Teenage passengers might influence risky driving, particularly in certain mental states. Notably, social exclusion could increase social conformity. Two studies examined simulated intersection management among young drivers after a social exclusion activity . In Study 1 [112 males (mean = 17.3 years)], risky driving was significantly greater among excluded males driving with a risk-accepting vs. passive passenger; no effect of social exclusion. In Study 2 [115 females (mean = 17.1 years)], risky driving was significantly greater among excluded females driving with a risk-accepting vs. a passive passenger, and greater among those included (fair play) vs. excluded when driving with a risk-accepting passenger. Risky driving behavior among male and female teenagers may be influenced uniquely by passenger norms and social exclusion. High crash rates among novice teenage drivers are thought to be due to deficiencies in driving skill and judgment due to young age , inexperFatal crash risk is lower with adult passengers, but higher with teenage passengers, particularly among teenage drivers . A recenPassenger influences on teenage driver behavior are thought to occur through social influence and/or distraction . Teenagen = 30) and 21–24 (n = 20) year-old males and females. Key measures of risky driving included average speed and reaction time to a stop signal at variable time intervals. Among drivers in both age groups, red light running was greater in the presence of passengers. Also, among participants with low inhibitory control, speeding was more prevalent in the presence of passengers. However, passenger presence seemed to improve hazard management and reduced time in the intersection when the light was red, providing additional support for the contention that peer passengers can increase some risks and decrease others, possibly conditional on characteristics of the driver, passenger, and/or driving conditions.Simulation, even in an actual vehicle with high fidelity sounds and motion representing acceleration, braking, and turning; and realistic graphics of scenarios based on actual roads, cannot fully capture actual on-road driving experience. However, simulated driving performance has consistent been associated with on-road performance  and has n = 53) were randomized to drive with a young male passenger (a study confederate) who in the risk-promoting group presented himself as risk accepting and when riding as the passenger exercised mild peer pressure to complete the course quickly; those in the other group drove with the confederate passenger who presented himself as risk averse and when riding as the passenger exercised mild pressure to complete the drive safely by taking few risks. Risky driving  and distraction (failure to stop at an intersection with an occluded stop sign) were greater in the passenger compared to the solo drives, a main effect for passenger presence, consistent with theory and research indicating that adolescent reward sensitivity increases in the presence of peers (of peers . In addin = 66) were randomized to drive solo and with a confederate passenger portraying either risk-accepting or risk-averse social norms. The results confirmed the independent effect of passenger presence and significant interactions by passenger social norms, with those in the group exposed to the confederate passenger with risk-accepting norms, relative to those exposed to the passenger with risk-averse social norms, more likely to run the red light and spend more time in the intersection while the light was red. These findings are consistent with other research indicating that teenage risk taking is greater in the presence of peers, perhaps by sensitizing the brain’s reward system to risk taking  and Study 2 included 115 females 16 to 18 years old (mean = 17.1); participants had a Level 2 Michigan driver license . Participant assent and parent consent were obtained and participants were compensated according to the protocol approved by the University of Michigan IRB.In the between subject designs shown in , particiConsistent with prior research on peer driving norms , predrivCyberball has been validated as a reliable way of simulating the experience of social exclusion . The expA fixed-base high-fidelity simulator located in a dedicated lab space was used for this study. The simulator comprised a full vehicle cab (Nissan Versa) surrounded by three forward screens and one rear screen. The forward screens were projected at a resolution of 1400 × 1050 pixels each and the rear screen at 1024 × 768 pixels, providing a 120-degree forward field of view and a 40-degree rear field of view. The simulator runs RTI’s  SimCreator software. The simulator system included steering feedback, road vibration, a virtual LED instrument cluster, sideview mirrors, and simulated audio. The driving simulator recorded vehicle and driving performance data, up to six synchronized channels of video, and two channels of audio at 30 Hz  was passive with respect to risk and rode quietly to minimize variability in passenger behavior. Participants completed three drives: 5-min coaching/practice drive; 10-min baseline (solo) drive ; and 15-min experimental (passenger) drive. All three drives included typical roadway features  and a wide range of roadway geometries, speed limits, traffic conditions, and visual elements. The drives differed in the ordering of residential, rural, urban, and freeway road segments, with distinct layouts and alterations to surface features , but included identical driving scenarios for eliciting participant behavior, including a car passing task and multiple four-way signalized intersections. Construction barrels at intersections and junctions guided participants to the destination and included a lead vehicle that served to minimize variability in the speeds at which intersections were encountered.Intersection management, particularly when in the dilemma zone when the light turns amber as the driver approaches the intersection and must quickly decide to brake sharply or pass through the intersection as or after the light turns red, which is a traffic violation and dangerous behavior zone’ . AccordiThe average treatment group percent was calculated for three intersection management/risky driving measures: (a) stopping for the red light (% Failed to Stop) in the 10 (of 18) intersections with relatively shorter durations between yellow lights ; this measured the percent of appropriate stopping at the 10 short duration lights; (b) time vehicles were in intersections while the light was red light (% Time in Red); the measure assessed the average amount of time the was in the intersection while the light was red as a reflection of the duration of potential risk for a crash; and (c) passing the slowing lead vehicle (% Passed Slow Vehicle); passing the slowing vehicle represented greater acceptance of risk.To assess individual variability, the week prior to the exclusion task and simulation drives participants completed the following baseline measures: susceptibility to peer pressure , includeCyberball has been validated in behavioral and neuroimaging studies to simulate the experience of social exclusion . ImmediaIdentification with passenger was measured by six items that asked participants to indicate  their identification with the passenger . Passenger approval, a measure of subjective norms, was measured by five items asking participants how likely it was (1 = very unlikely to 5 = very likely) that the passenger would approve of the participant’s involvement in five risky driving behaviors such as driving 10 mph above the speed limit and closely following a slow vehicle.The following items, administered in a post-drive survey, were adapted or created for this study and provide additional information about the participants’ experience. Power analysis was based on data from previous simulation studies  for the post hoc comparisons with Tukey–Kramer adjustment. Between treatment groups psychological needs differences for excluded and included participants were assessed after the exclusion task using independent t-tests.Treatment group differences on the pre-drive randomization and post-drive assessment variables  were assessed using one-way ANOVA (2X2 ANOVA might bias against possible effects) and The primary driving performance comparisons were examined as the differences (passenger minus solo drive) of the treatment groups on each measure of risky driving. The solo drive served to control for individual differences in driving behavior. PROC GLIMMIX in SAS (version 9.4) was used to fit generalized linear mixed models (GLMMs) where the outcomes were % Not Stopping for Red light (average of binary outcomes generating odds ratios), % Time in Red . The GLIMMIX model follows:ib denotes a subject specific random effect, β0 denotes baseline value, and β1, β2, and β3 characterize the effect of each exclusion/inclusion and passenger risk comparison. There were two treatment group comparisons : comparison 1 was the effect of a risk accepting vs. passive passenger given exclusion, where 1 = Excluded + Passive passenger; 2 = Excluded + Risk accepting passenger; Comparison 2 was the effect of inclusion (fair play) vs. exclusion, given a risky passenger; 1 = Exclusion + Risk accepting and 2 = Inclusion (fair play) + Risk accepting Passenger. The models were then rerun adjusting separately for baseline self-esteem and susceptibility to peer pressure. Odds ratios are considered the effect size for GLIMMIX models with binary outcomes and the beta is the effect size for mixed models. In addition, we calculated the standardized mean treatment group differences.where In Study 1, 112 of the 134 recruited participants and in Study 2, 115 of the 137 recruited participants completed the protocol and were included in the analyses. Exclusions were due to simulator sickness or technical issues with the simulator, a rate that is consistent with other driving simulator studies . As a chThe top half of Shown in Shown in In In Study 2 female drivers were significantly more likely to pass the slowing vehicle in the presence of a risk accepting passenger, increasing from 10 to 21%, but not changing among those driving with the passive passenger see . Overallp = 0.11 and