Precise control and maintenance of population size is fundamental for organismal development and homeostasis. The three cell types of the mammalian blastocyst are generated in precise proportions over a short time, suggesting a mechanism to ensure a reproducible outcome. We developed a minimal mathematical model demonstrating growth factor signaling is sufficient to guarantee this robustness and which anticipates an embryo's response to perturbations in lineage composition. Addition of lineage-restricted cells both in vivo and in silico, causes a shift of the fate of progenitors away from the supernumerary cell type, while eliminating cells using laser ablation biases the specification of progenitors toward the targeted cell type. Finally, FGF4 couples fate decisions to lineage composition through changes in local growth factor concentration, providing a basis for the regulative abilities of the early mammalian embryo whereby fate decisions are coordinated at the population level to robustly generate tissues in the right proportions. Across metazoa, coordination between cell fate specification and population size ensures robust developmental outcomes. Integration of cell behavior at the population level allows a coordinated response to injury in both embryos and adults . The preThe blastocyst-stage embryo is a hallmark of mammalian preimplantation development. It comprises three cell types – the pluripotent epiblast, which gives rise to the fetus, and the extra-embryonic trophectoderm (TE) and primitive endoderm , which predominantly form supporting tissues . In the Nanog, Gata6, Fgf4 or Fgfr1 alter these proportions and cause peri-implantation lethality . In the mouse embryo, uncommitted ICM progenitors, which co-express NANOG and GATA6, adopt epiblast or PrE identity asynchronously and irreversibly over the course of blastocyst development . In wildethality . The ratethality . HoweverIn this study, we combine manipulations of ICM composition with predictions from in silico simulations to address the question of regulation of the number of cells allocated to each ICM lineage. We develop a minimal mathematical model in which cell fate decisions in the ICM are mediated solely by intercellular signaling. In this model, ICM cells spontaneously and robustly segregate into two lineages, which scale with embryo size as they do in vivo. The model has only two free parameters, which are adjusted to recapitulate the observed wild-type behavior. The robustness of this in silico decision is evidenced by the response of the system to perturbations that alter lineage composition. Specifically, the model predicts  that reducing or increasing the number of cells in one lineage, would change the pattern of progenitor differentiation to restore lineage composition. This effect is also observed experimentally by using two-photon laser excitation for ablation of specific cells in embryos, and by adding exogenous, lineage-restricted cells to embryos to generate chimeras. The ability to recover from these perturbations is reduced over time, as the number of uncommitted progenitor cells is depleted. Finally, we alter the size of the PrE by experimentally tuning the size of the epiblast compartment. Using this system, we show that FGF4 is the growth factor providing the feedback necessary to couple lineage size with cell fate decisions. Our results provide a mechanistic basis for the regulative and scaling abilities of the early mouse embryo and illustrate how a self-organizing system can develop robustly and reproducibly without the need for external inputs.Epiblast and PrE cells originate from a population of bipotent progenitor cells that co-express the lineage-associated transcription factors NANOG (epiblast) and GATA6 (PrE) , which wEpiblast and PrE size (with respect to cell number) scale with embryo size to maintain a consistent ICM composition . To dete−/−Gata6 cells, instead of wt cells , wt cells contribute almost exclusively to the PrE, whereas in chimeras with fewer mutant cells (>60% wt GFP+ cells), wt cells contribute to both the epiblast and PrE, and generate ICMs with a normal lineage composition (We first mixed labeled and unlabeled wild type-(wt) cells, which have equivalent developmental potential, in different proportions . The prowt cells , and monepiblast , left  of epiblast:PrE cells , charactBox 1.A minimal model of growth factor-mediated cell-fate decisions.We use the following model to represent the dynamics of a population of cells in which the transcription factors NANOG and GATA6 mutually repress each other through extracellular growth factor signaling:i, and x over the immediate neighborhood of cell i, including Here, This model can be derived from a specific molecular-level circuit explicitly involving NANOG, GATA6 and the growth factor FGF4, and implicitly ERK signaling downstream of FGF receptors, as detailed in Appendix 1. However, due to its minimal character, the model is not unique to the molecular interactions assumed to be involved in this cell fate decision; other molecular circuits can likely be reduced to it to achieve the same result.In the model, the level of NANOG is inhibited by that of its neighbors, in a manner that resembles lateral inhibition-mediated signaling. We ask whether such a model can sustain solutions in which cells cluster into two distinct cell types, expressing NANOG at two different levels (which we could interpret as epiblast and PrE cells). Epiblast and PrE cells display a salt-and-pepper distribution in the ICM at early blastocyst stages . Therefoa and b of the two clusters are interchangeable.The dynamics of this potential two-cluster solution can be examined via the phase-plane portrait shown in The existence of the two symmetric stable equilibria ensures that the two-cluster state is a solution of the system, and that the population splits spontaneously into two distinct fates, as we show by means of agent-based simulations (described in Appendix 1) throughout the text.ix per cell, which in our case can be considered to represent the amount of NANOG in that cell. FGF4 is assumed to be activated by NANOG and feeds back onto NANOG and GATA6 via ERK  and are rearranged as the embryo develops and grows. To that end, we implemented an agent-based model to simulate the growth of the ICM (see Appendix 1) in three dimensions, in which cells divide and interact with one another via a soft-sphere potential . The bioThe fate decisions ascribed by the model are also biologically robust, since altering the absolute cell number within the in silico ICM leads to scaling of lineage size to maintain ICM composition , in agreThe attractor solution found in the model described suggests that the cell fate decisions reached by the embryo are robust to perturbations, in particular to those affecting the size of each population. To probe the capacity of the system to perceive and adjust to changes in cell numbers, we expanded the epiblast by introducing increasing amounts of mouse embryonic stem cells (ESCs) into embryos . ESCs ar+/−Gata6 or +/−Fgf4 blastocysts, which exhibit a reduction in PrE cell numbers relative to epiblast  , althougWe next asked whether this experimental perturbation would be recapitulated by our mathematical model in silico. To do so, increasing amounts of epiblast-equivalent cells (ESCs) were added to the system before activation of the molecular circuit  and a uWe used laser ablation  to test Pdgfra expression (Pdgfra and mKate2), as well the overrepresentation of PrE cells within the ICM. Progenitor cells in embryos where the PrE was targeted showed a comparable trend to control embryos, generally upregulating Pdgfra expression and acquiring a PrE identity  to recovery of ICM composition after cell ablation. In addition to changes in progenitor specification , we obseOverall, these results indicate that changes in ICM composition are primarily compensated for by a shift in the differentiation pattern of progenitor cells. Although the small sample size precludes any definitive conclusion regarding the roles of cell death and proliferation, both our cell ablation and chimera data suggest that cell death and division play only accessory roles in this process, and that uncommitted progenitors are the primary substrate for regulation.−/−Fgf4 embryos, which cannot make PrE . Embryos were obtained from natural matings of 4- to 12 week-old females to stud males. Alleles used and their original source are summarized in Embryonic days (E) were determined by considering noon of the day of detection of the copulation vaginal plug as E0.5. Higher-resolution staging based on total cell number was used throughout as previously . To deteEmbryos were flushed from dissected oviducts (8–16 cell stage) or uterine horns (blastocyst stages) using forcing Flushing and Holding Media , as previously described . Live emEmbryos were lysed for genotyping in 10 µl of lysis buffer  for 50 min at 50°C, followed by 10 min at 90°C to inactivate Proteinase K . 2 µl of2 atmosphere. Prior to culture, embryos were rinsed 3x in drops of KSOM-AA. For live imaging, embryos were cultured on 35 mm glass-bottom dishes (MatTek) within 5–10 µl drops of KSOM-AA under mineral oil.Embryos were cultured on 35 mm Petri dishes  within microdrops (10–15 µl) of Potassium Simplex Optimized Media with amino acids  under mineral oil (Sigma), at 37°C, in a humidified 5% COImages of live embryos were acquired using Zeiss LSM880 laser-scanning confocal microscopes. In cell ablation experiments, images were acquired using a Zeiss C-Apochromat 40x/NA1.1/WD0.21mm objective. For all other experiments, images were acquired using a Zeiss EC Plan-Neofluar 40x/NA1.3/WD0.17mm. GFP was excited using a 488 nm Argon laser at 20µW. mKate2 was excited using a 543 nm HeNe laser or a 561 nm DPSS 561–10 laser at 90µW. Laser power was measured through a Zeiss Plan-Neofluar 10x/NA0.3 objective prior to each imaging session with a light meter (Coherent) and the laser output adjusted to match laser power across experiments. 