist. According to present guidelines, tumor necrosis factor alpha (TNF-α) inhibitors are recommended for patients who fail to respond to steroid therapy and suffer from severe cardiac toxicity, although evidence-based studies are lacking. On the other hand, TNF-α inhibitors are contraindicated in patients with moderate-to-severe heart failure. This review summarizes real-world data from TNF-α inhibitors and other biologic agents for ICI-induced myocarditis to provide more evidence of the efficacy and safety of TNF-α inhibitors and other biologic agents. The approved ICIs on the market include programmed death-1 checkpoint inhibitors (PD-1LAG-3i) . Novel ii . Novel(LAG-3i) , CD47 6i, PD ligAlthough these drugs represent a major milestone in the area of cancer treatment, they are associated with a variety of immune-related adverse events (irAEs) that may affect almost all organs, lead to drug discontinuation, and finally mitigate overall therapeutic efficacy . Among tBiologic agents have been widely used in various autoimmune disorders due to their targeted inhibition of signaling to alleviate the systemic side effects of steroids and improve efficacy. Tumor necrosis factor-α (TNF-α), interleukin-17 (IL-17), interleukin-6 (IL-6), interleukin-4 (IL-4) antibodies and other drugs have been proven useful in treating rheumatoid arthritis (RA), psoriasis, cytokine release syndrome (CRS), atopic dermatitis (AD) and other autoimmune diseases –18. ICI-Therefore, we searched for studies focusing on TNF-α antibodies and other biological agents that were prescribed to patients with ICI-induced myocarditis, aiming to provide more clinical information for the selection of biological agents, time of intervention, efficacy and safety.The TNF superfamily consists of 19 ligands and 29 receptors. Among them, TNF-α represents one of the most potent proinflammatory cytokines . TNF-α fvia TNFR2. Although the mechanism is not well understood, mTNF-α can activate the MAPKs JNK and p38 and downstream transcription factors in the nucleus , TNF-α receptor associated factor 2 (TRAF2), receptor-interacting protein (RIP) kinase, and inhibitors of apoptosis proteins (IAPs). The TNFR1/TRADD/TRAF2/RIP/IAPs complex activates MAPK- and inhibitor of kappa B kinase (IKK)-dependent pathways. MAPKs, mainly c-Jun N-terminal kinase (JNK) and p38 isoforms, activate activation protein-1 (AP-1) and other transcription factors. IKKs transduce the signal through the activation of NF-κB , 23. Alt nucleus , 29  at baseline, and the inflammatory process was likely not the core reason for heart failure in this patient group. Additionally, a physiological dose of TNF-α might exert a protective effect on hypoxic injury in the heart, whereas overinhibition of TNF-α would eliminate this effect . EtanercIn contrast, TNF-α antibodies have been used to treat millions of patients with RA, but their association with cardiovascular events has not been confirmed. Compared to normal subjects, the risk of cardiovascular events is approximately 1.5-2-fold higher in patients with RA . AccordiAs mentioned above, anti-TNF-α therapy failed in clinical trials and was even associated with a deterioration of heart failure in patients who were mainly diagnosed with ischaemic cardiomyopathy. Therefore, TNF-α antibodies should be administered to patients with ICI-induced myocarditis with caution. However, a similar condition was not observed in patients with RA, in which inflammatory activation might play a more important role in the development of cardiovascular disease, such as ICI-induced myocarditis, and the risk of cardiovascular diseases was not increased after anti-TNF-α therapy . We next-/- mice have diffuse deposition of immunoglobulin G on the surface of cardiomyocytes, causing severe dilated cardiomyopathy and sudden death by congestive heart failure . In all patients with available echocardiographic data, the EF was significantly lower than 50%, and these patients should be classified as having moderate-to-severe heart failure, but anti-TNF-therapy was still adopted. In a previous study by Cautela and colleagues, the authors raised a concern that infliximab was associated with a greater risk of cardiovascular death (50%) than other intensified immunosuppressive therapies (19%) when first-line steroid treatment failed . HoweverComparison between the patients who finally survived and died from ICI-induced myocarditis progression or other causes was shown in In addition to second-line treatment in patients in whom steroid failed, anti-TNF-therapy was also proposed to be used earlier in first-line treatment or even for the prevention of ICI-induced cardiotoxicity. Michel and colleagues documented that TNF-α blockade may prevent the detrimental effect of anti-PD-1 therapy while preserving the anticancer efficacy in tumor-bearing mice . In 2021In summary, although infliximab does not increase cardiovascular risk in RA patients, the current evidence is not strong enough to support neither the conclusion that infliximab is also safe in steroid-resistant and steroid-refractory ICI-induced myocarditis, nor it will