p = 0.10) had moderate effect sizes favoring increased risk in the inclusion (fair play) group, with slight declines in the exclusion group and slight increases or little change in the inclusion (fair play) group. Analyses adjusted for susceptibility to peer pressure resulted in negligible differences in the estimates (available upon request).The right half of B = 0.60, p = 0.04) was significant, with declines in the socially excluded group (from 47.7 to 44.9%) and increases in the socially included (fair play) group (from 55.9 to 59.4%), and % Time in Red was significant  with a decline from 29.3 to 25.4% in the excluded group and an increase from 33.6 to 34.1% in the socially included group.For females in Study 2, shown in the right half of This research examined influences of peer passengers and social exclusion on simulated risky driving among male and female teenagers. Identical trials were conducted separately with males and females, in which participants were randomized to one of three treatment conditions allowing evaluation of the following research questions about simulated risky driving: (a) What is the effect of exposure to a risk accepting or passive passenger after social exclusion? (b) What is the effect of social exclusion or social inclusion (fair play) when exposed to a risk accepting passenger? We discuss the findings for each trial  in relation to each research question.To test the possible effect of peer influence on risky driving, it was necessary for participants to perceive differences in the risk acceptance of the confederate passengers. In post-treatment analyses, both males and females identified more strongly with the risk accepting passenger and perceived that the risk accepting passenger was more approving of risky driving than the passive passenger, consistent with successful manipulation of confederate passenger norms, thus allowing for logical interpretation of passenger effects. Accordingly, the findings generally support increased simulated risky driving in the presence of a risk accepting passenger after social exclusion. For males, 2 of the 3 risky driving variables, not stopping for the red light and passing the slowing vehicle, indicated significantly greater risk among those exposed to a risk accepting passenger relative to those exposed to a risk passive passenger. For females, 1 of 3 measures, passing the slowing vehicle, indicated significantly greater risk among those exposed to a risk accepting passenger, consistent with conformity to social norms. Hence, 2 of 3 variables for males and 1 of 3 for females indicated greater risky driving in the presence of a risk accepting passenger relative to a passive passenger after social exclusion, with moderate or large effect sizes.These findings are generally consistent with social norms theory , with paBoth male and female participants in the social inclusion (fair play) group reported being passed the Cyberball more than those in the exclusion group, consistent with successful manipulation of exclusion. In addition, both male and female participants in the inclusion (fair play) groups reported consistently higher scores than those in the exclusion groups on need threat variables  consistent with previous research indicating similar post-Cyberball need threat scores for both males and females . The finSome literature suggests that teenage males exert greater peer influence than females on both teenage males and females  and femaAlternatively, other research has shown that being rejected does not always cause affiliative behaviors, but instead can cause antisocial responses, not only toward the excluder but also toward neutral others . A largeStudy strengths include experimental design using a high-fidelity simulator and proven risky driving protocol applied in separate studies with males and females. Moreover, our experimental manipulations were successful in that participants who were excluded reported lower values on the need threat measures; participants perceived the risk accepting passenger to be more accepting of risk than the passive passenger; and participants identified post-treatment with the risk accepting confederate passenger relative to the passive passenger.The primary study limitation is the lack of a full factorial design (due to budget and time limitations), which would have provided a more elegant and complete test of passenger  and exclusion vs. inclusion (fair play) effects. We did not actually manipulate inclusion by allowing the participants who experienced fair play that others were being excluded, so our inclusion condition was actually a fair play or not-exclused condition. Also, the protocol called for the passive passenger to be neutral with respect to risk, but it is possible the participants interpreted passiveness as rejection, which could have affected their behavior, although we found no evidence of this possibility.After being socially excluded, male and female teenage study participants engaged in relatively greater risky simulated driving in the presence of a risk accepting compared to a passive passenger, consistent with social norms theory and previous research. Teenage female study participants in the presence of a risk-accepting passenger engaged in more risky driving after experiencing fair play, compared to those who had been socially excluded, contrary to prevailing theory; males exhibited similar but non-significant trends. These findings provide additional support for the contention that social norms influence teenage risky driving behavior, indicate that inclusion might increase and exclusion might reduce risk taking behavior in the presence of a risk-accepting male peer, suggesting that social relationships among teens matter with respect to their influence on risk behavior. The findings suggest important new avenues for research on gender differences with respect to the effects of social exclusion on adolescent risk behavior.The study was reviewed and approved by the University of Michigan IRB. Written and informed consent was obtained from the parents of all participants and assent was obtained from all participants.BS-M, CB, and JS conceptualized the study, obtain funding, and participated in the conduct, data analyses, and manuscript preparation. KL and CZ participated in methods development, analyses, and manuscript preparation. LB and EF participated in analyses and manuscript preparation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Approaches to manipulating disease resistance in plants is expanding exponentially due to advances in our understanding of plant defense mechanisms and new tools for manipulating the plant genome. The application of effective strategies is only limited now by adoption of rapid classical genetic techniques and the acceptance of genetically engineered traits for some problems. The use of genome editing and cis-genetics, where possible, may facilitate applications that otherwise require considerable time or genetic engineering, depending on settling legal definitions of the products. Nonetheless, the variety of approaches to developing disease resistance has never been greater. Genetic resistance represents the most economical approach to crop protection, and one goal of understanding plant/pathogen interactions at the molecular level is to facilitate disease resistance in crop species. Disease resistance is often the most dynamic component of the crop breeding process, requiring continual updating owing to pathogen adaptation to plant genotypes. An ancillary goal is to engineer resistance that is broad  and durable (lasting through many cropping seasons). Research continues to unveil details and mechanisms that function to enable pathogens to parasitize plants and how plants defend themselves against parasitism. Increasingly, the knowledge is being implemented in strategies to enhance resistance to pathogens in crop species and to expedite resistance breeding in the field. Creative transgenic approaches continue to be explored, and the more recent genome editing tools have expanded the approaches to engineering resistance. Nonetheless, advances are needed in understanding how basic endogenous defense components work together and in generating novel resistances with components of the defense system.1.Elongation Factor–Tu Receptor (EFR) is a PRR that was identified inArabidopsis and recognizes EF-Tu, a highly conserved abundant protein in prokaryotes2. Many crop species apparently lack this specific or analogous receptor despite the conserved nature of EF-Tu. Transfer ofAtEFR fromArabidopsis to tobacco and tomato, in particular, reduced disease severity in the field due to two bacterial pathogens with very different life styles, namelyRalstonia solanacearum andXanthomonas perforans, the causal agents of bacterial southern wilt and bacterial spot, respectively4. Similar results have been reported for potato5.AtEFR also triggered immunity in wheat upon challenge withPseudomonas syringae pv.oryzae6. However, transfer ofAtEFR to rice did not make the lines more resistant to the most prevalent pathogen,Xanthomonas oryzae pv.oryzae (Xoo), unless the elicitor portion of EF-TU was applied prior to infection8. Transfer of another PRR,Flagellin Sensitive 2 (FLS2) fromNicotiana, to sweet orange reduced susceptibility to citrus canker9. Use of the PRR geneXa21 continues to be expanded.Xa21 of rice produces a receptor kinase that recognizes a small sulfonated peptide (RaxX) synthesized by Xoo and some related species and confers resistance to bacterial blight of rice11. The effectiveness ofXa21 is limited to diseases caused byXanthomonas species that produce RaxX10. Fortunately, transfer ofXa21 to banana provides resistance to bacterial wilt, which is threatening banana and enset production in east Africa, because the pathogen,Xanthomonas vasicola pv.musacearum, also makes and processes RaxX12.Heterologous expression of pattern recognition receptors (PRRs), which recognize conserved molecules in pathogens or the products of pathogen-mediated degradation of host molecules and trigger immune responses to a wide range of pathogens, continues to be applied to a widening range of species, particularly those with recalcitrant disease issuesEFR andFLS2 were originally identified