80 µm stacks were acquired through embryos, at 2 µm intervals, every 15 min. Although these stacks do not capture an entire blastocyst, they encompass the ICM while limiting laser exposure to about 30–40 s per time point and embryo. Time lapse movies were 16–20 hr long.Tg/+CAG:H2B-EGFP R1 ESCs  used were  R1 ESCs  and HexVE14 ESCs . ESCs weTg/+CAG:H2B-EGFP (Tg/+CAG:H2B-EGFP) were used in the generation of both wt ↔ GFP+ and −/−Gata6 ↔ GFP+ chimeras  and those with four or fewer cells were discarded. Morulae (8–16 cells) from CD1 x EGFPTg/+  crosses chimeras , whereasGdf9iCre  and a fle  attached to an Eppendorf CellTram microinjectiong system was used to assist with injections. Blastocyst-stage embryos were collected in the morning of the 4th day of development and monitored for development to the mid-blastocyst stage (chimeric embryos were retrospectively estimated to have had ~60–80 cells at the time of injection). Individual embryos were held with a holding pipette  by the ICM end, while a Piezo Drill flat tip needle (Eppendorf) was used to introduce ESCs into the blastocyst cavity . After iPdgfra expression    , in whicteTg/Tg) . For ablud males . For ablTg males .H2B-GFPPdgfra allele was markedly higher than in their neighbors on each optical plane  to cover the period of PrE and epiblast specification  in which a number of ICM cells equivalent to the total number of PrE or epiblast cells found in GFP+ littermates  was randomly selected and ablated using the same settings.Cells were eliminated by repeatedly focusing the beams of an 800 nm Ti:Sapphire femtosecond laser at 10–12% output (Coherent), onto the central region of each nucleus to be ablated (approximately 30–50% of the nuclear area in the section). The number of pulses was determined empirically on a test embryo for each experiment, so that a clear wound was observed on the fluorescent nucleus, but no obvious damage was inflicted to the neighboring cells  . AlthougAfter cell ablation, live intact and targeted embryos, were imaged for 16–20 hr, as described above. Laser ablation generated a visible wound in nuclei expressing H2B-GFP, which allowed tracking of targeted and intact cells and assessing nuclear fragmentation as a mark of cell death ; Video 4Whole-mount embryo immunofluorescence was performed as described previously . PrimaryImmunolabeled embryos were mounted on 35 mm glass-bottom dishes (MatTek), within micro drops of a 5 µg/ml solution of Hoechst 33342 in PBS and imaged using a Zeiss LSM880 laser-scanning confocal microscope, equipped with an oil-immersion Zeiss EC Plan-Neofluar 40x/NA1.3/WD0.17mm. Z-stacks were acquired through whole embryos with 1 µm step between optical slices. Laser power was measured for each laser line prior to each imaging session and parameters adjusted so as to keep laser power consistent for each primary-secondary antibody combination across experiments over time – except for the 405 channel, which was used to excite the nuclear label (Hoechst 33342), and was solely used for image segmentation.https://github.com/therealkatlab/MINS (requires MATLAB license). Missing nuclei, or multiple nuclei segmented as one (under-segmentation) were measured manually using ImageJ  and the measured values for each channel, as well as XYZ coordinates, added to the data table.Nuclear image segmentation of still images and manual image correction was performed using the MINS software  as previPdgfra expression and allow the classification of cell types over time (Time lapse images were processed using Imaris (Oxford Instruments). Individual ICM cells were identified based on the presence of H2B-GFP and/or nuclear mKate2 and tracked manually over the course of the movie using the spots function. Cell death or mitotic events were labeled as such for each individual cell . GFP levver time . Cell idver time  and verihttps://github.com/nestorsaiz/saiz-et-al_2020/blob/master/notebooks/_data-processing-wf.pdf and in https://github.com/nestorsaiz/saiz-et-al_2020/blob/master/notebooks/Z-correction.ipynb , PrE (GATA6+), double positive  and NANOG-low or NANOG- epiblast (NANOG-lo) . A fifthon.ipynb .Pdgfra expression) for ICM cells were used to determine changes in cell identity over time in time-lapse movies. To reduce noise in the data, a moving average was calculated for each cell, with a window of 4 timeframes (1 hr). A simple classifier was thus devised to assign cell identity automatically to individual cells based on these GFP levels. Thresholds for fluorescence were manually determined for each litter analyzed, both at the start and the end of the movie, which were used to determine a threshold slope for PrE and epiblast identities. The classifier followed two rules: (1) cells classified as PrE or epiblast maintain that identity for the remainder of the movie – based on Corrected fluorescence values of H2B-GFP .Thank you for submitting your article "Growth factor-mediated coupling between lineage size and cell fate choice underlies robustness of mammalian development" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:In this study Saiz et al. explore the mechanism responsible for the balanced generation of epiplast and primitive endoderm cells from the inner cell mass of the mouse embryo. The authors use a combination of molecular and embryological assays coupled with mathematical modelling to conclude that non-autonomous regulative feedback, mediated by FGF4, is responsible for robustly maintaining Epi and PrE ratios.Essential revisions:The reviewers think this is a very well done study, which can be published without any further experiments. However they all commented on the extend of the modelling presented and suggest that this could be strengthened in 3 specific areas with regard to:– FGF spread;– How geometry affects the behaviour;– How asynchrony in decisions is implemented in the model and relates to the biology. Essential revisions:The reviewers think this is a very well done study, which can be published without any further experiments. However they all commented on the extend of the modelling presented and suggest that this could be strengthened in 3 specific areas with regard to:– FGF spread;– How geometry affects the behaviour;– How asynchrony in decisions is implemented in the model and relates to the biology.Our mathematical model was designed to capture the basic interactions posited to underlie the Epi-PrE decision taking place in the early mouse embryo. In spite of its simplicity, the model allows us to examine the influence of a variety of biological factors, in particular those highlighted by the reviewers: (i) the signaling range of FGF, (ii) the geometry of the population, and (iii) the role of cell division variability on the asynchronicity of cellular decisions.We have systematically studied the influence of these factors in the behavior of our model, by performing a sensitivity analysis of the final cell fate ratio exhibited by the model, when all model parameters are varied independently by increasing or decreasing their values 20%, 30% and 50% beyond the nominal values given in Appendix 1—table 1. The results are shown in the new Figure 2—figure supplement 1F. We describe these results in what follows.i) FGF signaling range (spread)An increase in the range of signaling  does not noticeably change the decision pattern exhibited by the model, which reproduces qualitatively the experimental observations. Only when the signaling range is substantially reduced  is the decision prevented. This is to be expected, since the latter case a cell only detects the FGF molecules that it itself secretes. In that limit the decision cannot occur, since in our model the decision is fully mediated by intercellular FGF signaling.We have also systematically varied the range of FGF signaling, from the autocrine limit , in which cells only sense the FGF they produce, to the all-to-all coupling limit , in which the FGF secreted by a cell is sensed by all cells in the embryo. The results show that as soon as cells are coupled with their nearest neighbors , the decision is reached with appropriate cell-fate ratios, which are maintained even when signaling extends over the entire population. Local nearest-neighbor coupling is thus not necessary for the correct decision to arise.ii) GeometryThe decision is also robust to most perturbations in the mechanical parameters of the model, which partly define, and are defined by, the geometry of the system. This can be seen again in Figure 2—figure supplement 1F, which shows that changes in the effective friction and adhesion between cells, and in the adhesion range, do not for the most part affect the cell fate decision ratios produced. Only when the effective friction decreases substantially  is the decision lost. In this latter case, cell mobility becomes too high and intercellular signaling stops taking place efficiently, which mimics the autocrine limit discussed in the previous point.A stronger geometrical constraint is the dimensionality of the system. The main results reported in this manuscript were obtained in a three-dimensional configuration, which corresponds to the experimental situation studied here. However, we have also examined how our model behaves in a more constrained two-dimensional topology. The response of the system when cells are restricted to move  on a 2D plane are displayed in Figure 2—figure supplement 1E. Simulations show that the cell fate decision process is maintained in these conditions, which manifests the robustness of the mechanism to the dimensionality of the system.iii) Variability and asynchrony of the decisionGiven the key role of cell-cell signaling set forth in this study, it is important for our model to describe cell proliferation adequately: when cells divide the population is reorganized, and thereby the signals sensed by cells from their neighbors change. Experimental observations from ourselves and others show that, as the early embryo develops, cells divide in an asynchronous manner.Following previous modeling approaches ), we implemented this asynchrony in our model, by considering that the cell division time of each cell varies randomly (according to a uniform distribution) with respect to a baseline in which cells would divide at times proportional to an average division time Both In the revised manuscript we discuss the results described above, which we have included in new panels (E-H) that have been added to Figure 2—figure supplement 1. We have also taken the opportunity to revise the text in order to discuss the similarities and differences between our model and previous pattern formation frameworks, such as the Turing and lateral inhibition mechanisms. While our model has conceptual similarities to both scenarios, its specific form, and the way in which it derives from a minimal set of assumptions regarding the regulation of the Epi-PrE cell fate choice, is to our knowledge, new. We have also attempted to clarify further the way in which FGF signaling affects NANOG and GATA6 in our model.


Scientific Reports 10.1038/s41598-020-60654-7, published online 27 February 2020Correction to: The original version of this Article contained an error in Figure 1 where the two maps were overlapping. This has now been corrected in the PDF and HTML versions of the paper.


Scientific Reports 10.1038/s41598-020-65327-z, published online 26 May 2020Correction to: The version of this Article previously published contained a corrupted version of Figure 5. This has now been corrected in the PDF and HTML versions of the paper.