accelerate or worsen heart failure. Further studies regarding TNF-α inhibitors are necessary. If the concern about its cardiovascular risk can not be solved, other newly developed immunosupressive biologic agents should be investigated more in the future.CTLA-4 and CD28 are homologous receptors expressed by T cells, sharing CD80/CD86, a pair of ligands expressed on the surface of antigen presenting cells (APCs). However, they mediate opposing functions in T cell activation. CD28 mediates T cell co-stimulation, while CTLA-4 serves to inhibit T cell responses . AbataceIn mouse models of ICI-induced myocarditis, the intervention of abatacept led to a reduction in cardiac immune activation and an increase in survival . Cases rTherefore, use of abatacept in ICI-induced myocarditis is worth expecting, especially considering its efficacy in concurrent myositis and myocarditis. However, the risk of tumor relapse should also be noted. Two patients experienced tumor relapse 3 months and 4 months after abatacept treatment, respectively , 97. TheH17 from naive T cells, and inhibit the Foxp3+ regulatory T cells (IL-6 is recognized as another proinflammatory cytokine that is produced in the pathogenesis of various inflammatory diseases. Formation of the IL-6/IL-6R/gp130 hexamer leads to the initiation of Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling, a pathway involved in many crucial biological processes, including cell proliferation, differentiation, apoptosis, and immune regulation , 99. IL- T cells . Tociliz T cells . In 2017 T cells , 105. Ad T cells . The oth T cells . For irA T cells . ConsideCD52 is expressed on peripheral mature immune cells, mainly on T and B lymphocytes, while it is expressed at lower levels on innate immune cells. Its antibody alemtuzumab leads to antibody-dependent cell-mediated cytolysis, complement-mediated destruction, and apoptosis of these cells . AlemtuzJAK inhibitors on the market are mainly small molecules rather than biologics . JAKs arvia JAK1/2, finally resulting in an upregulation of MHC-I molecules and PD-L1 to the cancer cell outer membrane. JAK mutations, causing a loss-of-function phenotype, can lead to the absence of PD-L1 expression and subsequent inefficacy of anti-PD-1/PD-L1 therapy . Cardiovascular risk seems to be the Achilles’ heel of JAK inhibitors, and it has received great attention from regulatory agencies. Meanwhile, JAK loss-of-function mutations were found responsible for primary and acquired resistance to anti-PD-1 therapy in patients with solid tumor. IFNγ binds to the interferon gamma receptor (IFNGR1/IFNGR2) and activates downstream signaling  therapy , 118. Th therapy .Other untargeted immunosuppressive agents, such as antithymocyte globulin (ATG), intravenous immunoglobulin (IVIG), and mycophenolate are also recommended by current guidelines for ICI-induced myocarditis, but are not the main focus of this review. They are empirical strategies logically recommended by the experts, since the histological lesions observed in ICI-induced myocarditis are similar to those observed during acute cardiac transplant cell rejection. Several case reports with favorable outcomes support the use of them in ICI-induced myocarditis , 121. Invia regulatory T cells (Researchers have not clearly determined which biologic antagonist would be optimized for ICI-related myocarditis, especially in steroid-resistant or steroid-refractory patients. According to the United States National Institutes of Health registry ClinicalTrials.gov, several trials are underway and will be informative Table 4. T cells . Rituxim T cells , and thi T cells . A compaAlthough the risk of aggravating heart failure is a potential safety concern, infliximab has been used in patients with ICI-induced myocarditis with more reported cases than other biologic agents Table 1.XL collected materials and wrote the paper. WW, YL, and WC provided the idea and reviewed the manuscript. LF, YL, and WC helped with the final revision of the paper. All authors contributed to the article and approved the submitted version.This work was supported by the Beijing Natural Science Foundation [grant number 7192156], the Capital’s Funds for Health Improvement and Research, CFH [grant number 2020-2-40110], and the CAMS Innovation Fund for Medical Sciences  to WC; and the National Natural Science Foundation of China [grant number 82000470] to YL.