by the response to the isolated elicitor and do not provide complete resistance to infection due to bacterial virulence factors, which are capable of suppressing defense signaling by receptors13. Perhaps it is not surprising that the introduction of heterologous PRR genes corresponding to highly conserved elicitors does not always provide protection to crop species, as their pathogens may have already adapted to defense responses elicited by infection. On the other hand, it is striking, as noted above, that some bacterial pathogens do not suppress host immunity in the presence of the PRRs. Crop species, including tomato, may have lost some PRRs in the domestication and breeding process.The application of heterologous transfer of PRRs among species will be interesting.Bs2 gene for resistance to bacterial spot disease in pepper to tomato, which suffers disease from related pathogens14. The NLR geneRGA2 for resistance toFusarium was transferred from a resistant diploid banana species to Cavendish banana15. Another example is the transfer of an NLR from pigeon pea to soybean for resistance to soybean rust16. The success of transferring NLR genes between species has led to more extensive efforts of extracting NLR homologs  from resistant species. R gene enrichment sequencing, or RenSeq, is a sequence capture technique for the enrichment of NLR sequences17. The underlying goal is to recover NLR family members from plants with known resistance and transfer candidates to the desired variety. Several variations of this method have been reported, including chemical mutagenesis of a line followed by sequence capture and association genetics of wild populations followed by sequence capture19. NLR capture from related species will facilitate the stacking of R genes against common core effectors of all extant pathogen genotypes and, consequentially, provide broad resistance. For example, theBs2 gene, which targets the ubiquitous AvrBs2 effector ofXanthomonas, can be combined withRoq1, a new R gene fromNicotiana directed at the common effector XopQ, and other as-yet-unidentified NLRs directed toward other conserved effectors as identified in sequencing of large strain collections from infected plants21. Advances in NLR gene mining and gene transfer may have come none too soon and can be applied to wheat blast outbreaks22. Advancements in NLR applications will come from new methods to identify novel R genes in existing NLR libraries.The largest family of resistance (R) genes encode the nucleotide binding site and leucine-rich repeat (NBS-LRR or NLR for short) proteins. Owing to their conservation and ease of identification, NLRs are closest to what might be called industrial-scale application, and NLR mining could potentially replace R-gene introgression from related but poorer quality germplasm and crosses with related species (wide crosses). NLR members  generally provide resistance against a specific subset of pathogens, or races, that express specific effector proteins, and the NLR complex often acts in a gene-for-gene manner. More problematic is that the genes tend to function only within closely related species, possibly because of the adaptation to other components of the specific NLR complex. An early example was the transfer of thede novo. Single NLR-related R gene transfer between distantly related species often fails, and broader application of NLRs will come from a greater understanding of NLR function. NLR-like R genes are simply the variable genetic component of NLR signaling complexes, which occur in a variety of forms. The complex can include a guarded protein, alternatively called guardee or sensor, and the sensor may be an integrated domain of the NLR23. The guardee may be a defense signaling protein or other component that is targeted by pathogens to enhance susceptibility. In some cases, the guardee has no apparent function other than to recognize the effector or effector activity of the pathogen. In the latter case, the guardee is referred to as a decoy. In a variety of cases, the components consist of a pair of NLR or NLR-like genes24. The NLRsRPS4 andRRS1, for example, are R genes fromArabidopsis and provide resistance against the bacteriaPseudomonas syringae andRalstonia solanacearum, respectively. The RPS4/RRS1 pair is a remarkable case where the variation between resistance and susceptibility toward each of the two pathogens was based on separate components. However, both genes are, in fact, required for the resistance to each pathogen25. Furthermore, tomato plants expressing bothRPS4 andRRS1 were resistant to both bacteria, and similar results were obtained for transgenic cucumber expressingRPS4/RRS1 against anthracnose26. Manipulation of the complexes may also allow changing the complex to recognize other pathogens. In theArabidopsis–P. syringae system, the R genePBS1 product is perceived as a decoy working in concert with the NLRRPS5 and cleaved by the secreted bacterial protease effector AvrPphB. The cleavage ofPSB1 is recognized by R-geneRPS5, resulting in the detection of bacteria and a resistance reaction27. A novelPBS1 gene was created by substituting the cleavage site with cleavage site sequences that are recognized by other viral and secreted bacterial proteases28. When expressed together, the modifiedPBS1 genes conferredRPS5-mediated resistance to new pathogens. Changes in the integrated sensor domain of the Pik group of NLRs directed at the rice blast pathogen effectors indicate that manipulation of the domain could produce new recognition motifs29. In the potato–Phytophthora infestans system, the resistance geneR3a is activated by the RXLR effector, AVR3aKI, but not by the allelic product AVR3aEM, and, through random mutagenesis, a mutant version of R3a was identified that recognized AVR3aEM30. Interestingly, mutation in the same position in theI2 gene, a homologue of theR3a gene in tomato, made the gene more responsive to AVR3a and conferred partial resistance toP. infestans and expanded the gene’s effectiveness to an additional fungal pathogen31. Recent studies of the structural changes that the NLR proteins themselves undergo upon elicitation may also provide insight for improved manipulation of this effector class33. Further utility of the NLR class will come from improved structural models and associated components and induced variation by gene targeting strategies23.Research is also providing clues that promise to broaden the application of NLR resistance strategies and even the promise of generating NLR libraries with novel pathogen recognition properties34. Secreted anti-microbial peptides (AMPs) are used in a variety of crop species35. Cecropin is an AMP naturally produced by a moth,Hyalophora cecropia, that confers a broad spectrum of protection against a wide range of pathogens. Rice seed expressing cecropin A from endosperm-specific promoter exhibited resistance to infection byFusarium verticillioides andDickeya dadantii36. A synthetic version of cecropin expressed in citrus was reported to be effective against the bacteriumCandidatus Liberibacter asiaticus, the causal agent of huanglongbing (HLB)37. Expression ofde novo designed AMP SP1-1 in tomato fused with secretion of the signal from radish defensin provided resistance to bacterial spot38. Using pathogen physiology against itself has also provided promising results. Diffusible signal factor (DSF) is a mobile extracellular signal molecule produced by bacterial pathogens which controls cell density-dependent patterns of gene expression. InXylella fastidiosa, DSF production is conferred by the generpfF. Ectopic production of DSF results in hypervirulence but decreases transmissibility by vectors owing to interference with bacterial gene regulation39. Susceptible scion grafted to transgenic rootstock also displayed resistance to the pathogen in field trials. Similar reductions in disease severity were also observed by ectopic expression ofrpfF in citrus and tobacco40.Ectopic expression of defense-related, toxin, and other miscellaneous genes has always been a major part of the toolbox in engineering resistance. The approaches have been applied to recalcitrant disease problems and, in a variety of cases, have reached or completed confined field trial stage41. The classic example ismlo, a recessive R gene of barley with resistance toward powdery mildew42. The null allele provides broad, durable resistance against the pathogenBlumeria graminis f. sp.hordei. Simultaneous editing of all three homoeoalleles ofMLO locus in hexaploid wheat conferred recessive resistance against powdery mildew in one generation43. Disruption ofdowny mildew resistance-6 (DMR6) was originally identified inArabidopsis and suppresses free salicylic acid (SA) levels44. Enhanced SA levels are associated with reduced susceptibility to a variety of pathogens, particularly bacterial pathogens. Mutations created by CRISPR–Cas9 inDMR6 orthologs in tomato were reported to confer resistance to a number of pathogens, includingP. syringae pv.tomato,Phytophthora capsica, X. perforans, andXanthomonas gardneri45. Modifications of the SA pathway adds to the widespread experimental utilization of ectopic expression of the SA receptornonexpressor of pathogenesis related genes 1 (NPR1), which has been reviewed extensively47.Strides in genome editing, particularly the CRISPR–Cas9 system, have increased interest towards the development of disease resistance through the modification of susceptible (S) genes of the plantXanthomonas, function by binding to specific DNA sequences, known as effector binding elements (EBEs), in the promoters of S genes and promote heroic levels of S gene expression and, consequently through S gene product function, enhance disease susceptibility. Polymorphisms in EBEs by preventing TALe binding can provide recessive resistance in cases where TALes play a critical role in disease development. The citrus geneCsLOB1 is a susceptibility factor for citrus canker induced by the TALe PthA4 ofXanthomonas citri. CRISPR–Cas9-mediated modification in the EBE in the promoter region ofCsLOB1 improved canker resistance and reduced canker development in sweet orange and grapefruit50. Bacterial blight of rice is highly dependent on TALe-mediated expression of sucrose transporters