First reported in China, the coronavirus responsible for coronavirus disease 2019 (COVID-19) has spread to 213 countries and territories around the world as of April 26, 2020. This study was designed to explore COVID-19 trends in the Eastern Mediterranean Region (EMR), with a particular focus on Pakistan. Daily reports and updates from the Ministry of National Health Services Regulations and Coordination COVID-19 Pakistan and the European Centre for Disease Prevention and Control were collected and study-specific data were extracted and analyzed. Our analysis revealed that, as of April 26, 2020, a total of 22 countries and territories in the EMR have reported COVID-19 cases. Iran had the highest number of cases  followed by Saudi Arabia , Pakistan , and the United Arab Emirates . Egypt (7.1%), Iran (6.3%), and Iraq (4.9%) had high case fatality rates; Lebanon (3.4%) and Pakistan (2.1%) had moderate case fatality rates; Saudi Arabia and the United Arab Emirates had low case fatality rates of 0.8% and 0.7%, respectively. Iran (76.3%) and Iraq (69.4 %) had the highest recovery rate followed by Pakistan (22.5%), the United Arab Emirates (19.2%), and Saudi Arabia (13.6%). If the current trend continues, based on the susceptible, infected, recovered (SIR) epidemiological model, we predict that EMR countries might experience a surge in the number of COVID-19 cases, resulting in as many as 2.12 million cases in Iran, 0.58 million in Saudi Arabia, and 0.51 million in Pakistan by June 20, 2020. Pakistan is the most populated country in the EMR and was the third most-affected country in terms of the number of cases with moderate case fatality and recovery rates. We predict that Pakistan’s weak healthcare system would not be able to sustain care if there is an explosive increase in the number of cases due to insufficient and inconsistent disease prevention and control policies. The best strategy for mitigating the COVID-19 pandemic is to strictly follow recommendations based on epidemiological principles. Coronaviruses have previously been known for causing Severe Acute Respiratory Syndrome (SARS-CoV) in China in 2003 and Middle East Respiratory Syndrome (MERS-CoV) in Saudi Arabia in 2012. The novel virus responsible for coronavirus disease 2019 (COVID-19), termed SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2), is more contagious than SARS and MERS-CoV [Scientific reports suggest that 81% of COVID-19 patients have a mild or asymptomatic disease, 14% have severe disease manifesting respiratory distress or pneumonia and require medical care, and 5% of hospitalized patients require transfer to intensive care units .After originating from China, the novel coronavirus has spread to 213 countries and territories throughout the world as of April 26, 2020. A World Health Organization (WHO) report confirmed the first 100,000 cases in two months , however, the number of cases increased to 200,000 in the next 12 days  .The available global data on April 26, 2020 reflects 2,804,796 COVID-19 confirmed cases with 193,710 deaths. In the Americas region, the United States of America has the highest number of confirmed cases  with 46,204 deaths, followed by Brazil with 52,995 confirmed cases and 3,670 deaths. In the Eastern Mediterranean region, Iran has reported 89,328 confirmed cases and 5,650 deaths followed by the Kingdom of Saudi Arabia with 16,299 confirmed cases and 136 deaths. Spain in the European region has reported 219,764 confirmed cases and 22,524 deaths, followed by Italy with 195,351 confirmed cases and 26,384 deaths. In the Western Pacific region, China has the highest number of confirmed cases  with 4,642 deaths, followed by Japan with 13,182 confirmed cases and 353 deaths. Reports from Southeast Asia showed 26,496 confirmed cases in India with 824 deaths, while Indonesia has reported 8,607 confirmed cases with 720 deaths. In the African region, South Africa has reported 4,361 confirmed cases with 88 deaths, and Algeria has reported 3,256 confirmed cases with 419 deaths [The Eastern Mediterranean Region (EMR) comprises 22 countries and territories and is home to over 679 million people .In mid-February 2020, the first nine confirmed cases of COVID-19 were reported in the Eastern Mediterranean region. Eight cases from the United Arab Emirates tested positive between January 29 and February 9, 2020. One case from Egypt was confirmed on February 14, 2020. The Ministry of Health and Prevention of the United Arab Emirates announced that three of the reported cases recovered and were discharged from the hospital during the second week of February 2020. The confirmed case in Egypt was asymptomatic and had a history of travel to China .On January 30, 2020, the WHO declared COVID-19 a Public Health Emergency of International Concern (PHEIC) . Since tData were collected daily from the Pakistan Ministry of National Health Services Regulations and Coordination COVID-19 dashboard and situation updates from the European Centre for Disease Prevention and Control -12. The The true numbers of cases in the EMR countries were calculated based on the estimation that a substantial number of COVID-19 cases do not get reported and only 14% of the cases have been reported . The numa) Case fatality rate =  x 100b) Recovery rate =  x 100The number of cases that EMR countries might expect by June 20, 2020, was estimated by using a calculator based on the susceptible, infected, recovered (SIR) model, a standard epidemiological model that is used to estimate disease dynamics . These ca) Every infected symptomatic person interacts with seven people a dayb) Every infected asymptomatic person interacts with 15 people a day As of April 26, 2020, Iran had the highest number of total of COVID-19 confirmed cases  in the EMR, with 15,485 active cases, 3,096 critical cases, 5,630 deaths, and 68,193 recoveries. The number of confirmed cases per million population was 1,064 , the number of confirmed COVID-19 cases was 9,813, with 7,457 active cases, 71 deaths, one critical case, and 1,887 recoveries. The number of confirmed cases per million population was 938 Table . The numIn Qatar, the number of confirmed COVID-19 cases was 9,358, with 8,419 active cases, 72 critical cases, 10 deaths, and 929 recoveries. The number of confirmed cases per million population was 3,248  followed by Bahrain , Iran , UAE (938), Kuwait (677), Saudi Arabia (468), Lebanon (103), and Pakistan (55) , Bahrain (229), Pakistan (187), Egypt (166), Saudi Arabia (133), Kuwait (130), Iraq (124), Lebanon (109), and UAE (98).Pakistan, along with many countries around the world, is facing a historical public health challenge. As of April 26, 2020, Pakistan had 12,723 COVID-19 cases. In Pakistan, the COVID-19 outbreak initially presented as sporadic. Two cases were reported on February 26, 2020, and the volume of cases remained quite low (53 cases) until March 15, 2020 . On MarcThe number of confirmed cases was doubled in Pakistan in 11 days Table , which wThe number of deaths doubled in Pakistan in 10 days. However, Iraq 27 days) and Iran (26 days) had a longer deaths doubling time , Iran (6.3%), and Iraq (4.9%) , which describes the average number of people who could be infected by a sick person. Currently, Pakistan has a local transmission rate of 80.6% . As showAll countries and territories in the EMR have reported cases of COVID-19, however, the number of cases and CFR varies among the EMR countries. Pakistan has the third-highest confirmed cases with moderate case fatality and recovery rates. If unswerving preventative and control measures are not adopted to prevent the widespread transmission of SARS-CoV-2, Pakistan could experience an explosive surge in the number of COVID-19 cases amounting to an estimated half a million cases by June 20, 2020, as determined by the SIR epidemiological model. This study highlights the need for strategic development and the implementation of policies and programs focused on enhanced testing, contact tracing, quarantine, and social distancing in Pakistan.


Phaeobacter virus MD18, a phage antagonist of Phaeobacter inhibens, a bacterium with promising use as a probiotic for aquatic farming industries. Genomic analysis suggested that Phaeobacter phage MD18 has evolved to enhance its replication in P. inhibens by adopting favorable tRNA genes as well as through genomic sequence adaptation to resemble host codon usage. Lastly, a high-throughput analysis of P. inhibens transposon insertion mutants identified genes that modulate host susceptibility to phage MD18 and implicated the type IV pilus as the likely receptor recognized for adsorption. This study marks the first characterization of the relationship between P. inhibens and an environmentally sampled phage, which informs our understanding of natural threats to the bacterium and may promote the development of novel phage technologies for genetic manipulation of this host.Bacteriophages are useful nonantibiotic therapeutics for bacterial infections as well as threats to industries utilizing bacterial agents. This study identified  Phaeobacter inhibens and determined its mechanism of infection. Phaeobacter virus MD18, a novel species of bacteriophage isolated in Woods Hole, MA, exhibits potent lytic ability against P. inhibens and appears to be of the Siphoviridae morphotype. The genomic sequence of MD18 displayed significant similarity to another siphophage, the recently discovered Roseobacter phage DSS3P8, but genomic and phylogenetic analyses, assessing host range and a search of available metagenomes are all consistent with the conclusion that Phaeobacter phage MD18 is a novel lytic phage. We incubated MD18 with a library of barcoded P. inhibens transposon insertion mutants and identified 22 genes that appear to be required for phage predation of this host. Network analysis of these genes using genomic position, Gene Ontology (GO) term enrichment, and protein associations revealed that these genes are enriched for roles in assembly of a type IV pilus (T4P) and regulators of cellular morphology. Our results suggest that T4P serve as receptors for a novel marine virus that targets P. inhibens.Bacteriophages have immense potential as antibiotic therapies and in genetic engineering. Understanding the mechanisms that bacteriophages implement to infect their hosts will allow researchers to manipulate these systems and adapt them to specific bacterial targets. In this study, we isolated a bacteriophage capable of infecting the marine alphaproteobacterium IMPORTANCE Bacteriophages are useful nonantibiotic therapeutics for bacterial infections as well as threats to industries utilizing bacterial agents. This study identified Phaeobacter virus MD18, a phage antagonist of Phaeobacter inhibens, a bacterium with promising use as a probiotic for aquatic farming industries. Genomic analysis suggested that Phaeobacter phage MD18 has evolved to enhance its replication in P. inhibens by adopting favorable tRNA genes as well as through genomic sequence adaptation to resemble host codon usage. Lastly, a high-throughput analysis of P. inhibens transposon insertion mutants identified genes that modulate host susceptibility to phage MD18 and implicated the type IV pilus as the likely receptor recognized for adsorption. This study marks the first characterization of the relationship between P. inhibens and an environmentally sampled phage, which informs our understanding of natural threats to the bacterium and may promote the development of novel phage technologies for genetic manipulation of this host. Viruses are the largest known reservoir of genetic diversity. As such, they have given rise to an incredible assortment of genetic tools and potential therapeutics used as sustainable substitutes for antibiotics , 2, targPhaeobacter inhibens is a marine bacterium and member of the Roseobacteraceae family of Alphaproteobacteria. Organisms in this clade are found worldwide, especially near coastal waters , which protects its natural algal symbiont from marine pathogens  and whole-genome sequencing. Based on the morphology and genomic comparison to other related phages, we found that this isolate represents a novel species of siphophage, which we name Phaeobacter virus MD18. Finally, we used a barcoded transposon insertion mutant library  to characterize the relationship between Phaeobacter phage MD18 and P. inhibens. We identified the type IV pilus system, the ChvI/ChvG two-component system, and regulators of cell division as key determinants of infection. This work characterizes the genetic basis of phage infection in an underexplored nonmodel host system.In this study, we isolated a siphophage capable of infecting P. inhibens. We enriched for phages infecting P. inhibens by incubating filtered environmental samples together