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Small Ruminant Research and Journal of the Hellenic Veterinary Medical Society. The most frequent general topics of study were animal health-welfare and animal products. The findings have indicated that research has focused on milk production and diseases of the udder of small ruminants; moreover, there was accumulation of relevant research in Greece in some establishments only. The findings of the study can be used by researchers; stakeholders and Government entities to improve relevant research and to better allocate resources in the country.The article studies the research output on sheep and goats from Greece; a country where small ruminant farming is the most important terrestrial animal farming business. Milk production from sheep and goats exceeds that from cattle and over 90% of total milk produced from sheep and goats is used for dairy products. Research output on sheep and goats have increased significantly from 1997 to 2022. The bulk of publications, 87.5% of relevant papers, has originated from four establishments: Aristotle University of Thessaloniki; University of Thessaly; Agricultural University of Athens and Hellenic Agricultural Organization—Dimitra. Papers were published most frequently in the journals sheep OR goat*] OR [Ovis aries OR Capra hircus]] (1997–2022)) and 1080 papers were considered in detail. Throughout the study period, there was a clear progressive increase in numbers of papers published. The papers originated from 39 different entities, most from Aristotle University of Thessaloniki (34.0%), University of Thessaly (28.0%), Agricultural University of Athens (21.2%) and Hellenic Agricultural Organization—Dimitra (13.6%). Papers were published in 318 different journals. Journals with more published papers were Small Ruminant Research and Journal of the Hellenic Veterinary Medical Society. The most frequent general topic of study in the papers was health and welfare (46.7% of papers); second most frequent topic was animal products (18.6%). The papers have received 16,558 citations, i.e., οn average 15.4 citations per paper; the h-index was 56, the 10i-index was 518 and the yearly citations per paper were 1.71. Papers on goats had higher impact than papers on sheep. There were 1711 individual authors, of which 728 were first or last authors. In total, 24 authors have each co-authored ≥2.5% of all papers; five authors were each first or last in that proportion of all papers. The findings have indicated that relevant research has focused on milk production and diseases of the udder of small ruminants; moreover, there was accumulation of relevant research in Greece in some establishments only. The findings of the study can be employed to initiate improved relevant research approaches in the country.The study is a scientometrics evaluation of published articles performed in Greece on sheep and goats during the last 25 years, a period coinciding with implementation of reforms to shape and consolidate tertiary education and research establishments in the country. Objectives were: evaluation of the relevant publications and presentation of quantitative characteristics regarding scientific content and bibliometric details. The Web of Science platform was used (search terms: [[ Scientometrics refers to ‘all quantitative aspects of science and scientific research’  and can scientometrics’ AND ] revealed only 12 published articles. Among these, Gupta et al.  OR [Ovis aries OR Capra hircus]] was performed (the asterisk served as a truncation symbol to include variations of the term (goat or goats). A topic search retrieved records that included the query terms in the title, the abstract or the keywords. The search was performed on 16 July 2022 (‘freeze date’).A topic search using the terms  among the papers previously detected.General topic(s) of study in the paper: (i) production systems and sustainability, (ii) genetics and breeding, (iii) physiology, (iv) nutrition , (v) animal products , (vi) health and welfare , (vii) human-related models and (viii) society and professionals. A subsequent search was performed using the term [Involvement of international collaboration .Total number of citations received.Number and names of all co-authors.Accessibility, i.e., whether there was open or subscription-only access to the paper.h-index, 10i-index. Moreover, the number of citations received by papers were also normalised by calculating the average citations per year after publication of each paper.For assessment of the impact of papers published, the following bibliometrics measures were employed: total number of citations received, r [p < 0.05.All data were entered into Microsoft Excel. Descriptive analysis was performed initially. The frequency of the various outcomes was evaluated in tables of cross-categorised frequency data by use of Pearson chi-square test as appropriate. Comparisons of proportions were performed by a two-proportion z-test. Comparisons between continuous data were performed by use of one-way analysis of variance. Pearson’s correlations were performed as indicated and significance of the result was evaluated according to the critical values for Pearson’s r . Linear All the 1080 papers individually assessed, were indexed in the Web of Science, fulfilled the search criteria, originated from establishments based in Greece and presented work on sheep or goats. In total, 649 papers presented work solely on sheep, 197 papers presented work solely on goats and 234 papers presented work on both sheep and goats.p < 0.0001)  .n = 984, 91.1%) and fewer ones from research establishments , central government structures , private sector companies  or local authorities . With regard to universities, most papers originated from the Aristotle University of Thessaloniki , the University of