known as SWEET genes. Three SWEET genes are known to be targeted by strains of Xoo, and natural polymorphisms occur in the SWEET gene promoters that provide recessive resistance to some strains of the pathogen Xoo.OsSWEET14 is an S gene in rice for infections by Xoo. Mutations in the promoter EBE region for the TALe effectors AvrXa7 and PthXo3 using TALe nucleases (TALENs) led to loss of expression of S gene and resistance to strains depending on those two TALes for virulence53. The S geneOsSWEET13 is upregulated by TALes, leading to bacterial blight54. Modification of the promotor ofOsSWEET13 rice by CRISPR–Cas9 treatment resulted in resistance against strains that requireOsSWEET13 expression54. Modification of all three genes simultaneously has been accomplished with CRISPR–Cas9 and provides broad resistance against extant strains55.Diseases that involve transcription activator-like effectors (TALes) are excellent candidates for genome editing. TALes, which are deployed by many members of the bacterial genus56. For example, a CRISPR–Cas9-mediated complete disruption of theOsSEC3A gene in rice caused enhanced defense response against blast caused byMagnaporthe oryzae. However, the resistant plant showed dwarf stature and lesion-mimic phenotype58. Similarly, the mutations in wheatMLO conferred resistance against powdery mildew, but some mutants have been reported to have a leaf chlorosis phenotype similar to what is sometimes observed in barley59.Disruption of S genes, by either editing or silencing, potentially comes with its own set of problems. First, these resistances are recessive, requiring homozygous mutant plants to screen for resistance. Second, these genes often have functional roles in the plant, and deletion or modification of expression may result in growth defects and/or loss of yield61. TAL EBEs can also be added to promoters to recruit TALe-mediated expression of autonomous R genes or avirulence proteins leading to resistance reactions63.The affinity of TALes to bind to specific EBE sequences can be hijacked for activation of resistance by TALes. R gene promoters have been modified to include a concatemer of EBE sites for TALes from multiple strains of a pathogen64. They are also vital for normal initiation of translation in host cells. Due to the presence of two isoforms, null mutants of any one isoform can result in resistance to a number of viruses66. However, simultaneous knockout of both isoforms can lead to lethality or impaired growth67. Genome editing can be used to create novel functional alleles by mimicking natural variation of eIF4E in resistant plant species68. These synthetic genes for elF4E confer resistance to viruses without affecting plant physiology.Because of their dependence on host functions, viruses are vulnerable to manipulations of host genes. Eukaryotic translation initiation factors (eIF4E) are essential for infection by single-stranded positive-sense RNA virusesin vivo69. The CRISPR–Cas9 system expressed transiently inNicotiana along with sgRNA that recognize the viral genome significantly reduced geminivirus accumulation not just in inoculated areas but also systemically71. The ability of viruses to overcome immunity is much lower when targeting intergenic regions compared to coding regions57. A powerful system to control multiple viruses in cotton leaf curl disease related to begomovirus complex can be achieved by multiplex CRISPR system, imitating CRISPR function in bacteria72.Site-specific nucleases have also been deployed as functional components in plants for resistance against viruses. This approach imitates the viral immunity in prokaryotes in which the CRISPR system recognizes and cleaves the viral genomeLr34 andLr67, provide broad partial resistance to wheat leaf rust (Puccinia triticina) and to other pathogens, for example, yellow rust (Puccinia striiformis f. sp.tritici). The two genes were discovered to encode ABC-type and hexose transporters, respectively74. Despite the novel properties of the gene products, transfer of the genes, at least within the cereal family, indicates that the genes can function similarly in related species79.Lr67 appears to be impaired in sugar transport, whileLr34 product was shown recently to be capable of transporting abscisic acid (ABA)80. Whether these attributes are relevant to the broad and multi-pathogen resistance is unknown. At the same time, the functionality in related species indicates a conserved function.The analyses of naturally occurring resistances, particularly cases of single gene broad R genes, has provided a remarkable variety of genes beyond the now-classic PRRs and NLRs. Wheat breeding and characterization of many wheat relatives has provided many broad, durable R gene candidates, and the characterization of a number of rust R genes of wheat warns us that knowledge of broad, durable resistance will likely come with considerable advances in our understanding of plant physiology. The broad R genes,81. Genome modification using site-specific nucleases and removal of vector sequences by crossing, or use of vector-free approaches, has been accepted in some agencies not requiring regulation as a transgenic event. However, acceptance has not been universally accepted. Regardless of regulatory issues, our understanding of plant resistance mechanisms has increased considerably in the last five years, and new insights into defenses against resistance that incorporate abiotic and other physiological pathways of plants will undoubtedly be forthcoming. The discoveries will inform mutational and traditional breeding strategies in the absence of adoption of gene transfer or gene editing technologies and help meet the future needs for food output for a growing world population and climate-challenged food production system.Success of many of the advances in engineering disease resistance in crop species, of course, depends on societal acceptance of various approaches to plant genome modification. Ectopic expression of heterologous transgenes or silencing of genes generally comes under the guise of foreign DNA transfer and still faces considerable headwinds owing to acceptance and regulatory protocols. Modification of transgenic classifications, for example, the concept of cisgenics  as opposed to transgenics (the addition of a gene or genes from a non-crossable species), may increase the workable space in crop modification


The purpose of this review was to provide detailed insights into the pathophysiology of myofibroblast-mediated fibrosis (scarring or late haze) after corneal injury, surgery, or infection.Literature review.The epithelium and epithelial basement membrane (EBM) and/or endothelium and Descemet's basement membrane (BM) are commonly disrupted after corneal injuries, surgeries, and infections. Regeneration of these critical regulatory structures relies on the coordinated production of BM components, including laminins, nidogens, perlecan, and collagen type IV by epithelial, endothelial, and keratocyte cells. Whether a cornea, or an area in the cornea, heals with transparency or fibrosis may be determined by whether there is injury to one or both corneal basement membranes (EBM and/or Descemet's BM) and delayed or defective regeneration or replacement of the BM. These opaque myofibroblasts, and the disordered extracellular matrix these cells produce, persist in the stroma until the EBM and/or Descemet's BM is regenerated or replaced.Corneal stromal fibrosis  occurs as a consequence of BM injury and defective regeneration in both the anterior (EBM) and posterior (Descemet's BM) cornea. The resolution of fibrosis and return of stromal transparency depends on reestablished BM structure and function. It is hypothesized that defective regeneration of the EBM or Descemet's BM allows key profibrotic growth factors, including transforming growth factor beta-1 (TGF-β1) and TGF-β2, to penetrate the stroma at sustained levels necessary to drive the development and maintenance of mature opacity-producing myofibroblasts from myofibroblast precursors cells, and studies suggest that perlecan and collagen type IV are the critical components in EBM and Descemet's BM that bind TGF-β1, TGF-β2, platelet-derived growth factor, and possibly other growth factors, and regulate their bioavailability and function during homeostasis and corneal wound healing. The role of Descemet's BM injury in posterior corneal fibrosis has only recently been recognized14 Transient haze or mild opacity associated with corneal abrasions, normal PRK,13 or routine riboflavin-UV cross-linking,15 for example, also cause disruption of the EBM, but in these situations the normal regeneration of the EBM halts the development of myofibroblast precursor cells and triggers apoptosis of the myofibroblast precursors and subsequent repopulation of the stroma with keratocytes after termination of the early injury-related wave of keratocyte apoptosis.14Injuries, surgeries, infections, and diseases that trigger myofibroblast-associated corneal fibrosis have in common disruption of the corneal BMs and delayed regeneration of the BMs.4 Thus, in normal LASIK surgery, fibrosis is typically noted only at the flap margin (16PRK and laser in situ keratomileusis (LASIK) provide readily reproducible models for the development of anterior corneal stromal fibrosis. An increased understanding of the factors that promote stromal fibrosis rather than stromal transparency in PRK and LASIK has been of critical importance in understanding fibrosis that occurs after other injuries, surgeries, and infections of the cornea. PRK causes disruptions of the EBM overlying the central cornea, whereas normal LASIK results in disruptions of only the peripheral EBM at the flap edge.p margin B. Howeve1720 Driven by TGF-β, platelet-derived growth factor (PDGF), and possibly other unidentified factors, the best-characterized precursors to these cells in corneal stroma (keratocyte-derived and fibrocyte-derived precursors) transition through convergent developmental programs from vimentin+, alpha-smooth muscle actin (αSMA)−, desmin− (V+A−D−) immature myofibroblasts to vimentin+, αSMA+, desmin− (V+A+D−) intermediate cells to vimentin+, αSMA+, desmin+ (V+A+D+) mature myofibroblasts.21 This developmental maturation