with exponentially growing liquid cultures of P. inhibens overnight. Then, we filtered the enrichment cultures and spotted each enrichment supernatant onto a lawn of P. inhibens grown on agar plates and monitored for the formation of plaques. Of the 20 environmental samples, only 1 sourced from a seashore environment produced a clear plaque, indicating the presence of a lytic phage. This enriched phage sample contained a high titer of PFU (∼1010 PFU/ml [Bdellovibrio spp.) were not responsible for the observed inhibition. Propagation of clear plaques indicated essentially complete bacterial host lysis and thus was likely the result of a lytic phage, henceforth referred to as Phaeobacter phage MD18.We obtained samples from 20 aquatic environments around Woods Hole, MA, to isolate a wild bacteriophage capable of infecting 0 PFU/ml ) and rep0 PFU/ml ). GrowthCaulobacter crescentus. MD18 was incapable of forming plaques on C. crescentus under conditions in which ΦCbK readily formed plaques  and C. crescentus (bottom). Phages demonstrated host-specific lytic potential. Download FIG S1, PDF file, 1.4 MB.Phaeobacter phage MD18 exhibits lytic specificity to Copyright © 2020 Urtecho et al.2020Urtecho et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Siphoviridae morphotype . Highlighted regions indicate sections exhibiting over 50% sequence similarity with Roseobacter phage DSS3P8. Despite sequence similarity, gene order appears to differ between these two phages  , eight (D4), and five (D6) species clusters. At the genus level, two (D0), five (D4), and two (D6) clusters resulted. The number of clusters determined at the family level were one (D0), five (D4), and one (D6). Download FIG S3, PDF file, 0.2 MB.Phylogenetic analysis of Copyright © 2020 Urtecho et al.2020Urtecho et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Phaeobacter virus MD18 corresponds to a unique species that has not previously been identified.Finally, we assessed whether MD18 had been previously identified in metagenomes. We used BLAST to search the MD18 genome against 27,346 marine virome contigs assembled from 78 marine viromes . This seN-acetylmuramoyl-l-alanine amidase, which have been demonstrated to facilitate bacterial cell wall degradation and cell lysis  . Phage genomes frequently encode tRNA genes, which facilitate the translation of phage transcripts in host bacteria  and less significantly correlated with a distantly related nonhost bacterium, Escherichia coli   . These tRNA gene   .10.1128/mSphere.00898-20.6TABLE S2Table S2, XLSX file, 0.02 MB.tRNA genes identified in Phaeobacter phage MD18 genome. Download Copyright © 2020 Urtecho et al.2020Urtecho et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. inhibens by identifying host gene products that confer susceptibility to infection. Recent work has used transposon insertion sequencing to rapidly perform reverse-genetic screens in hosts and identify genes that contribute to phage susceptibility (–P. inhibens transposon insertion mutants with decreased susceptibility to phage by exposing a previously constructed barcoded transposon mutant library (P. inhibens genes annotated using RAST (8 PFU) for 8 h, conditions which allowed for significantly delayed growth in wild-type P. inhibens  was originally isolated in Galicia, Spain, and is available through the Leibniz Institute DSMZ. P. inhibens DSM 17395 was grown in Difco marine broth 2216 liquid (MB), on plates supplemented with 1.5% agar, or in 0.5 to 0.7% top agar overlays at 30°C. The P. inhibens-infecting bacteriophage was isolated from Woods Hole Waterfront Park in Woods Hole, MA. A sterile 15-ml tube was loaded with a sample of seagrass and filled to 15 ml with seawater. After vigorous vortexing for 5 s, 5 ml of this sample was filtered using a 0.22-μm polyethersulfone (PES) syringe filter and divided into 1-ml aliquots. A single aliquot was incubated overnight at room temperature with 5 ml of log-phase  P. inhibens in MB, centrifuged for 2 min at 2,000 × g, and filtered using a 0.22-μm PES syringe filter to obtain the enriched bacteriophage population. This enriched sample was used for all downstream experiments with no further purification. The presence of the isolated phage was confirmed by spotting onto 0.7% top agar inoculated with mid-exponential-phase P. inhibens. The isolated phage was named MD18 because it was isolated as part of the microbial diversity course at the Marine Biological Laboratory in Woods Hole, MA, during the summer of 2018.The P. inhibens, and this mixture was spread evenly across a petri dish containing MB agar. Once solidified, 5 μl of each dilution was spotted and incubated for 16 h at 30°C. As a control to exclude the presence of a cellular predator, 10 μl of the undiluted phage stock was incubated with 1 μl of 2.0% chloroform for 1 h with shaking at 4°C before spotting on the same plate. Medium control was prepared by incubating 10 μl of MB with 1 μl of 2.0% chloroform for 1 h with shaking at 4°C. To perform the liquid growth assays in the presence of bacteriophage, a log-phase culture of P. inhibens was diluted 1:100 in MB and 190 μl was aliquoted into a 96-well Corning clear-bottom plate. To each well, 10 μl of each bacteriophage dilution or sterile MB was added  in triplicate. The plate was grown for 24 h at 30°C with shaking at 150 rpm, and the OD600 of each culture was monitored using a Promega GloMax Explorer multimode microplate reader.Top agar spotting and liquid growth assays were used to measure the concentration of PFU in the lysate and to characterize the plaque phenotype of the enriched phage MD18 isolate. Eight 10-fold dilutions of the enrichment were prepared by serial dilution in MB. To perform the spotting assay, 4 ml of MB with 0.5% molten agarose was cooled to ∼50°C before mixing with 1 ml of log-phase 10 PFU/ml) was incubated with a glow-discharged Formvar-coated 200-mesh copper grid for 3 min before being washed three times with sterile 0.22-μm-filtered water. The sample was negatively stained by incubation in a 2% uranyl acetate solution under darkness for 1 min before undergoing another series of washes. For imaging host cells and phage MD18 together, 5 μl of the same phage preparation was preincubated with 5 μl of exponential-phase P. inhibens culture for 10 min and adhered to the grid and stained as described above. Samples were imaged using a Zeiss 10CA transmission electron microscope with help from the Marine Biological Laboratory Central Microscopy Core.To prepare the bacteriophage for transmission electron microscopy, 10 μl of the enriched bacteriophage isolate lysate (∼1010 PFU/ml) in 100 ml of mid-log-phase P. inhibens in MB. This culture was grown for 24 h with shaking before centrifugation and 0.22-μm filtration of the supernatant. This phage fraction was concentrated to ∼1 ml with Corning 30,000-molecular-weight-cutoff (MWCO) Spin-X UF 20 concentrators .Phage MD18 was prepared by inoculating 100 μl of the phage enrichment  purification kit . Briefly, DNase I was added to 300 μl of phage sample at a final concentration of 1 μg/ml and incubated at room temperature for 1 h. To the virus sample, 480 μl of 50 mM EDTA and 900 μl of cell lysis solution were added, vortexed, and incubated for 30 min at 30°C. To this mixture, 600 μl of nucleus lysis solution was added, vortexed, and incubated for 5 min at 80°C. To this mixture, 3 μl of RNase A solution was added, vortexed, and incubated for 30 min at 37°C. To this mixture, 200 μl of protein precipitation solution was added, vortexed for 20 s, and incubated for 5 min on ice. Cellular debris was pelleted by centrifugation at 13,000 × https://rast.nmpdr.org/rast.cgi) to identify putative genes and the tRNAscan-SE 2.0 (http://lowelab.ucsc.edu/tRNAscan-SE/). Genome feature tables were generated by converting GenBank (.gbk) files using the GB2sequin online server . The DNA was sequenced on an Illumina NovaSeq 6000 with a NovaSeq SP reagent kit, yielding 647,478 250-nucleotide (nt) read pairs. Reads were trimmed using Trimmomatic version 0.38, using the parameters SLIDINGWINDOW:4:15 LEADING:2 TRAILING:2 MINLEN:35, resulting in 623,304 reads. These reads were assembled using SPAdes (version 3.11.1) using default parameters. The primary assembled contig was submitted to the RAST , 21 onlin-SE 2.0 , 25 onlie server . The cire server .Phaeobacter virus MD18 and potentially related species was performed using the Virus Classification and Tree Building Online Resource web server (VICTOR). C. crescentus phages were selected from reference JX100813; CcrMagneto, JX100812; CcrSwift, JX100809; CcrKarma, JX100811; CcrRogue, JX100814; and CcrColossus, JX100810. These accession numbers, along with those for Phaeobacter phage MD18 and Roseobacter phage DSS3Ф8 (accession no. KT870145), were submitted to the Virus Classification and Tree Building Online Resource web server .Phylogenetic analysis of VICTOR processed these genomes in the following manner. All pairwise comparisons of the nucleotide sequences were conducted using the genome BLAST distance phylogeny (GBDP) method  under sehttps://genoplotr.r-forge.r-project.org/). Genomes were aligned using NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using discontiguous megablast, which is optimized for more dissimilar sequences. The hit table (text) file from the alignment along with the genome GenBank records (.gb) were used as input for GenoPlotR.Genome maps between Phaeobacter phage MD18 and Roseobacter phage DSS3Ф8 were generated in R (version 4.0.2) using the GenoPlotR package (version 0.8.9) .To determine whether MD18 had been previously identified in metagenome libraries, the MD18 genome was searched against a data set of 27,346 marine virome contigs assembled from 78 marine viromes . BLAST , 60 commP. inhibens library was originally produced by Wetmore et al. , and 1.5 ml of 100-fold-diluted plaque-purified bacteriophage lysate (∼1010 PFU/ml) was added to three of these cultures. These six cultures were grown for 8 h, then 1 ml of each culture was centrifuged at 2,000 × g for 2 min, and the pellets were frozen for later DNA preparation.The e et al.  and geneIn total, nine samples were processed for BarSeq. Three of these samples represented the original library, three represented the library grown without phage, and three more represented the library grown in the presence of phage. Genomic DNA from all samples was harvested using a Promega Maxwell RSC PureFood GMO and authentication kit. To prepare these samples for Illumina sequencing, sequencing primer sites, Illumina flow cell adapters, and sample indices were added using two successive PCRs. Primers used are described in 10.1128/mSphere.00898-20.8TABLE S4Table S4, XLSX file, 0.01 MB.Primers used for BarSeq libraries (PCR 1 and PCR 2). PCR 1 added sequencing primers, whereas PCR 2 added indices and flow cell adapter sequences. Download Copyright © 2020 Urtecho et al.2020Urtecho et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. inhibens DSM 17395 genome (GenBank no. CP002976.1) was annotated using the RAST (https://rast.nmpdr.org/), and barcodes were grouped by which RAST-identified gene they overlapped. Barcode transposon insertion sites were previously described by Wetmore et al. . The the RAST , 21 onlie et al. . All insFirst, the gene enrichment scores with and without phage were calculated. This was determined by dividing the RPM-normalized counts of all barcode insertions overlapping each gene after treatment by the RPM-normalized counts before treatment.RPM-normalized counts were calculated from the mean of three biological replicates. Then, the fitness score of each gene disruption was calculated by dividing the sum of all enrichment scores within the phage treatment by the sum of all respective enrichment scores without phage added (see equations).https://string-db.org/) and searched against Phaeobacter inhibens DSM 17395. Default settings were utilized and online STRING tools were used to identify three gene clusters using K-means clustering.Fitness scores were considered significant if they were greater or less than 3 standard deviations of the bootstrapped mean fitness score. All gene annotations were generated using RAST with default parameters and viewed using SEED to acquire Gene Ontology terms. To generate the gene network, amino acid sequences of the genes whose knockouts exhibited significantly greater fitness scores were uploaded as multiple sequences to the STRING online tool (MT270409). P. inhibens transposon mutant fitness data are available on NCBI Gene Expression Omnibus (accession no. GSE148502). Code to analyze BarSeq data and generate figures is available on Github (https://github.com/gurtecho/PhaeobacterphageMD18).The Phaeobacter phage MD18 genome is available in GenBank (accession no.