Thessaly  and the Agricultural University of Athens ; with regard to research establishments, most papers originated from the Hellenic Agricultural Organization—Dimitra . Cumulatively, from these four establishments, originated 87.6% (n = 946) of all papers.Papers originated from 38 different entities in the country . Most pap = 0.019) in the origin of papers according to animal species, as some establishments published proportionately more papers on sheep than on goats , whilst others published more papers on goats than on sheep . Details are in There was a significant difference  and fewer ones by the Faculty of Agriculture  . Within the University of Thessaly, most papers were also published by the Veterinary Faculty  and fewer ones by the Faculty of Medicine . Cumulatively, 465 papers  originated from the two veterinary faculties of the country, with fewer ones from faculties of agriculture  and faculties of medicine . At department level, most papers originated from the Department of Obstetrics and Reproduction of the Veterinary Faculty, University of Thessaly   .Small Ruminant Research (n = 166 papers) and Journal of the Hellenic Veterinary Medical Society (n = 41 papers). The journals (n = 19) in which at least 10 papers  have been published, are listed in In total, the 1080 papers were published in 318 different scientific journals. The journals in which most papers were published, were p < 0.0001).In total, 471 of the 502 papers in these 19 journals (93.8%) originated from one of the four establishments with the most papers published . This prp < 0.0001) and of papers on animal products for goats . Details are in The most frequent general topic of study in the papers was health and welfare, which was covered in 504 (46.7%) papers. Second most frequent general topic was animal products  . There wp < 0.0001), as papers from the Aristotle University of Thessaloniki and the University of Thessaly focused on health and welfare of sheep and goats , whilst papers from the Agricultural University of Athens focused on nutrition and animal products . In general, however, output from the four main establishments referred to over 80% of all papers in six of the eight general topics   .Small Ruminant Research; differences were seen for the second and third most popular journal across the topics, although Livestock Production Science / Livestock Science, and to a lesser degree Sustainability, frequently published papers in a variety of topics  . There was a clear progressive increase in the number of papers published annually: of the 26 papers, 4 (15.4%) were published before 2011 , 15 (57.7%) were published from 2011 to 2020  and 6 (23.1%) were published in 2021 and 2022   .n = 93) or diseases of the udder (n = 79). Most diseases described in the papers were of bacterial (n = 164) or endo-parasitic (n = 81) nature . International co-authors were affiliated with establishments in 38 countries, more frequently in the United Kingdom (n = 89 papers), France (n = 38) and Italy (n = 36)   .p < 0.0001).Involvement of international collaborations was more frequent among the four establishments with the most published papers than among other ones: 27.0% versus 11.7% of papers with origin from respective establishments  and on human-related models (33.3% of papers on that topic) had more frequently involvement of international collaborations than papers on other topics  citations per paper, 44 and 49 h-index (p = 0.002), 206 and 406 10i-index (p = 0.63) and 1.79 ± 0.09 and 1.51 ± 0.06 yearly citations per paper (p =0.018), respectively.In total, the 1080 papers have received 16,558 citations, i.e., οn average (± standard error of the mean) 15.4 ± 0.6 citations per paper; overall, the There were no significant differences between the citations received by papers with origin the four establishments with the higher number of papers . There wp = 0.006 for differences in average citations and p < 0.0001 for differences in average yearly citations).Papers on animal products and on human-related models were the ones with the most citations: 20.4 ± 2.0 and 19.0 ± 4.6 average citations, respectively, and 2.42 ± 0.18 and 1.65 ± 0.49 average yearly citations, respectively. The differences between the various general topics of the papers  were sigCumulatively, in the 1080 papers, there were 5770 co-authors, i.e., on average 5.3 ± 0.1 co-authors per paper . In total, there were 1711 individual authors of the papers; among these, 25 authors have co-authored at least 27 papers each , of which 7 authors at least 54 papers each ; one author has co-authored 162 papers . Of these 25 authors, 11 were affiliated with University of Thessaly, 9 with Artistotle University of Thessaloniki, 3 with Agricultural University of Athens and one with Hellenic Agricultural Organization—Dimitra.Cumulatively, these 25 authors were first or last authors in 53.1%, 36.8%, 28.4% and 6.1% of all papers published by the establishments with which they were affiliated, respectively.Of the above authors, 728 individuals were first or last authors in the papers; among these, 5 authors were first or last in