process, and the secretion of disordered extracellular matrix once the myofibroblasts are mature, explains why fibrosis typically does not appear until 2 to 4 months after PRK in humans and why corneal fibrotic scars tend to intensify over time after corneal infections, trauma, or surgery. Myofibroblasts are contractile cells that express decreased intracellular crystallins17 and that produce disordered extracellular matrix components that alter the normal organization of the corneal stromal collagen lamellae to diminish transparency.17 This contrasts with corneal fibroblasts  that are also generated by injuries, surgeries, infections, or diseases of the cornea. Corneal fibroblasts also show decreased expression of corneal crystallins and an area relatively opaque compared to keratocytes,17 but they tend to be transient, produce only small amounts of disordered extracellular matrix, and are generated whether or not there are persistent EBM defects. Thus, transient haze noted after normal PRK, rapidly healing corneal abrasions, or normal riboflavin-UV cross-linking is attributable to corneal fibroblast generation without the production of mature myofibroblasts.1315The development of mature myofibroblasts in the corneal stroma, and the persistence of these cells, is the critical stage in the development of fibrosis of the stroma .4,17–20 2225 Although stromal cells themselves produce TGF-β, especially after injuries, the amount produced is insufficient to drive myofibroblast generation from either keratocyte-derived or bone marrow–derived precursors.23 Sufficiently large amounts of TGF-β can, however, be provided by the epithelium .28 Clearly, there are mechanisms in place to regulate the penetration of epithelial- and aqueous humor–derived TGF-β, PDGF, and possibly other profibrotic cytokines and growth factors into the stroma to maintain corneal transparency and normal function. The EBM and the corneal EBM (Descemet's BM) are the critical regulatory structures limiting the access of activated TGF-β and PDGF into the stroma via the anterior and posterior corneal surfaces, respectively.14 Thus, normal EBM effectively limits the passage of profibrotic heparin-binding TGF-β1, TGF-β2, and all isoforms of PDGF, but importantly, not antifibrotic, non-heparin-binding TGF-β3.30 The mechanisms of posterior corneal fibrosis mediated by endothelial and Descemet's BM injury are likely similar to those that occur with anterior corneal fibrosis mediated by epithelial and EBM injury,7 but this review will primarily focus on anterior stromal fibrosis since more extensive research has been performed on the latter.Myofibroblast development and persistence is dependent on ongoing sufficient concentrations of critical growth factors—especially TGF-β and PDGF.31 After most superficial injuries to the cornea, where the epithelium heals in a timely manner, such as abrasions, riboflavin-UV cross-linking, and normal PRK, the EBM is regenerated within 8 to 10 days  PRK in one individual when the EBM regenerates normally with maintenance of stromal transparency in another individual with the exact same PRK procedure? Thus, even in the absence of mitomycin C treatment, the incidence of stromal fibrosis after PRK corrections greater than 5 to 6 D of myopia in humans is only approximately 2% to 6% of eyes.13 Similarly, why does the EBM nearly always regenerate normally and the stroma remain transparent after PRK for low myopia when abnormal EBM regeneration and stromal fibrosis occur commonly after the same exact PRK procedure performed for higher myopia?13 Finally, why was corneal fibrosis (late haze) found to be much more common when PRK was performed with some excimer lasers  than with other brands of excimer lasers  prior to the routine use of mitomycin C in PRK?14Thus, defective EBM regeneration is a critical factor leading to myofibroblast generation and persistence and resulting anterior corneal stromal fibrosis.20 demonstrated that there was a direct correlation between the level of surface irregularity after PRK and (1) the myofibroblast density in the anterior stroma and (2) the severity of stromal opacity. Thus, lasers such as the Summit excimer laser or Autonomous excimer laser, which have been shown to produce greater surface irregularity during stromal ablation,39 also trigger greater anterior stromal fibrosis than lasers such as the VISX STAR S4/IR  or the WaveLight Allegretto Wave Eye-Q  excimer lasers that generate smoother stromal surfaces.40The first factor to be appreciated was the mechanical effect of stromal surface irregularity retarding EBM regeneration  made the clinical observation that including a corneal surface-smoothing PRK treatment after PRK for high myopia decreased the incidence of late haze  in human corneas. Subsequently, using a rabbit PRK model, Netto and colleagues4143 and other organs4451 have demonstrated that fibroblastic or mesenchymal cells produce BM components such as laminins, nidogens, and perlecan that contribute to the development or regeneration of BMs needed to maintain normal tissue morphology and function. In a recent study, Santhanam and coworkers32 used laser capture microdissection-quantitative reverse transcriptase polymerase chain reaction to study EBM component mRNA production in the anterior stroma of rabbit corneas after −4.5 D PRK or −9 D PRK. The authors found that the messenger RNAs for EBM components nidogen-2 and laminin alpha-3 (LAMA3), but not nidogen-1 or perlecan, were significantly reduced in the subepithelial anterior stroma of the −9 D PRK corneas at 4 and 7 days after surgery—the time interval just prior to ultrastructural appearance of normal lamina lucida and lamina densa in −4.5 D PRK corneas (8 to 10 days after surgery) that did not develop fibrosis.32 Torricelli and coworkers33 showed that rabbit corneas that develop anterior fibrosis, or late haze, after PRK had no detectible EBM lamina lucida and lamina densa when the ultrastructure was examined with transmission electron microscopy (TEM).There are, however, other factors besides stromal surface irregularity that can lead to defective EBM regeneration since corneas that have PTK smoothing after PRK may still develop anterior stromal fibrosis without postoperative mitomycin C treatment. Another factor involves important cellular interactions involved in EBM regeneration after injury. Many studies in the cornea45 Thus, nidogens have a strong affinity for perlecan, laminins, and collagen type IV,45 and deficiencies in nidogens in skin-organotypic coculture models lead to defective EBM assembly in vitro.52Nidogens and perlecan have critical roles in linking other BM components, acting as bridging molecules between components.53 Dimeric TGF-β family members are excreted from the cell of origin and interact with extracellular matrix proteins, including perlecan, to regulate their bioavailability and function.53 Thus, perlecan in EBM and Descemet's BM likely has a critical role in modulating the function of TGF-β1 and TGF-β2 excreted by corneal epithelial cells or endothelial cells, or is present in the aqueous humor. Similarly, TGF-β1 and TGF-β2 bind to collagen type IV, a major component of EBM and Descemet's BM.54 Finally, both PDGF-AA and PDGF-BB bind to perlecan, collagen type IV, and nidogen present in EBM and Descemet's BM.55 Therefore, at least three components of EBM and Descemet's BM likely bind TGF-β1, TGF-β2, and PDGF and thereby control their bioavailability to stimulate the development of myofibroblasts from precursor cells. We hypothesize that when no EBM or Descemet's BM is present, or when these BMs are defective, TGF-β1, TGF-β2, and PDGF from corneal epithelial cells, endothelial cells, or aqueous humor penetrate the stroma at sufficiently high levels to drive development of myofibroblasts and maintain their viability. BM components have similarly been shown to regulate the bioavailability of other growth factors and cytokines.5658 It is important to point out that this regulatory role of the EBM is likely bidirectional since keratinocyte growth factor (KGF or FGF-7) produced by keratocytes and corneal fibroblasts to regulate the proliferation, motility, and differentiation of corneal epithelial cells60 also specifically bind to the perlecan protein core.61What components in the EBM and Descemet's BM are likely involved in binding TGF-β1, TGF-β2, PDGF, and other cytokines and growth factors to modulate their bioavailability in the stroma and therefore corneal fibrosis? All TGF-β family members, including TGF-β1 and TGF-β2, are synthesized as precursor molecules with a N-terminal prodomain, C-terminal mature domain and a signal peptide for extracellular transport.62 followed by addition of laminin-332, this layer creates a barrier to penetration of more posterior EBM components from the epithelial cells, and at least some of these EBM components  must be provided by keratocytes in the cornea. Thus, if there are insufficient keratocytes in the anterior stroma in proximity to the nascent EBM, then full regeneration of the mature EBM is delayed and myofibroblast precursors are driven by TGF-β and PDGF  to develop into mature myofibroblasts.The working hypothesis is that once the self-assembling nascent laminin-511 and/or -521 layer is laid down beneath basal epithelial cells,32 the authors hypothesized that increased anterior stromal keratocyte apoptosis that occurs immediately after high correction −9 D PRK compared to low correction −4.5 D PRK8 contributes to decreased anterior stromal keratocytes and the EBM components they produce in the early postoperative period when the EBM normally regenerates. Differences were also noted in the intracellular localization of EBM component proteins, perhaps related to secretion, in keratocytes and myofibroblasts cultured in vitro.43 Thus, it was hypothesized that keratocytes must be in proximity to the nascent EBM during regeneration for a fully functional, mature EBM to be regenerated by active, coordinated function of the epithelium and the keratocytes.32 It is likely that studies that found correlations between the stromal volume ablated by PRK and the incidence and severity of stromal haze63 were also detecting a correlation between the availability of keratocytes in the