Acute restraint stress (ARS) is an unavoidable stress situation and may be encountered in different clinical situations. The aim of the current study was to investigate the effects of ARS on the hippocampus and cerebellum, assess the impact of these effects on the behavior and cognitive function, and determine whether pretreatment with ceftriaxone would attenuate the damages produced by ARS on the hippocampus and cerebellum. Four groups of male mice were included in this study: The control group, ARS group, ceftriaxone group, and ARS + ceftriaxone group. Tail suspension test, Y-maze task, and open field tests were used to assess depression, working spatial memory, and anxiety. The biochemical analyses included measurements of serum cortisol, tumor necrotic factor (TNF), interleukin-6, hippocampal expression of bone morphogenetic protein 9 (BMP9), lysosomal-associated membrane protein 1 (LAMP1), glutamate transporter 1 (GLT1), heat shock protein 90, cerebellar expression of S100 protein, glutamic acid decarboxylase (GAD), and carbon anhydrase. Histopathological examination of the brain sections was conducted on the hippocampus and cerebellum by hematoxylin and eosin stains in addition to ultrastructure evaluation using electron microscopy. Our results suggested that ceftriaxone had neuroprotective properties by attenuating the effects of ARS on the hippocampus and cerebellum in mice. This effect was demonstrated by the improvement in the cognitive and behavioral tests as well as by the preservation of the hippocampal and cerebellar architecture. Acute restraint strain (ARS) is an inevitable stress situation that might cause autonomic and behavioral alternation . The hipThe cerebellum is the area of the hind brain that plays an important role in cognitive and emotional processes . The dysCeftriaxone is an antibiotic that has shown neuroprotective effects in animal models of stroke , traumatThe aim of the current study was to investigate the effects of ARS on the hippocampus and cerebellum, assess the impact of these effects on the behavior and cognitive function, and evaluate whether pretreatment with ceftriaxone would attenuate the damages produced by ARS on the hippocampus and cerebellum.In total, 24 male albino mice aged 10 weeks (25–30 g) constituted the animal model of the current study. The guidelines of the Ethical and Scientific Committee of the Department of Medical Physiology at the Faculty of Medicine, Cairo University were followed. Rats were divided into 4 groups with 6 mice per group:Vehicle control group: Where mice received normal saline (0.9% NaCL solution) intraperitoneally (i.p), with a 24-h interval for 3 consecutive days.-Acute restraint stress (ARS) group: Where each mouse experienced a single stress session following 18 h of fasting (food deprivation). Each stress session consisted of 2.5 h of immobilization by firmly securing all four limbs of each mouse to a grid using a quartz tape [-rtz tape .Ceftriaxone group: Received ceftriaxone , i.p., as a single dosage of 200 mg/kg/day dissolved in normal saline [-l saline  for 3 coARS + ceftriaxone group: Where each mouse experienced ARS (in a similar way to that in the ARS group) and received 3 doses of ceftriaxone .-The following tests were performed before drug administration and grouping of mice and after acclimatization to the laboratory environment to avoid previously reported problems in the literature. The tests were then repeated 24 h after exposure to ARS.Open field test: Open field testing measures locomotion, exploration, and anxiety. A wooden box (1 m × 1 m × 0.5 m) was divided by a marker pen into equally spaced squares. A mouse was positioned in the center of the open field and was examined in a quiet room illuminated by controlled light for 5 min [or 5 min . The behTail suspension test (TST): It tests depression-like behavior in mice. Mice were held 50 cm above the floor by adhesive tape positioned about 1 cm from the tip of the tail. Immobility time was recorded for a period of 6 min. Mice were considered immobile only when they hung passively and became completely immobile [immobile .Y-maze test: Mice were placed in the middle of the maze and were allowed to explore the three arms for 8 min to check the working memory of the space. The three successive decisions taken by the mouse with three different arms were counted as a correct choice. The alternation score was calculated by dividing the total number of alternations by the total number of choices minus 2 multiplied by 100 [d by 100 . After 24 h, serum samples were received from the retro-orbital sinuses. The mice were euthanized by cervical decapitation. The cerebella and hippocampi were dissected, and their tissues were used for biochemical analyses and histopathological examination.Serum was analyzed for cortisol, tumor necrotic factor-alpha (TNF-α), and interleukin-6 (IL-6) and cortisol levels by ELISA (Quantikine R&D system USA) as instructed by the manufacturer. Hippocampus tissue was assessed for bone morphogenetic protein 9 (BMP9), lysosomal-associated membrane protein 1 (LAMP1), glutamate transporter 1 (GLT1) and heat shock protein 90 and cerebellar tissue was evaluated for S100 protein levels, GLT1, glutamic acid decarboxylase (GAD) and carbonic anhydrase by RT-PCR .Total RNA extraction: Total RNA was extracted from the homogenized tissue using the SV Total RNA Isolation System  according to the manufacturer’s instructions. RNA concentrations and purity were calculated using an ultraviolet spectrophotometer .2, 2 μL of 0.1 M DTT, and 1 μL of SuperScript® III RT (200 U/μL) was applied to the total mixture and incubated at 25 °C for 10 min followed by another 50 min at 50 °C.The cDNA was synthesized from 1 μg of RNA using the SuperScript III First-Strand Synthesis System according to the manufacturer’s protocol . To summarize, 1 μg of total RNA was combined with 50 μM oligo (dT) 20, 50 ng/μL random primers, and 10 mM dNTP. The total mixture volume was 10 μL. The mixture was incubated at 56 °C for 5 min, then put in ice for another 3 min. The total mixture comprising 2 μL of 10× RT solution, 4 μL of 25 mM MgClReal-time PCR amplification and analysis were performed using the Applied Biosystem software version 3.1 StepOne . The reaction included SYBR Green Master Mix , a gene-specific primer pair , which wTo summarize, proteins were extracted from tissue homogenates using ice-cold radioimmunoprecipitation assay (RIPA) buffer supplemented with phosphatase and protease inhibitors , then centrifuged at 12,000 rpm for 20 min. The protein concentration for each sample was determined using Bradford assay. Equal amounts of protein  were separated by SDS/polyacrylamide gel electrophoresis (10% acrylamide gel) using a Bio-Rad Mini-Protein II system. The protein was transferred to polyvinylidene difluoride membranes  with a Bio-Rad Trans-Blot system. After transfer, the membranes were washed with PBS and were blocked for 1 h at room temperature with 5% (w/v) skimmed milk powder in PBS. The manufacturer’s instructions were followed for the primary antibody reactions. Following blocking, the blots were developed using antibodies for GLT1and beta actin supplied by Thermoscientific  and incubated overnight at pH 7.6 at 4 °C with gentle shaking. After washing, peroxidase-labeled secondary antibodies were added, and the membranes were incubated at 37 °C for 1 h. Band intensity was analyzed by a ChemiDocTM imaging system with Image LabTM software version 5.1 . The results were expressed as arbitrary units after normalization for β-actin protein expression.The dissected mice brains were fixed in a 10% formalin solution and enclosed in paraffin. Sections of 5 µm thickness were obtained at the level of the hippocampus and cerebellum, stained with hematoxylin and eosin, and examined by a light microscope for histological examination .Morphometric measurements were performed in Cairo University-Research Park (CURP), Faculty of Agriculture, Cairo University using a Leica Qwin 500 image analyzer computer system . The image analyzer consisted of a colored video camera, Panasonic wv. GP 210, colored monitor, and a hard disk of a Leica IBM personal computer connected to a BX41 Olympus microscope  and controlled by Lecia Qwin 500 software. Each parameter was measured by two observers blinded to the experimental group by using 10 non-overlapping readings from each animal in different groups.The following structures were measured :(1) Measurements of the molecular cell layer (M) and granular cell layer (G) thickening; at the mid sagittal section of the cerebellum; at 3 different areas; cortical thickness of the folium surface (1), cortical thickness facing the fissure (2), and cortical thickness at the fissure base (3) . They we(2) Morphometric analysis of the Purkinje cell count at higher magnification .(3) Morphometric analysis of pyramidal neurons counts in the CA3 region of the hippocampus .Small blocks (1–2 mm thick) of the brain at the level of the hippocampus and cerebellum were fixed in 2.5% glutaraldehyde at 0.1 M sodium cacodylate buffer at 4 °C for 6 h. All procedures for the preparation and analysis of the samples were conducted according to Grimaud and Borojevic .p ≤ 0.05 [Using SPSS 21 , data presented as mean ± standard deviation (Mean ± SD), a comparison of quantitative variables between groups was made using variance analysis (ANOVA) with Bonferroni post-hoc test. Results were considered statistically significant for p ≤ 0.05 . p ≤ 0.05) in the number of line crossings, the duration of central square entry, and the time spent in the central square. The ARS group also experienced increased anxiety compared to the control group as indicated by a significant increase in rearing, grooming, freezing, and stretching frequencies (p ≤ 0.05) compared to the control group. Ceftriaxone pretreatment significantly (p ≤ 0.05) improved exploratory activity and reduced anxiety relative to the ARS group  in the ARS group compared to the control group  compared to the control group and the same test was significantly decreased (p ≤ 0.05) in the ARS + ceftriaxone group compared to the ARS group (The length of TST immobility suggesting depression in the ARS group was significantly increased (RS group .p ≤ 0.05) in the ARS group compared to the control group. Serum cortisol was decreased and serum IL-6 and TNF-α were significantly decreased in the ARS + ceftriaxone group compared to the ARS group. The same markers were significantly increased (p ≤ 0.05) in the ARS + ceftriaxone group compared to the control group.The biochemical analyses in blood, hippocampal, and cerebellar tissues are presented in p ≤ 0.05) in the ARS group and the level of hippocampal HSP90a was significantly increased (p ≤ 0.05) in the same group compared to the control group. The levels of BMP9, LAMP1, and GLT1 were significantly (p ≤ 0.05) increased in the ARS + ceftriaxone group and the level of HSP90 was significantly decreased in the same group to levels that were similar to those of the control group  increased in the ARS group compared to the control group. The levels of cerebellar S100B and GAD were significantly (p ≤ 0.05) decreased in the ARS + ceftriaxone group compared