at least 27 papers each (max: 122 papers). Cumulatively, these 5 individuals were first or last authors in 237 papers . There appeared to be limited interaction and collaboration between them in publishing papers jointly .p < 0.0001) (The average (± standard error of the mean) number of co-authors per paper progressively increased from 4.6 ± 0.4 and 3.6 ± 0.4 in 1997 and 1998, respectively, to 6.9 ± 0.5 and 6.7 ± 0.7 in 2021 and 2022, respectively  . In 36 pp = 0.42). Moreover, there was a mild correlation between the number of authors in a paper and yearly citations per paper . Finally, there was a clear difference in the number of authors of papers in accord to the general topic of the paper (p < 0.0001) (The average number of authors in papers with origin in any of the four establishments with largest number of papers did not differ significantly to that in papers with origin in any of the other establishments: 5.3 ± 0.1 versus 5.4 ± 0.1 ( 0.0001) .p = 0.011). Proportionately, more papers in the general topics of society and professionals (37.5% of those in the topic) and genetics and breeding (35.0% of those in the topic) were published with open access than papers in other topics (p = 0.0001) (p = 0.003).In total, 237 (21.9%) papers were published with open access. There was clear evidence that, after 2019, papers published with open access outnumbered those published with subscription-only access . Respect 0.0001) . Papers Small ruminant farming is currently the most important animal farming business in Greece and generates approximately 18% of the total primary sector income ), revealA comparison with Turkey, another country in the area with high sheep and goat populations (42.0 M sheep and 11.9 M goats ), revealThe above points indicate that possibly the size of a country  and the extent of its educational system  seem to be more also important for the total research output additionally to the relative importance of the livestock sector within the country.Nevertheless, research output from Greece has increased progressively. There was a stability in the number of papers published during 2011 to 2015, which coincided with the financial problems of the country, a consequence of which was the significant reduction in funding of public activities, including research. Increase has resumed after 2015 and again a steady growth has been established.The findings indicate that research work on sheep and goats and consequent output have centred around four establishments in the country. These are the two veterinary faculties of the country, the Agricultural University of Athens  and an agricultural research institute.These establishments have attracted more frequently international collaborations, as they can be ‘seen’ more often in the international scene. International collaboration, which is an important branch of bibliometric indicators , can proFive persons affiliated with the above establishments have authored (as first or last authors) most papers. Moreover, most authors with a large number of papers were affiliated with these establishments, which indicates the presence of large groups active in the topic within these institutions. However, few collaborative inter-establishment papers were found. Possibly, international collaborations might be preferred over work with other establishments within the country. Differences in research interests  or rules for funding projects may also potentially account for this limited national collaboration. However, science and research can provide solutions to various problems, and thus collaborations need to be established at various levels, nationally and internationally .Reasonably, in the country, research on sheep and goats is focused on dairy production. Most papers on the general topic of animal products have dealt with dairy products technology and safety, whilst only few papers have dealt with meat products. Further, Greece has already been identified to have a high output on sheep mastitis . Those fThe present findings are aligned with the production systems prevailing in the country. Nevertheless, further work needs to be performed on meat production and this is an area identified for future studies. In this respect, the results of scientometrics studies could be of use and could be considered by governments, grant-giving bodies, and researchers to direct resources of research and perform relevant studies accordingly.Small Ruminant Research, an international journal with a thematic approach to sheep and goat research. Such journals are favoured by researchers, as they address their work to a specialised audience with greater interest in relevant studies. Moreover, scientists working on a topic possibly read systematically thematic journals, thus become aware more easily about developments in research in their work area. The Journal of the Hellenic Veterinary Medical Society is a journal published by a national society; the journal, further to the international audience, is also widely distributed to local veterinarians, members of the Society; hence, authors can use publications in that journal