anterior stroma to facilitate regeneration of the EBM.In the corneal laser microdissection study of EBM component expression,64 Thus, surgeons who unnecessarily use ethanol in these procedures may unwittingly increase the risk of anterior stromal fibrosis (late haze) after PRK, even during PRK surgery for lower corrections of myopia or when mitomycin C is used to reduce fibrosis  after PRK.9 Also, rare patients who have PRK for corrections as low as 1 D of myopia develop bilateral anterior stromal fibrosis (late haze).14 Specific genetic factors involved in the development of anterior stromal fibrosis have not been reported. However, subtle genetic anomalies in EBM components or other genes involved in the mechanics of BM regeneration or repair are likely candidates for study.There are likely genetic factors that influence the regeneration of the EBM and the development of anterior stromal fibrosis after PRK and other injuries, infections, and treatments. Thus, patients who develop fibrosis (late haze) in the cornea of one eye after PRK typically develop similar fibrosis in the opposite eye, although unilateral cases have been described.32 Prior LASIK surgery provides a potential space beneath the original LASIK flap that facilitates penetration of bone marrow–derived myofibroblast precursor cells from limbal blood vessels into the central subepithelial stroma that promotes the development of corneal fibrosis analogous to the increased risk of diffuse lamellar keratitis following corneal abrasion months to years after LASIK.66Finally, corneas that have had prior PRK or other corneal surgeries (such as LASIK) are at increased risk of developing anterior stromal fibrosis after retreatment with PRK, even years after the original surgery—especially if mitomycin C treatment is not included at the time of the retreatment. Repeat PRK or PRK enhancement following LASIK could result in a greater decrease in anterior stromal keratocytes available to participate in the EBM regeneration process.Pseudomonas aeruginosa keratitis in rabbits8 demonstrated that defective regeneration of the EBM and Descemet's BM is associated with anterior and posterior corneal fibrosis, respectively , and Descemet's membrane endothelial keratoplasty (DMEK).74 One study of DSAEK in cats did not find myofibroblasts associated with the graft-host interface,75 but this study did not study sufficiently late time points because αSMA+ myofibroblasts would not be expected to develop by 9 days after the surgical injury—the latest time point examined by the investigators. Recent experiments on fibrosis after microbial keratitis demonstrated that the important role of the EBM in modulating fibrosis in the anterior stroma is paralleled by the role of Descemet's BM in the modulation of fibrosis in the posterior stroma.7 Thus, when Descemet's BM and the endothelium was damaged in the setting of severe Pseudomonas aeruginosa keratitis, posterior corneal myofibroblasts and fibrosis persisted in the stroma, even after the EBM regenerated and myofibroblasts disappeared in the anterior to midstroma , it was found that when an 8-mm diameter circle of Descemet's BM and endothelium was excised without replacement, nearly 100% of the corneas developed myofibroblast-mediated posterior fibrosis (77 This experiment confirms that Descemet's BM is the critical modulator of posterior corneal fibrosis, as the EBM is for anterior corneal fibrosis. Further study is needed to fully understand the role Descemet's BM plays in the modulation of posterior corneal fibrosis that occurs after corneal infections, diseases, trauma, and surgeries.Fibrosis of posterior cornea can occur after infections such as severe microbial infections,idstroma C. The sofibrosis , althoug68The corneal BMs are likely critical modulators of the corneal fibrosis response. Following injury, if the EBM is not fully regenerated and its function restored, then epithelium-generated TGF-β and PDGF persistently penetrate into the stroma at sufficient levels to drive the development of myofibroblasts from precursor cells. If TGF-β and PDGF continue to penetrate into the stroma, these profibrotic cells persist indefinitely. Once established, the opaque myofibroblasts produce disordered extracellular matrix that generates fibrosis and reduced stromal transparency. Myofibroblasts also block keratocytes from repopulating the fibrotic area of the stroma. Fibrosis persists until normal EBM is regenerated, likely by the coordinated response of keratocytes that finally penetrate through the fibrotic band to cooperate with epithelial cells in restoring the EBM structure and function in localized areas within the fibrosis. Deprived of adequate levels of epithelial TGF-β and PDGF, underlying myofibroblasts undergo apoptosis and repopulating keratocytes produce lacunae of more transparent anterior stroma. These zones progressively enlarge and coalesce as more keratocytes invade, contribute to regeneration of normal EBM, and reabsorb disordered extracellular matrix produced by the myofibroblasts. Descemet's BM serves a parallel function in the posterior cornea, and damage to Descemet's BM can lead to the development and/or persistence of posterior myofibroblasts and fibrosis. The association of BM injury and defective regeneration with myofibroblast-mediated fibrosis may be relevant to fibrosis in other organs such as lung, liver, kidney, heart, and skin.Supplement 1Click here for additional data file.


Transcranial Magnetic Stimulation (TMS) is a non-invasive brain stimulation method used worldwide to make causality-based inferences about brain-behavior interactions, assess cortical reactivity, and map functionally relevant brain regions inducing a controlled current pulse in a specific cortical area. Clinical applications of TMS have shown promising results in the treatment of a vast number of psychiatric and neurological conditions such as headache disorders - migraine being one of the most encountered. In patients with migraine, the pharmacologic therapy is divided in urgent/ abortive treatment of the attack and prophylactic one. As first-line drugs simple analgesics and non-steroidal inflammatory are preferred. Nevertheless, many individuals continue to have attacks refractory to various prophylactic and/or abortive therapies, while others are at high risk of developing medication overuse headache. Among non-pharmacologic therapies TMS has been broadly studied as a preventive migraine treatment with good outcome results.Abbreviations: DLPFC - Dorsolateral prefrontal cortex, FDA – United States Food and Drug Administration, HF-TMS – High frequency transcranial magnetic stimulation, TMS – Transcranial magnetic stimulation, rTMS – Repetitive transcranial magnetic stimulation This technique opened up new ways of studying the functionality, morphology, and connectivity of various cortical regions, especially the motor cortex [3]. In 1990, technological advances introduced generators capable of producing rapid, repetitive pulses of magnetic stimulation at frequencies of up to 30 Hz known as repetitive TMS (rTMS) or high-frequency TMS (HF-TMS) [4]. Currently, rTMS is considered a useful tool in the treatment of several conditions originating in the cerebral cortex including pain, dystonia, epilepsy, headaches, Parkinson’s disease, stroke, and tinnitus. Although the exact mechanisms through which rTMS works are not completely known at the moment, it is considered to be multifactorial. The applied electric charges induced by the magnetic field cause various neurochemical changes such as increased dopamine levels in the hippocampus, reduction in Raclopride C11 binding in the caudate nucleus, fluctuations in glutamate/glutamine levels at the site of rTMS stimulation and increased plasma β-endorphin levels [5][6].Single-pulse transcranial magnetic stimulation (TMS) was introduced for the first time in 1985, as a method of noninvasive and painless stimulation of the human cortex [7], very often being associated with affective disorders such as anxiety and depression, there has been an increased interest in using rTMS in the management of these patients applying different TMS paradigms and protocols [8][9]. There are several studies that confirmed the fact that people with migraine (“migraineurs”) had 2.2 to 4.0 times increased fold of developing depression than non-migraineurs [10]. Regarding the effects of TMS in migraine, previous studies have also suggested that blood flow [11] and metabolic changes [12] at the stimulation site, brain-derived neurotrophic factor upregulation, improvements in synaptic plasticity [13] and changes in the activity of the neural circuitry of the dorsolateral prefrontal (DLPFC) - cingulate cortex, including both the DLPFC and the anterior cingulate cortex [14], are involved. There is also research data that confirms a positive effect of HF-TMS on cognitive tasks in healthy adults [15] suggesting that TMS can not only recruit more neural resources by inducing an electrophysiological excitatory effect but can also enhance the efficiency of resource deployment during multiple stages of cognitive control processing. In a position statement for the neuromodulation of chronic headaches, the European Headache Federation states that the application of noninvasive rTMS in chronic headaches is not yet evidence-based, given the limited amount of randomized controlled data and can’t be used on a regular basis for all migraine patients [16]. Although there are multiple positive and promising conducted studies regarding the use of TMS in migraine prophylaxis and FDA approved devices indicated for the acute treatment of pain associated with migraine headache with aura [17], evidence on the efficacy of TMS for the treatment of migraine is still limited in quantity, demanding randomized controlled trials to analyze safety of long-term or frequent use of TMS. Therefore, TMS should only be used with special arrangements for clinical governance, consent and audit or research until proven otherwise [18].Considering the pathophysiology of migraine and the fact that the incidence of migraine is gradually rising and becoming one of the most common nervous system diseases in the world [The authors declare that there is no conflict of interest.