to the ARS group to levels similar to those of the control group. Cerebellar S100 B, carbonic anhydrase, and GAD were significantly (p ≤ 0.05) decreased in ARS + ceftriaxone group compared to the ARS group, although it was still significantly increased (p ≤ 0.05) compared to the control group. Carbonic anhydrase was significantly (p ≤ 0.05) decreased in the ARS group and significantly (p ≤ 0.05) increased in the ceftriaxone group compared to the ARS group  were uniform in size and evenly arranged. Each neuron had a rounded central vesicular nucleus with a prominent nucleolus. The cytoplasm contained prominent basophilic cytoplasmic Nissel’s granules and was surrounded by thin neuropil. The molecular layer (M) contained many glial cells (G) among the neuronal processes A. The H&E-stained sections of the mouse hippocampus in the ARS group demonstrated pathologic changes in most of the nuclei of the pyramidal neurons as well as pathologic cytoplasmic changes in the form of vacuolated cytoplasm (V). Some pyramidal cells had vesicular nuclei with clogged marginated chromatin and prominent nucleoli (P) while others had pyknotic nuclei (white arrow). Few neurons had homogenous nuclei with eosinophilic cytoplasm (arrows), and others had ghost changes (G) B. The H&E-stained sections of the mouse hippocampus in the ARS group demonstrated other specimens with markedly affected pyramidal cells of the CA3 region. Most of the neurons were shrunken, with hyperchromatic nuclei (thick arrows) and vacuolated cytoplasm; others had pyknotic nuclei (thin arrows) or karyolysis (K). Areas devoid of pyramidal neurons (asterisk) were detected C. The H&E-stained sections of the mouse hippocampus in the ARS + ceftriaxone group demonstrated marked improvement of the pyramidal neurons similar to the normal architecture of the control group. They were heavily crowded with thin neuropil in between and had basophilic cytoplasm, well-formed Nissel’s granules, and vesicular nuclei (P). Few pyknotic nuclei with vacuolated cytoplasm were detected (arrows) compared to other groups D.The electron micrographs of the control group demonstrated a normal ultra-structure of the pyramidal cell as the neuron had an euchromatic, central, and rounded nucleus (N) with a smooth bilaminar nuclear envelope and finely dispersed chromatin. Well-formed rough endoplasmic reticulum (R) and intact mitochondrial (M) were seen A. The ARS group demonstrated a distorted dense neuron with an irregular outline. The nucleus (N) had marginated chromatin, chromatin clumps, and was surrounded by perinuclear space (thick black arrow). The cytoplasm was shrunken and showed deposition of electron-dense bodies (e). Some mitochondria were distended with disrupted cristae (black arrow) while others were comparable to the control (white arrow). The rough endoplasmic reticulum was markedly dilated (r) B. The ARS group demonstrated markedly affected pyramidal cells. The nucleus was irregular in shape (N) and the cytoplasm contained multiple ballooned mitochondria (arrows) with disrupted cristae C. The ARS + ceftriaxone group demonstrated a marked improvement of the pyramidal cell architecture; the nucleus (N) was regular in shape and was surrounded by the bilaminar nuclear membrane. The cytoplasm revealed multiple mitochondria comparable to the control group (black arrow) while others were ruptured (white arrow) D.The H&E-stained sections in the mouse cerebellar tissues of the control group demonstrated the characteristic pattern of the cerebellar cortex formed of three layers: The molecular cell layer (M), Purkinje cell layer (P), and granular cell layer (G). The medulla formed of white matter fibers (W) was also seen A. The ARThe H&E-stained sections in the mouse cerebellar tissues of the control group demonstrated the molecular cell layer (M) and showed small scattered basket cells and stellate cells. The Purkinje cell layer (P) showed large pyriform-shaped cells having vesicular open-face nuclei, eosinophilic cytoplasm, and prominent Nissl’s granules. The granular cell layer (G) showed crowded small deeply stained cells A. The ARS group demonstrated few Purkinje cells (P) with deeply stained nuclei and eosinophilic cytoplasm, surrounded by vacuolated neuropil. Other Purkinje cells were shrunken with pyknotic nuclei and vacuolated cytoplasm (arrows) B.The ARS group demonstrated other specimens with marked degeneration of the Purkinje cells, exhibiting vacuolated cytoplasm (V) and pyknotic nuclei (black arrows). Basket cells and stellate cells were surrounded by perineuronal spaces (white arrows) C. The ARS + ceftriaxone group demonstrated a remarkable improvement and restoration of the normal architecture of the three cortical layers of the cerebellum; Purkinje cells were increased in number, and most of them were comparable to the control group with open face nuclei (P) while few cells had darkly stained nuclei and eosinophilic cytoplasm (arrow) D.The electron micrographs of the control group demonstrated Purkinje cells with an euchromatic nucleus (N) with finely dispersed chromatin and well-formed bi-laminar nuclear envelope. The cytoplasm contained scattered cell organelles. A primary dendrite (D) was also seen projecting from the cell membrane A. The ARS group demonstrated Purkinje cells with a dark electron-dense nucleus (N) and corrugated nuclear membrane. The cytoplasm contained multiple swollen mitochondria with rarified matrix and fragmented or lost cristae (M) B.The ARS group demonstrated markedly affected Purkinje cells with signs of degeneration. The nucleus was shrunken, darkly electron dense, and irregular in shape with indentation of its nuclear membrane (I). Many dispersed irregularly shaped chromatin aggregates were seen throughout the nucleus. The cytoplasm contained multiple ballooned mitochondria (arrows) with disrupted cristae C. The ARS + ceftriaxone group demonstrated an improvement of the Purkinje cell architecture, with an euchromatic nucleus (N) and finely dispersed chromatin. The cytoplasm revealed multiple mitochondria comparable to the control group (M) while others were disrupted with separated outer and inner mitochondrial membranes (arrows) D. p < 0.05)  .p < 0.05)  were significantly restored in the ARS + ceftriaxone group ( < 0.05) .p < 0.05) (There was a significant decrease in the number of cerebellar Purkinje cells of the ARS group compared to the control and ceftriaxone groups. The number of Purkinje cells was significantly restored in the ARS + ceftriaxone group ( < 0.05) .Stress leads to the production of mental, emotional, and cognitive alterations . Acute rIn the current study, ceftriaxone (CTX)  demonstrated a substantial neuroprotective impact on plasma stress markers and tissue markers in the hippocampus and cerebellum. In accordance with our results, Wei et al.  demonstrThe pathological changes in the hippocampal and cerebellar cells in the untreated ARS group can be attributed to the high level of serum cortisol, which is involved in stress reactions. High levels of serum cortisol have previously been found to lead to multiple structural and physiological changes in the nervous system .Cortisol has also been shown to influence cognition and decrease activity in the cerebellum. For example, cerebellar volume has been shown to be similarly decreased in individuals with Cushing’s disease . The conDysmetria is thought to be related to the role of the cerebellum in sensory, cognitive, and emotional regulation . Stress Cerebellar activation in response to acute stress involves the expression of the c-fos gene , which was associated the action potential of the neurons . Stress Stress impairs long term potentiation (LTP) in the hippocampus. High doses of cortisol in vivo or in vitro have also been shown to impair LTP , indicatThe effect of ceftriaxone on improving behavior, cognition, and depression in the current study can be explained by the fact that ceftriaxone and corticosterone modulate specific frequency bands in opposite directions, and reveals the potential role of ceftriaxone in counteracting the effects of corticosterone . CeftriaThe number of crossed lines of the mice in the ceftriaxone group was significantly decreased compared to mice in the control group and those in the ARS + ceftriaxone group. This could be attributed to the upregulation of GLT1, and this is in agreement with Matos-Ocasio et al. .The results of the current study showed that mice in the ARS group showed reduced hippocampal BMP9 and LAMP1 and increased hippocampal HSP90 compared to the control group. Mice in the ceftriaxone group showed increased BMP9 and LAMP1 and reduced HSP90 to levels similar to those in the control group.Basal forebrain cholinergic neurons (BFCNs) project into the hippocampus and cerebral cortex where the activation of their neurotransmitter, acetylcholine (ACh), is essential for attention, learning, and memory processes . Bone moThe lysosomal membrane is implicated in the cell death pathway. Lysosomal-associated membrane protein-1 (LAMP-1), an abundant lysosomal membrane protein, produces a sugar coat or glycocalyx on the inner side of the lysosomal membrane and protects the membrane from hydrolytic enzymes and degradation . LAMP1 iCells react to environmental stress through the synthesis of several molecular chaperones. One of the amplest molecular chaperones is the heat shock protein 90 (Hsp90). The expression of HSPs is a sensitive marker for metabolic activation or oxidative stress . RegardiIn the current study, mice of the ARS group showed increased cerebellar S100 B and carbonic anhydrase relative to those in the control group. Mice in the ceftriaxone group showed normalized cerebellar S100B and decreased carbonic anhydrase compared to those in the ARS group; however, it was still upregulated compared to those in the control group. These findings may be explained by the affinity of S100 proteins to bind with calcium as a calcium-binding protein The conformational change caused by calcium binding results in the display of two adjacent hydrophobic-binding surfaces. S100 proteins also interact with calcium-dependent partner proteins, such as . S100B aCarbon anhydrase in the nervous tissue is considered a marker for glial cells. pH has a strong modulatory effect on the function and excitability of the central nervous system. Calcium, even if catalytically inactive, may function as ‘proton-collecting antennas’, thus increasing cerebellar S100 B and carbonic anhydrase in the net transmembrane proton flux and suppressing the development of HC microdomains . CA III γ-aminobutyric acid (GABA) from glutamate. GABAergic neuron dysfunction impairs the cerebellar output [Glutamic acid decarboxylase (GAD) catalyzes the formation of glutamate to r output . The neuIn conclusion, the harmful effects of ARS on the hippocampus and cerebellum  could be ameliorated by ceftriaxone, which appears to have neuroprotective properties. This deduction is supported by the improvement in the cognition and behavior of the mice, and was confirmed structurally by the preservation of the hippocampal and cerebellar architecture, as revealed by tissue markers’ expression, and microscopic and ultrastructural evaluations.