as a means to inform the national community about matters of interest. The journal has been published continuously since 1950 and has been listed in the Web of Science since 2010, after confirmation that it met the inclusion criteria.Most papers have been published on The sharp increase in numbers of articles published in open access is compatible with the increasing support of the European Commission for this mode of publication and the requirement for using this approach for all publications derived from EU-funded research . ReasonaScientometrics takes into account objective measurements and indexes to assess independently and objectively scientific research. First, the results can be used to better orientate relevant research, e.g., by detecting little-studied topics, by identifying researchers with relevant outcomes, by classifying establishments, ultimately by exploring the broader impact of research.Moreover, the results can be used in the evaluation of research performance and research outputs by research groups and academic establishments of various levels for many purposes, e.g., assessment or resource allocation. Certainly, the results of scientometrics studies are not meant to replace peer-reviewed assessments. However, these evaluations can be supported by scientometrics results, so-termed ‘informed per-review’ , and canThe findings have indicated focusing of research on sheep and goats in Greece in areas associated with dairy production. It is noted that research in meat production in all its facets  will also be of benefit for the country and should be instituted to support this part of production from small ruminant farms. Such research can also attract further international collaborations and funding. Moreover, the findings have indicated accumulation of research on sheep and goats in Greece in only some establishments. These institutions and the respective researchers have accumulated expertise on this scientific area and have been contributing on it by expanding their work. The presence of large groups in these establishments favours scientific output. The findings of the study can be employed to initiate improved relevant research approaches in the country.


Background: The level of competition achieved following biosimilars market availability varies by country, care setting and molecule. Hence, biosimilars contribution to attaining price reductions and extended access to treatments can also vary.Objective: The aim of this study is to capture market dynamics for tumor necrosis factor (TNF)-alpha inhibitors and competing molecules in Southern European markets (2011–2020), and to evaluate the benefits of the competition generated by the availability of biosimilars.Methods: This study is based on a literature review examining market characteristics for TNF-alfa inhibitors and competing immunomodulator molecules, and on the quantitative analysis of market data for these molecules in Italy, Portugal and Spain.Results: Following biosimilars availability in Italian, Portuguese and Spanish markets, there has been an expansion in the overall access to TNF-alfa inhibitor pharmaceuticals. Further, savings have been generated within the TNF-alfa inhibitors class even after the increased use of these molecules. However, the potential of infliximab, etanercept and adalimumab biosimilars to generate price competition outside of their own drug class appeared limited in the studied markets. Considering this limitation and that shifts towards on-patent and higher-cost therapies have occurred after TNF-alfa inhibitor biosimilars availability, the importance of investing in biosimilars development for still on-patent immunology biologics is emphasized.Conclusion: This study highlights the need for policies that do not only seek higher utilization of biosimilars, but that also support a sustainable market for these products. This is expected to foster the future development of biosimilar medicines. The market entry of biological medicines has revolutionized care for patients diagnosed with immune-mediated inflammatory conditions. The timely access to targeted biologic disease-modifying therapies -alfa inhibitors) has allowed to improve patients’ outcomes, by reducing disease activity and facilitating disease remission . HoweverBesides complying with the same quality, efficacy, and safety standards as originator biologics , biosimivia centralized procedures in Portugal. Since then, National Health Service’s expenditure  associated to the purchase of on-patent biologic immunomodulators such as secukinumab, ixekizumab and vedolizumab has been on the rise  within the group of products purchased the rise . Similarthe rise . Along tthe rise ; c. Basethe rise .For an exhaustive understanding of the real impact of the market availability and use of biosimilars in Europe, one should consider country-, care- and product-specific scenarios. This study aims to capture market dynamics following TNF-alfa inhibitor biosimilars availability in a sample of countries representative of Southern Europe . Here, biologics are primarily prescribed/dispensed at the