Six families of terminal oxidases are currently recognized, all of which occur in alphaproteobacterial genomes, two of which are also present in mitochondria. Many alphaproteobacteria encode several different terminal oxidases, likely reflecting ecological versatility with respect to oxygen levels. Terminal oxidase evolution likely started with the advent of O2 roughly 2.4 billion years ago and terminal oxidases diversified in the Proterozoic, during which oxygen levels remained low, around the Pasteur point (ca. 2 μM O2). Among the alphaproteobacterial genomes surveyed, those from members of the Rhodospirillaceae reveal the greatest diversity in oxygen reductases. Some harbor all six terminal oxidase types, in addition to many soluble enzymes typical of anaerobic fermentations in mitochondria and hydrogenosomes of eukaryotes. Recent data have it that O2 levels increased to current values (21% v/v or ca. 250 μM) only about 430 million years ago. Ecological adaptation brought forth different lineages of alphaproteobacteria and different lineages of eukaryotes that have undergone evolutionary specialization to high oxygen, low oxygen, and anaerobic habitats. Some have remained facultative anaerobes that are able to generate ATP with or without the help of oxygen and represent physiological links to the ancient proteobacterial lineage at the origin of mitochondria and eukaryotes. Our analysis reveals that the genomes of alphaproteobacteria appear to retain signatures of ancient transitions in aerobic metabolism, findings that are relevant to mitochondrial evolution in eukaryotes as well.Oxygen reducing terminal oxidases differ with respect to their subunit composition, heme groups, operon structure, and affinity for O The alphaproteobacteria are a large and diverse group of prokaryotes, members of which can grow under chemotrophic, phototrophic, lithotrophic, organotrophic, autotrophic, heterotrophic, parasitic, aerobic, anaerobic, or diazotrophic conditions . Some alAlthough the fossil record of prokaryotes is scant, alphaproteobacteria can be estimated to have a minimum age of roughly 2 billion years (Ga) from molecular dating of eukaryotes, since mitochondria originated from this class of proteobacteria. Fossil data place a minimum age on eukaryotes of 1.45 Ga . CurrentAlphaproteobacteria not only participated in the origin of eukaryotic organelles , they ha2 appeared in the geochemical record approximately 2.4 billion years ago, it took almost 2 billion years to accumulate to current levels in either the ocean or the atmosphere H oxidases (diaphorases)   or ferreproteins , rather y chains   anaerobes invented enzymes for protective O2 detoxification, (iv) anaerobes invented enzymes for bioenergetic O2 utilization, and (v) anaerobes integrated O2 reduction into the broader scheme of bioenergetic evolution. During that process, anaerobes became facultative anaerobes  life started out anaerobically, (ii) anaerobes invented Onaerobes , obligat2 to water, can use quinols, reduced cytochrome c, copper proteins, or even high-potential iron-sulfur proteins  typical of Pseudomonas  containing a mixture of high, intermediate and low affinity oxidases, and; (IV) containing only low affinity oxidases of the A1 family (with/without AOX)  without oxidases; (II) containing only high affinity oxidases, out AOX) . An exceAzospirillum contains organisms whose metagenomic records indicate absence of all terminal oxidases and therefore can be categorized as strictly anaerobes, as shown at the top of Azospirillum sp. CAG:239; see also Azospirillum brasilense, broadly categorized as facultatively anaerobes as shown in the middle of Azospirillum is thought to reflect pervasive contributions of LGT to their genomes  anaerobic capacities within alphaproteobacteria . Additioquinones . The prequinones . These equinones  or in plquinones . The solquinones . Among tquinones . As expeper se does not imply lack of functional association nor different evolutionary history along the broad time scales discussed in here. In the course of this work we encountered several operon subtypes of both A and B families of HCO that can be distinguished by the different sequence of the catalytic and accessory subunits. Some of these subtypes corresponded to previously introduced COX operon types  oxidase present in Magnetospirilli  regarding their nitric oxide activity exist, such as the increase in transcription levels under conditions that favor nitric oxide production  in the presence of sulfide (2. Although Proterozoic deep ocean chemistry following the GOE is thought to have been dominated by anoxic conditions (2-dependent enzyme) and in enzymes of the denitrification pathway  first characterized in Bacillus (CtaG_Cox11 family of periplasmic Cu assembly proteins (PFAM family PF04442), which are close homologs of eukaryotic Cox11 (Deinococcus proteins involved in the CopCD system used for eliminating Cu toxicity (The insertion of the Cu atom in the oxygen-reducing binuclear center requires the action of Cu assembly proteins (chaperones) that belong to two different families: the Bacillus  and the ic Cox11 . Differeic Cox11 . CtaG prtoxicity  and are toxicity . However3bo ubiquinol A1 oxidases, present in many alphaproteobacteria other than Magnetospirilli  and have been inherited vertically along microbial lineages during evolution, such that their distribution is the result of lineage divergence . The othKm values ranging from nanomolar to micromolar  or less (2 concentration at which facultative anaerobes like E. coli start to respire O2 (The terminal oxidases of alphaproteobacteria differ in terms of oxygen affinity along a scale of cromolar . For comf 250 μM . Modern  or less . Oxygen spire O2 .2 affinity might have arisen in accordance with oxygen availability, with high affinity enzymes having emerged first and low affinity enzymes coming later in evolution. Because lateral gene transfer has substantial impact on terminal oxidase evolution (2 to low O2 environments could have been significantly impacted by bioenergetic factors. They pointed out that the four electron O2 reduction reaction is sufficiently exergonic at 1 nM O2 such that the low proton pumping stoichiometries typical of B- and C-type families is not a result of thermodynamic constraint, and furthermore that improved O2 diffusion to the active site of the B- and C-family enzymes might maintain physiologically relevant reaction rates at low O2 concentrations. 2 adaptation that we propose — from low to high O2 environments during geological time (Here we have suggested that oxygen reductases of differing Ovolution , straighcal time  — differ2 affinity arose in a temporal sequence, from high affinity to low affinity following the gradual emergence of O2. LGT has distributed terminal oxidases from bacteria to archaea, yet the different Cu assembly proteins figure into evolutionary transitions in oxygen utilization within alphaproteobacteria. The distribution of terminal oxidases of differing oxygen affinity, often co-occurring with enzymes of anaerobic fermentations (Both alphaproteobacteria and eukaryotes arose and diversified during a phase of Earth history in which oxygen levels were lower than today . Both lintations , reveals-10 (Sequence searches were undertaken with two independent complementary approaches that were then merged in a consensus picture for the presence or absence of aerobic and anaerobic traits in bacterial and eukaryotic genomes. The first approach consisted in genome-wide, computer-assisted analyses using reference proteins that are representative of metabolic traits, or each subunit of the various terminal oxidases as queries in Blast searches . As a ge-10 . HCOs we-10 .1 (bd oxidase in genomes more than 90% complete, were rendered in light gray in display material such as The second approach was based upon searches in the NCBI protein and gene webpages using multiple keywords that had empirically shown to retrieve most of the homologs of a given protein in previous works . Protein1 . In seve1 . SearcheA initial literature search identified around 200 genomic records to be analyzed. The initial set of alphaproteobacterial genomes was compiled from taxa that are predominantly uncultured, unclassified or derived from metagenomic assemblies, have around 1000 or more coded proteins, and could represent all known families and genera of the alpha class . This seThe presence of terminal oxidases was color coded with increased intensity of blue following their decreased affinity for oxygen , 2, 4. OMultiple sequence alignments were calculated using Clustal  and maxiMDE and WM contributed conception of the study. MDE, WM, MM, and FS designed the study. MDE and FS performed bioinformatics analysis. All the authors contributed to the writing and revision of the manuscript, and read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


PRS mice) showed biochemical and behavioral abnormalities consistent with a schizophrenia-like phenotype  protein, a molecular player of DNA demethylation, induced by the activation of mGlu2/3 receptors. This effect was mimicked by clozapine and valproate but not by haloperidol. Treatment with the selective mGlu2/3 receptors agonist LY379268 also increased the amount of Gadd45-β bound to specific promoter regions of reelin, BDNF, and GAD67. A meta-analysis of several clinical trials showed that treatment with an orthosteric mGlu2/3 receptor agonist improved both positive and negative symptoms of schizophrenia, but only in patients who were early-in-disease and had not been treated with atypical antipsychotic drugs (Kinon et al., Mice subjected to prenatal restraint stress ( PRS) in mice showed epigenetic changes and behavioral abnormalities consistent with a schizophrenia-like phenotype. Prenatal restraint stress  represents a suitable non-pharmacological model to study schizophrenia and to develop novel antipsychotics. Activation of mGlu2/3 receptors corrects the altered epigenetic and behavioral changes induced by prenatal stress in mice. Both clozapine and the mGlu2/3 receptor agonist LY379268 acted as epigenetic agents targeting specifically DNA methylation reversing the molecular and behavioral alterations in PRS mice.2A serotonin receptors, showing good clinical efficacy in improving positive symptoms, and moderate activity in improving negative symptoms of schizophrenia. None of these drugs has significant activity on cognitive symptoms associated with schizophrenia, with the possible exception of clozapine  of the role of metabotropic glutamate 2/3 receptors in prenatally stressed mice (PRS mice) as potential targets for novel antipsychotics; and (iii) to show our more recent observations on the epigenetic effects induced by the mGlu2/3 receptors agonist, LY379268, and by clozapine.Schizophrenia is a major psychotic disorder which affects one percent of the world's population and usually leads to a severe mental disability  alterations of glutamic acid decarboxylase 67 (gene symbol = GAD1), DNMT and reelin in GABAergic neurons, and (ii) of brain derived nerve growth factor (BDNF), vesicular glutamate transporter (VGLUT1) and (iii) alterations in mGlu2/3 receptors in glutamatergic neurons , altered fear conditioning, object recognition, and