The coronavirus disease 2019 (COVID-19) is an epidemic caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). Populations at risk as well as those who can develop serious complications are people with chronic diseases such as diabetes, hypertension, and the elderly. Severe symptoms of SARS-CoV-2 infection are associated with immune failure and dysfunction. The approach of strengthening immunity may be the right choice in order to save lives. This review aimed to provide an overview of current information revealing the importance of bee products in strengthening the immune system against COVID-19. We highlighted the immunomodulatory and the antiviral effects of zinc and polyphenols, which may actively contribute to improving symptoms and preventing complications caused by COVID-19 and can counteract viral infections. Thus, this review will pave the way for conducting advanced experimental research to evaluate zinc and polyphenols-rich bee products to prevent and reduce the severity of COVID-19 symptoms. On 11 March 2020, the World Health Organization recognized the new coronavirus disease 2019 (COVID-19) as a pandemic. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was detected for the first time in Wuhan, China, at the end of 2019; the virus has not reported before in humans . Most in® combined with standard care  were used for hospitalized COVID-19 patients in a trial registered under the number NCT04480593 ,1918,19]+ cytotoxic T lymphocytes and then induce their restriction by the release of the effectors’ molecules such as perforin and granzymes , which are subsequently recognized by antigen-specific CD8ranzymes C 19]. L. L+ cyto+ helper T lymphocytes with the transition into a T helper (Th) which has a double function: cytokines produce and promote B Cells to generate antibodies. Being stimulated by intracellular pathogens, T helper lymphocytes 1 (Th1) phenotype promotes the cytotoxic T lymphocyte activity by releasing IL-2 and enhances the differentiation of B lymphocytes to plasma cells which produce specific antiviral antibodies by liberating IFN-γ  via MHC I (MHC I); [Human Leukocyte Antigen (HLA) in humans] and MHC II (HLA-Cw∗08) . Furtherng IFN-γ B.T helper lymphocytes (Th2) activate and promote the degranulation and the release of chemokines, proteases, and histamines by innate immune system cells including mast cells and basophils, which increased vascular permeability and enhanced the recruitment of macrophages and other inflammatory cells . Besides+ and CD4+ memory T cells in SARS-CoV-2 patients were effective in prompting a specific immune response from 3 months to 6 years without the presence of antigens ..194].Meanwhile, there is evidence that viral infections are associated with an increase in reactive oxygen species (ROS) levels and a decrease in antioxidant defenses . ExposurPolyphenols are natural compounds well known for their antioxidant and anti-inflammatory effects. The antioxidant activity of polyphenols is related to their capacity to react with ROS using different reaction pathways such as the inhibition of the enzymes responsible for ROS production, the up-regulation of endogenous antioxidant defenses, or scavenging reactive oxygen species . 2 pathways. Resveratrol exerted an anti-inflammatory effect by inhibiting NF-κB and TPA-induced COX-2 expression. Similarly, caffeic acid phenethyl ester (CAPE) inhibits inflammatory reaction via the reduction of NF-κB [It seems that the main pathways, by which polyphenols-rich bee products can attenuate SARS-CoV-2 infection, are the modulation of the immune system, the prevention of inflammatory responses, and the reduction in oxidative stress . It was of NF-κB .pro with efficacy and binding energies equivalent to an already claimed N3 protease inhibitor [Recently, numerous published reports revealed that phenolic compounds found in bee products can react with SARS-CoV-2 and blocks its entry into human cells . It was nhibitor .In addition, the molecular docking of ten flavonoid compounds: apigenin, chrysin, fisetin, galangin, hesperitin, luteolin, morin, naringin, quercetin, revealed that all of the flavonoids studied have a high binding affinity with the active site of the spike protein of SARS-CoV-2 .pro and replication of the virus [The antiviral effect of 6 compounds present in honey and propolis  was studied in silico. The results showed that four compounds  had a strong binding affinity with a good glide score and may inhibit the COVID-19 Mhe virus .pro) and Spike (S) protein) revealed that cyanidin may suppress rdrp by binding at asp761 catalytic residue and halting the viral replication process. In addition, the following flavonoids can interact on the spike proteins’ key rbd and inhibit the spread to receptors and thus limit viral spread: daidzein, eriodictyol, fisetin, genistein, kaempferol, myricetin, quercetin, arbutin, chalconaringenin, phloretin, and liquiritin. In a study conducted by Vijayakumar et al. , the molO-glucoside can be considered lead compounds in the inhibition of SARS-CoV-2 papain-like protease (PLpro) [A total of 220 phenolic compounds were tested in a study published by Pitsillou et al. The results of in silico and in vitro using an enzymatic inhibition assay indicated that hypericin, rutin, and cyanidin-3- (PLpro) .p-cumaric acid, and benzoic acid revealed that rutin and caffeic acid phenethyl ester have the highest affinity to COVID-19 3CL-protease and S1 Spike [In the same vein, docking studies of rutin, caffeic acid phenethyl ester, quercetin, kaempferol, pinocembrin, pinobanksin, galangin, chrysin, S1 Spike  . Antioxidant compounds and other food micronutrients with valuable additional functionalities are important in resolving other COVID-19-related issues. For instance, human trial studies documented that ferulic acid and resveratrol have strong antidiabetic, cardioprotective, and renoprotective effects ,206. Thuhttps://clinicaltrials.gov/ct2/home, accessed on 10 February 2022). For instance, the NCT04578158 trial, a randomized, open-labeled, and controlled study, investigates the adjuvant benefits of quercetin Phytosome  in community-based subjects with confirmed SARS-CoV-2 infection. In addition to quercetin, resveratrol, a stilbenoid that highly presents in bee products, has been the object of 4 clinical trials . The NCT04400890 trial aims to evaluate the safety and explore the effectiveness of resveratrol treatment  against COVID-19 infection. Published data support that resveratrol inhibits coronavirus replication through the down-regulation of mRNA and nucleocapsid protein expressions of SARS-CoV-2 [Furthermore, as of 10 February 2022, quercetin has been approved in 14 clinical trials for profylaxis and managing COVID-19 symptoms (available online at RS-CoV-2 ,208.In the absence of specific antiviral drugs against SARS-CoV-2, natural remedies such as apitherapy could alleviate the complications associated with COVID-19. Considering the crucial role of the immune system in fighting SARS-CoV-2 infection, boosting immunity was highly recommended. In this direction, the present review highlights and provides an overview of zinc and phenolic compounds found in bee products and their direct and indirect actions on the immune system in fighting against this emergent public health crisis. The problems which arise are that the nature, quality, and quantity of these bioactive molecules in bee products differ from one country to another. To overcome this major drawback, an international norm, ISO/TC 34/SC 19, is currently in progress to standardize the whole process and circulation of bee products.


In the two studies presented here, we explore the significance of word class for concreteness and imageability in human and computationally obtained ratings. The observed correlations in the CPD indicate correspondences between psycholinguistic measures expected from the literature. Word classes exhibit differences in subjective frequency, age of acquisition, concreteness and imageability, with significant differences between nouns, verbs, adjectives and adverbs. In the computational study which focused on concreteness and imageability, concreteness obtained higher correlations with human ratings than imageability, and the system underpredicted the concreteness of nouns, and overpredicted the concreteness of adjectives and adverbs. Overall, this suggests that word class contains schematic conceptual and distributional information. Schematic conceptual content seems to be more significant in human ratings of concreteness and less significant in computationally obtained ratings, where distributional information seems to play a more significant role. This suggests that word class differences should be theoretically explored.Psycholinguistic databases containing ratings of concreteness, imageability, age of acquisition, and subjective frequency are used in psycholinguistic and neurolinguistic studies which require words as stimuli. Linguistic characteristics  are frequently coded, but word class is seldom systematically treated, although there are indications of its significance for imageability and concreteness. This paper presents the Croatian Psycholinguistic Database , containing 6000 Croatian nouns, verbs, adjectives and adverbs, rated for concreteness, imageability, age of acquisition, and subjective frequency. Moreover, we present computationally obtained extrapolations of concreteness and imageability to the remainder of the Croatian lexicon (available at:  Psycholinguistic databases containing human ratings of characteristics such as concreteness, imageability, age of acquisition, and subjective frequency are routinely used in a variety of psycholinguistic and neurolinguistic studies which require words as stimuli  are often based on subjective ratings which have been found to correlate with objective measures of AoA , will thus have similar vector values and appear as close in the spatial representation of that particular corpus. What is even more interesting for our work, the N values of the resulting n-dimensional vectors were proven to encode various linguistic properties of words.In machine learning studies, word embeddings are often used. Word embeddings are numerical representations of words, usually in the form of n-dimensional vectors. Considering that vectors can mathematically be represented as positions in an n-dimensional Cartesian space, every word-related vector assigns a unique “address” in space to every word. Word vectors are calculated using the positions of words within large language corpora, based on their neighboring words and in line with observations first made by Harris , and latThere have been warnings that extrapolation may create semantic artifacts absent from the human ratings for AoA, concreteness and affective variables, even if correlations are high  entities defined in time and being diffuse in space  from the object (table). Adverbs are more varied than adjectives, and may refer to the spatial or temporal domain, as well as various other circumstances .This last view hinges on treating word class and other grammatical information as meaningful, which is in line with cognitive linguistic treatments . Finally, adjectives and adverbs have the grammatical valence of exactly 1, because the way they describe qualities and circumstances requires a noun/verb, respectively, whose quality/circumstance is realized. In this sense, adverbs are further removed from adjectives, because they work with verbs which have their separate grammatical valence of at least 1.This information is also related to grammatical valence as conceptually defined by Croft . GrammatThese explanations shed new light on the three leading models that have been developed to account for the processing differences of concrete and high-imageable words on the one hand, and abstract and low-imageable words on the other: the context availability theory  and the University of Rijeka . No monetary compensation was offered, but some students were awarded class credit for their participation. The mean age of the participants was 21.18 (SD = 2.61), and their age range was 18–50. There were 78.45% females and 21.55% of males. Most participants (69.32%) reported speaking two or more foreign languages . Most participants (79.67%) reported that they spent between 1 and 4 hours reading per day on average. Every word was rated by an average of 30 participants (see Table hrast “oak”) and groups of people (e.g. razred “class”) were marked as inanimate, whereas supernatural, anthropomorphic and dead entities as animate , based on the differences in morphological marking of masculine nouns. Preliminary results of the first round of data collection, focusing on the dimensions of concreteness and imageability and testing the dual-coding theory, were published in Croatian  to control for the method (order) effect. The participants rated two variables on the same sheet (concreteness and subjective frequency or imageability and AoA).tuljan “seal”, plivati “to swim”, slan “salty”; abstractness: pravda “justice”, morati “to have to”, poetski “poetic”; high imageability: kuća “house”, grmjeti “to thunder”; low imageability: nedosljedan “inconsistent”, smjeti “be allowed to”, aspekt “aspect”).The instructions see  were forSubjective frequency was operationalized using a Likert scale, where the participants were asked to report how frequently they encounter a word: almost never (1), once a year (2), once a month (3), once a week or (4) once/several times a day (5). AoA was defined as “understanding the meaning of a word at a certain age”, so the participants were asked to estimate the age at which they could say they knew the meaning of a word. Such a continuous measure of AoA correlates highly (above .80) with more frequently used Likert-like rating-scale measures, where the participants have to mark a number indicating