hospital level and incur high costs for National Health Services . An addeThe study methodology was designed according to published research analyzing markets for TNF-alfa inhibitors prior to and following biosimilars availability . We firsApart from screening academic databases, we searched gray literature repositories within websites of Spanish, Italy and Portuguese health institutions , AIFA’s Medicines Utilization Monitoring Centre (OsMed), Portuguese National Authority of Medicines and Health Products (Infarmed), Portuguese Central Administration of the Health System (ACSS), Spanish Medicines Information Centre (CIMA), Spanish Medicines Agency (AEMPS), etc.). In the light of findings from the literature review, we then conducted a quantitative analysis of market data for TNF-alfa inhibitors and competing immunomodulator molecules in Italy, Portugal and Spain.Our analysis is based on consumption volume and National Health Services’ acquisition costs evolutions for TNF-alpha inhibitors, in relation to competing biologic  -inhibitors) and targeted synthetic disease-modifying molecules  -inhibitors). The inclusion of molecules in the analysis  or number of units . Acquisition cost data for the three countries of study are based on National Health Service’s expenditure (in euros) and do not account for confidential discounts or rebates following tender negotiations. Volume and cost data were provided by the Portuguese National Authority of Medicines and Health Products (Infarmed) and the Spanish Ministry of Health Department of Pharmaceuticals and Health Products . In the We examined the growth of the market (fold change in consumption volume) for TNF-alfa inhibitors in the years 2017–2020, and we compared the evolution of consumption volume for each marketed TNF-alfa inhibitor product, and for the whole class in the three countries of study see . We thenData provided by The Rheumatic Diseases Portuguese Register (Reuma.pt)  allowed We analysed the evolution in National Health Services’ costs associated with the acquisition of TNF-alfa inhibitor therapies and competing products  in relation to the evolution in consumption volumes. Based on available data for the years 2018–2020 for Italy, Portugal and Spain we represented the yearly percent units change in costs in relation to the yearly percent units change in consumption volume see –C. PositNational Health Services’ expenditure associated to the purchase of on-patent biologic immunomodulators in Italy, Portugal and Spain has increased in the past years. The market presence of off-patent TNF-alfa inhibitor molecules and the availability of biosimilar versions for these molecules , even after biosimilars availability.The consumption volume of TNF-alfa inhibitor products has increased in the past years . This represents an expansion in the overall access to TNF-alfa inhibitor pharmaceuticals see . AdalimuFollowing TNF-alfa inhibitor biosimilars availability, the market presence of adalimumab-containing products augmented with respect to other TNF-alfa inhibitor molecules. In Italy for instance, the volume share of adalimumab increased by 5.31% in the first year or adalimumab biosimilars availability. In Portugal and Spain, a similar trend was observed, although less pronounced See . ConversWe characterized shifts in utilization trends for TNF-alfa inhibitors and for competing classes in Portugal, based on data provided by the Rheumatic Diseases Portuguese Registry  for the For the three indications of study, RA, PsA and AS, we observed a shift in utilization trends from off-patent TNF-alfa inhibitors  towards certolizumab pegol and golimumab and towards other competing products . This is despite the market entry of infliximab and etanercept biosimilars, available at lower acquisition costs. The biggest shift in drugs utilization was reported in the indication ankylosing spondylitis See . In a 6-We examined the evolution in costs incurred by National Healthcare Systems to fund TNF-alfa inhibitors and competing pharmaceuticals in relation to the evolution in consumption volumes for these products. More precisely in Italy See  and PortData presented in Immunosuppressant and immunomodulating agents belong to the highest expenditure category of hospital-use pharmaceuticals in Italy, Portugal and Spain. Within this category, patent-covered biologic and targeted synthetic disease-modifying agents account for most of the expenditure ; d. It h® and Simponi®), and IL-inhibitors in the rheumatology area.The availability of TNF-alfa inhibitor biosimilars in Southern European markets has supported the earlier and expanded access to these therapies . This haEven though costs per DDD for infliximab, etanercept and adalimumab biosimilars have been generally lower than originator costs in the studied markets, we detected 1) high intra-country heterogeneity in biosimilars uptake and 2) areas where TNF-alfa inhibitor biosimilars uptake was lower than 25%. After 6 years of biosimilars availability in the TNF-alfa inhibitors market and considering European-level uptake data, a higher biosimilars adoption could have been expected from Southern European markets . The het® and Simponi®). This confirms the notion that the scope of the competition between different active molecules is limited and showcases the need for strategies that can fully leverage biosimilars competition . For these molecules, National Health Services’ costs in 2020 were lower than in previous years despite stable or increased consumption volume. However, the extent of cost savings is rather limited for on-patent molecules within the drug class , Cimzia® (certolizumab pegol), Simponi® (golimumab) and Stelara® (ustekinumab). Although the main patents for Actemra® and Cimzia® already expired, biosimilar candidates for these molecules are not yet being evaluated for approval by the EMA. The fact that there are multiple biosimilar candidates in development for tocilizumab and ustekinumab is encouraging. However, it is unclear whether the market entry of these products will result in strong competitive pressure within the category of immunomodulator/immunosuppressant drugs. Also, estimates indicate that various originator biologic immunosupressants/immunomodulators whose patents are set to expire in the coming years, may not face biosimilar competition in the short-term . It is noteworthy that we studied molecules with different indications and formulations profiles. Consumption trends were compared for a broad range of subcutaneous formulations and for a more limited range of intravenous formulations. Unlike for intravenous formulations, dosage of subcutaneous formulations can be modulated by patients. The effect of dose modulation on consumption trends has not been discussed within this manuscript. Furter, differences in consumption trends for the molecules studied could be partly due to the different indications profile of these molecules . Also, registered indications for originator products do not necessarily coincide with registered indications for the respective biosimilars. These differences increase the complexity of analysis of volume trends evolution. Volume trends can in fact reflect prescribers’ lack of familiarity with newly granted indications for specific molecules. Based on our data, it was not possible to analyze these factors.Regarding the inclusion of molecules in the analysis, we aimed to incorporate all marketed TNF-alfa inhibitors and all relevant competitors. Although the list of molecules selected is not complete, we managed to include most relevant market competitors  in the rheumatology area in Portugal. Here, it would have been optimal to provide data at least up to 2020 . However, this was not possible due to missing information from 2017 onwards. Future research would benefit from expanding our Portuguese case study on drug utilization shifts in rheumatology to Italy and Spain. Future research would also benefit from analyzing the interplay of volume and cost evolutions for TNF-alfa inhibitors and competing products at the regional level in the countries of study. Due to procurement activities organized at the regional level in these countries, these dynamics have varied regionally.®, Enbrel® and Humira®. In light of these data, the need for policies that do not only seek higher utilization of marketed ‘best-value’ biologics, but that also support the sustainable development of new biosimilar molecules is highlighted.Our analysis showed that the availability of TNF-alfa inhibitor biosimilars in Southern-European markets led to lowered National Health Services’ costs within the drug class, even after an increased consumption volume for these therapies. However, the potential of marketed TNF-alfa inhibitor biosimilars to generate price competition outside their drug class has been limited. This limitation becomes especially relevant in the context of observed drug utilization shifts towards on-patent and less-affordable therapies, even after loss of exclusivities for Remicade


Community‐based cohort studies have shown that low levels of N‐terminal pro‐B‐type natriuretic peptide (NT‐proBNP) increase the risk of cardiovascular events and death.et al.In a recent issue of the mother journal, Verbrugge special patient population, and it is unclear whether the findings can be applied to patient populations that were excluded, even with the same disease phenotype. The treatment effects of some drugs that have not been shown to be effective in these special HFpEF populations are also uncertain in non‐special HFpEF populations.Recently, clinical trials of newer HF drugs, such as sacubitril valsartan and sodium‐glucose cotransporter 2 inhibitors, have pried open the tightly closed doors to effective treatments for the HFpEF population.special patient populations are often used to develop treatment recommendations in relevant guidelines and consensus statements. We currently have little evidence to manage the clinically important but overlooked HFpEF population, including patients with lower NP concentrations.Importantly, we need to be reminded of the current framework in which the findings of clinical trials conducted on et al.,cannot‐miss HFpEF phenotype.In the study by Verbrugge