an age range (defined in advance by researchers) in which they acquired a word  for the psycholinguistic variables . Moreover, linguistic characteristics of each word are also coded, i.e. word length in number of characters, word class , animacy and gender for nouns and raw frequency in the hrWaC corpus. Currently, the rated words appear only in Croatian, but English translations will be provided by the end of the project.M = .88) for concreteness, from .81 to .88 (M = .85) for imageability, from .89 to .93 (M = .91) for AoA, and from .87 to .90 (M = .89) for subjective frequency. Moreover, there were no significant correlations between the place on the list and ratings for any of the variables, except for imageability ratings on list A and AoA ratings on list B which were both very small at .04 (p < .01). This corroborates the reliability and validity of the collected ratings.The reliability of the ratings for concreteness, imageability, AoA and subjective frequency was calculated by randomly dividing the participants into two subgroups of equal size and computing the correlation between averaged estimation by item. The reliability indexes were calculated on 5000 different randomizations of the participants. The obtained split-half correlations ranged from .86 to .90 . The correlation for concreteness with Ćoso et al. .Results of ANOVAs revealed significant differences between word classes in all the psycholinguistic word features, as well as in word length (p < .001) in all pairs of word classes in mean concreteness ratings, with nouns being rated as the most concrete, followed by verbs (nouns – verbs Cohen’s d = 0.51), adjectives (verbs – adjectives Cohen’s d = 0.42), and finally, adverbs as the most abstract words (adjectives – adverbs Cohen’s d = 0.38). The same pattern was observed for imageability, with nouns rated as the most imageable, followed by verbs (nouns – verbs Cohen’s d = 0.36), adjectives (verbs – adjectives Cohen’s d = 0.31), and finally, adverbs as the least imageable words (adjectives – adverbs Cohen’s d = 0.41).Post hoc Bonferroni tests revealed a statistically significant difference (p < .001) in subjective frequency except for the difference between nouns and adjectives (p = .052). Adverbs were rated as the most frequent, followed by verbs (adverbs – verbs Cohen’s d = 0.65), and nouns and adjectives as the least frequent (verbs – nouns Cohen’s d = 0.29). The same pattern was observed for the AoA. The Bonferroni test indicated a statistically significant difference (p < .001) in all pairs of word classes, except for the difference between adverbs and verbs (p = .205). Adverbs and verbs were rated as the earliest acquired words, followed by nouns (verbs – nouns Cohen’s d = 0.23), and adjectives as acquired the latest (nouns – adjectives Cohen’s d = 0.20) (see Table p < .001). The shortest word class—adverbs—were shorter than nouns (Cohen’s d = 0.27), nouns were shorter than adjectives (Cohen’s d = 0.41), and finally, adjectives were shorter than the longest word class—verbs (Cohen’s d = 0.23). We report effect sizes for the smallest differences obtained in the post hoc tests .As for subjective frequency and AoA, a Bonferroni test indicated that all pairs of word classes differed statistically significantly , novčić “coin” , normalizacija “normalization”  and neshvatljiv “incomprehensible” . However, given the .819 correlation, the CPD also contains words that diverge from this pattern exhibiting high concreteness and low imageability, e.g. primatelj “recipient” , kositar “tin” , and low concreteness and high imageability, e.g. mišljenje “opinion” , obećanje “promise” . Reflecting the high association between imageability and concreteness, the pattern of relations of the two variables with other word features was similar. Thus, words rated as more concrete and more imageable tended to be estimated as acquired at a younger age, as being subjectively and objectively more frequent, and as being shorter. AoA had the highest correlation with subjective frequency, suggesting that words reported to be acquired at a younger age also tended to be rated as more subjectively frequent. Although a lower correlation was obtained between AoA and objective frequency, it reveals that words estimated as being acquired at a younger age are objectively more frequent than those reported to be acquired at an older age. Furthermore, words rated as being acquired at a younger age tended to be shorter than those rated as being acquired at an older age. In addition to the mentioned correlations between subjective frequency and the psycholinguistic variables, subjective frequency had a high positive correlation with objective frequency, and a low negative correlation with word length. Finally, the two linguistic word features correlated moderately negatively: words which are objectively more frequent tend to be shorter. Pearson correlations calculated separately in the subsamples of the four word classes showed that the overall pattern and the magnitude of associations between the word features were very similar across the four word classes.Pearson correlations between all word features included in the CPD, calculated for all 6000 words, are presented in Fig. The values of all word features are comparable to values available in databases for other languages cited earlier. AoA means in our study may seem somewhat higher than those obtained in other studies using a continuous AoA measure for French , with all post hoc differences significant (p < .001), except for the difference between nouns and adjectives. This indicates that an explanation based on word class-related factors may be worth exploring, although the overall effect size of word class on AoA is small.All the correlations obtained in the data follow the expected pattern found in previous studies and databases  to which extent we can extrapolate concreteness and imageability ratings in our lexicon of 6000 entries to the remainder of the Croatian lexicon and (2) to extend the existing body of research on extrapolation of psycholinguistic variables by focusing on (a) variation between word classes and (b) the interaction between concreteness and imageability.Extrapolations in this work were performed by using word embeddings as explanatory variables and one of the two variables, concreteness or imageability, as response variable. In line with Ljubešić et al. , we usedDifferent to our previous work , rs =.87 (MRC) and rs = .89 (BWK), whereas they were rs = .68 (MEGAHR) and rs = .80 (MRC) for imageability. A larger dataset seems to facilitate machine learning, which is why English extrapolations are better than Croatian. The correlation for concreteness for the MRC database for our model is slightly better than the one published by Hollis et al. , and in the case of MRC . Differences in predicting concreteness and imageability between MEGAHR and MRC are also significant .Concreteness predictions correlate with human predictions better than predictions for imageability, and the difference between them is significant, both in the case of MEGAHR  with a clear cline between them. The results for imageability show the strongest correlation for nouns (r = .68), and weaker correlations for the other word classes .We performed a separate evaluation of the models trained and evaluated on the MEGAHR dataset, by separating the predictions by word class. The results show that the overall correlations for concreteness for nouns are stronger than those for verbs, adjectives and adverbs (r = .93) than they are in human ratings (r = .82). We then compared the direction of the predictions for a set of 5301 nouns, verbs, adjectives and adverbs. The sample was obtained by removing the training set and manually discarding any obvious errors in the predicted data . We subtracted the predicted concreteness or imageability mean ratings from the human-rated concreteness or imageability mean ratings separately for each variable, obtaining a score that shows the direction of the prediction. Negative scores indicate that the mean of the predicted value is greater than the mean of the human rating; i.e. in the case of negative scores, extrapolated values are overpredicted in relation to human ratings. Positive scores represent underprediction by machine learning. We performed an ANOVA on the means to test for word class differences.Finally, we compared concreteness and imageability extrapolations with human ratings for all the word classes. The results show that the overall correlations between concreteness and imageability are higher in machine predictions . Post hoc Bonferroni tests revealed that all pairs of word classes except for nouns and verbs differed statistically significantly (p < .001), with nouns being consistently underpredicted, and adjectives and adverbs consistently overpredicted . The results for imageability also showed a significant difference between word classes, but the effect size was minimal .The results for concreteness showed a significant difference between word classes, with a small effect size (imagine comes to mind).Concreteness was easier to predict than imageability, which may mean that concreteness—i.e. availability to the senses—is captured more easily using distributional data. Notionally, concreteness is verifiable by our senses; it refers to entities that are present in our environment and is textually related to a number of lexical items . In contrast, imageability is an “internal” (“one’s minds’ eye”) capacity of a human being to invoke a mental image. The mental image is based on the knowledge of an external stimulus; however, invoking a mental image makes sense only if the stimulus is not present (you do not need to imagine a desk or a foul smell when you can see it or smell it). Imageability is not as clearly related to a number of basic and readily available lexical items . In the remaining cases, distributional evidence may not be sufficient. This is evident from a comparison of predictions of concreteness and imageability for words that participants rated as abstract and highly imageable. A total of 98 such words, with concreteness rated below 2.5 and imageability above 3.5, were found in our set (some of them mentioned in the first study). Among them, imageability was predicted within .5 of the mean of the human rating in four cases, and in 94 cases going beyond this. In contrast, in the same set, concreteness was predicted within .5 of the mean of the human rating in 59 cases, with the remaining 39 cases going beyond the arbitrary .5 limit. This suggests that distributional evidence works better for concreteness than for imageability. This is in line with Crossley et al.’s  study whThe results of the human rating study showed expected correlations between psycholinguistic variables, as well as an effect of word class on the psycholinguistic variables of AoA, subjective frequency, concreteness and imageability. For concreteness and imageability, there was a cline nouns > verbs > adjectives > adverbs. In the machine learning study, we focused on concreteness and imageability and obtained results which, in the whole sample, correlated with human predictions. However, the size of the correlations across word classes followed the same cline. Finally, a similar cline was obtained for the direction of the predictions for concreteness. The system underpredicted the concreteness of nouns, and overpredicted the concreteness of adjectives and adverbs. Imageability predictions followed suit, but the effect size was minimal.These results are in line with the conceptual definition of word classes given in the introduction, which suggests that word class should be taken as a shorthand for semantic and distributional characteristics, at least within a single language. Simply put, nouns typically denote objects, verbs denote relations between objects, and adjectives and adverbs denote qualities and external characteristics. There is a clear conceptual decrease in concreteness and imageability: nouns are most highly concrete and imageable because they are conceptually related to entities that are less diffuse and have a spatial basis, and the decrease in the spatial basis of verbs, adjectives and adverbs, and their increasingly relational nature leads to lower concreteness and imageability. In other words, word class adds a schematic conceptual frame to the lexical meaning of words.This is also visible from the differences in the direction of the prediction for concreteness in the machine learning experiment, where this “conceptual frame” seems to be more significant for humans than when using machine learning based on collocational evidence. It seems that, at least for some nouns, humans give more weight to the conceptual factor of “nouniness” (i.e. thing-like representation) rather than to distributional evidence. By the same token, people see some adjectives and adverbs as more diffuse and hence less concrete, perhaps because adjectives and adverbs require conceptual separation of the quality from the object it describes. In contrast, relying on collocational evidence (where no such separation occurs), the system predicts them as more concrete than humans.Regarding the theoretical significance of word class for the context availability theory, the dual-coding theory and grounding theories, it seems that all of them should incorporate grammatical information as part of their program. It seems that various flavors of grounding theories  convincingly, so that the artifacts are not a result of using only one type of evidence (and the wrong kind of evidence at that). Characteristics other than imageability that require more than just textual evidence seem to include emotional valence, arousal and dominance . Given the role of word class in this work, and the relation between affective variables and concreteness and imageability (Kousta et al.,