hypersensitivity to N-methyl D-aspartate (NMDA) receptor blockers , associated with an alteration in these gene expression.Urged by the need to use a neurodevelopmental animal model to study the epigenetic status at each neurodevelopmental stage of schizophrenia, we investigated the molecular and behavioral abnormalities found in the brain of the offspring of dams stressed during pregnancy (PRS mice). PRS mice showed a marked and long-lasting increase in DNMT enzymes (both 1 and 3a), and TET enzymes and a significant increase in 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC) in the promoters of putative schizophrenia-related genes, such as natural course of major psychosis including schizophrenia compared, for example, to the phencyclidine (PCP) model which is a well-established pharmacological-induced model for schizophrenia reflecting the positive symptoms through the blockade of the NMDA receptors. Schizophrenia and autism spectrum disorders are considered diseases of neurodevelopment, characterized by a natural course, starting with a prodromal phase, first episode during adolescence or early adulthood, followed by relapses/remitted periods and eventually leading to brain function deterioration that ensues over subsequent adult years. Hence, the epigenetic history of such complex neurodevelopmental disorders cannot be adequately studied only in the postmortem brains of chronic SZ patients. We then have focused on studying the epigenetic signature of schizophrenia in offspring of PRS mice.To the best of our knowledge, the PRS model represents a promising model to study the PRS mice for the study of the role played by mGlu2 and mGlu3 receptors in the pathophysiology of schizophrenia based on clinical findings with pomeglumetad methionyl, an oral prodrug of the mGlu2/3 receptor agonist, LY404039. This drug showed an efficacy similar to the comparator, olanzapine, on positive and negative symptoms of schizophrenia in a phase-2 clinical trial . Interestingly, the biochemical and behavioral abnormalities of PRS mice were corrected by the treatment with LY379268 (Matrisciano et al., As summarized in Table Little is known on the action of antipsychotics on specific epigenetic mechanisms in GABAergic or glutamatergic neurons. Thus, PRS mice represent a valid and suitable model for drug testing and development.bdnf , reln, and gad1 (Dong et al., gad1, bdnf , and reln and the ensuing increase in their mRNA expression levels. In addition, LY379268 induced a decrease of MECP2 binding at the mGlu2, Gad1, and Bdnf gene promoters, whereas clozapine reversed DNMT binding at the promoters of schizophrenia-related genes. Both clozapine and LY379268 reversed the increase in locomotor activity in PRS mice and the deficits showed by these mice in social interaction tasks. These findings are consistent with the previous evidence that a combined treatment with clozapine and valproate reversed the downregulation of GAD67 expression induced by repeated methionine administration in mice (Guidotti et al., Gad1, Reln, and Bdnf-ix regulatory regions in PRS mice was considerably reduced by clozapine treatment whereas haloperidol failed to reduce the increased DNMT1 binding in PRS mice, in agreement with previous results (Matrisciano et al., Clozapine, the prototype of atypical antipsychotics, is considered the drug of choice in patients with treatment-resistant schizophrenia due, in our opinion, to its unique chromatin remodeling properties. We have shown that clozapine reversed the behavioral deficits and induced chromatin remodeling in PRS mice that are resistant to haloperidol treatment (Dong et al., PRS mouse model has construct and face validity as an experimental epigenetic model of vulnerability for neurodevelopmental disorders such as schizophrenia, schizoaffective disorders, and autism. This mouse model is highly reproducible and useful for novel anti-psychotic drug screening acting on altered epigenetic mechanisms. Early-life stressors, even during pregnancy, in mice lead to alterations of some molecular players of epigenetic mechanisms that are translated into a schizophrenia-like phenotype. A potential glutamate-based pharmacotherapy for schizophrenia remains, at least in part, a possibility that requires the identification of an appropriate subgroup of patients that satisfy specific criteria such as no previous history of atypical antipsychotic treatments and treatment onset in early phases of the disease. For preclinical studies, PRS mice represent a valid epigenetic “endophenotype” model for drug testing and development and for studying the pathogenesis of the disease. In our opinion, mGlu2/3 receptors, based on the peculiar role as modulators of glutamate transmission in the frontal cortex, can still represent a suitable target for novel antipsychotic medications targeting specific high-risk population with dysregulation of brain glutamatergic tone. Then, ligands acting on mGlu2 and 3 receptors, either orthosteric agonists or PAMs, require further experimental studies in PRS mice and other epigenetic models to identify the optimum receptors target and time window of intervention in the treatment of psychosis.This review underlies the concept that the All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Dictyostelium, through to the demonstration that medium-chain fats play a direct role in seizure control, and finally the identification of a mechanism of action of these fats and related congeners leading to reduced neural excitability and seizure control.Since the original report of seizure control through starvation in the 1920s, the ketogenic diet has been considered an energy-related therapy. The diet was assumed to be functioning through the effect of reduced carbohydrate intake regulating cellular energy state, thus giving rise to seizure control. From this assumption, the generation of ketones during starvation provided an attractive mechanism for this altered energy state; however, many years of research has sought and largely failed to correlate seizure control and ketone levels. Due to this focus on ketones, few studies have examined a role for free fatty acids, as metabolic intermediates between the triglycerides provided in the diet and ketones, in seizure control. Recent discoveries have now suggested that the medium-chain fats, delivered through the medium-chain triglyceride (MCT) ketogenic diet, may provide a key therapeutic mechanism of the diet in seizure control. Here we describe an unusual pathway leading to this discovery, beginning with the use of a tractable non-animal model— Identifying the key therapeutic target of drugs is of great importance for biomedical science, since this enables rapid screening to develop improved compounds . It has been used to demonstrate a common mechanism of action of bipolar disorder treatments through inositol depletion  bicarbonate transporter  in a picrotoxin-induced seizure model in mice, with small but significant effects of decanoic acid in delaying the onset of clonic convulsions without an effect on survival time. The opposite effect was found for decanoic acid with subcutaneous PTZ induced seizures, where survival time was increased but clonic convulsions were not delayed  and GluA1/2 (IC50 = 1.16 mM). This inhibitory effect was voltage-dependent, where potency against GluA2/3 receptors at −80 mV (IC50 of 1.11 mM) was elevated following depolarization to −40 mV (to IC50 of 0.43 mM), suggesting stronger inhibitory activity during prolonged seizure activity.Two molecular mechanisms for decanoic acid have recently been proposed. As an acute mechanism for seizure control, decanoic acid has been shown to reduce excitatory postsynaptic currents (EPSCs) using whole cell patch clamp recordings from CA1 pyramidal neurons, likely through inhibition of excitatory AMPA receptors , MW (UCL) and RW (RHUL) contributed equally to the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Haematological tests are an important diagnostic tool for animal diseases. However, little is known beyond the standardly used haematological methods in sheep, especially those in which the detection of sheep red blood cells (RBCs) shape changes is crucial. Our goal is to obtain sheep RBC morphometric parameters as well as RBC subpopulations based on their morphometric parameters. Morphometric parameters of RBC size and shape were determined from stained blood smears using SFORM, a computer-assisted program for automated cell morphometric measurements. Based on their morphometric parameters, three RBC subpopulations were obtained using principal component and cluster analysis: the smallest and most elongated RBCs, the biggest and most rounded ones, and RBCs of average size and shape. When the values of RBC haematological parameters were higher or above the physiological range, a significantly higher proportion of both average size and shape RBCs, as well as the biggest and most rounded RBCs, was obtained. Since they were obtained from healthy animals, these results indicated the importance of determining morphometric parameters of RBCs and the proportion of each RBC subpopulation, which could serve as a basis for future possibilities in the diagnostic interpretation of haematological disorders in sheep, especially those for which the detection of shape changes in ovine RBCs is crucial.Data concerning the morphometric parameters of sheep red blood cells (RBCs) obtained using computer-assisted image analysis have not yet been investigated, and there are no data on any analyses of ovine RBC subpopulations based on their morphometric parameters. The aims of this study are to determine the values of RBC haematological and morphometric size and shape parameters, to form groups according to the obtained values of haematological parameters; to determine the differences in RBC morphometric parameters between the formed groups, and to determine RBC subpopulations and their respective proportions in the formed groups. Thirty-six blood samples were collected from the jugular vein of clinically healthy Lika pramenka sheep, aged between 2 and 5 years. Haematological parameters including haemoglobin (HGB), haematocrit (HTC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), and RBC distribution width were analysed using a haematology analyser. Haematological parameters were categorized into two groups: those with lower values or values below the physiological range (Groups 1) and groups with higher values or values above the physiological range (Groups 2). Morphometric parameters of RBCs were determined from stained blood smears using SFORM, a computer-assisted program. Significantly higher values of RBC area, outline, convex, minimal and maximal radius, as well as length and breadth were established in Groups 2 compared to Groups 1 of HGB, HCT, MCV, MCH, and MCHC, respectively. Based on the morphometric parameters of RBCs, three RBC subpopulations were obtained using principal component and cluster analysis: ES 1—the smallest and most elongated RBCs, ES 2—the biggest and most rounded RBCs, and ES 3—average size and shape RBCs. Significantly higher proportions of ES 2 and ES 3 subpopulations, as well as a significantly lower proportion of ES 1 subpopulation, were established in Groups 2 compared to Groups 1 of HGB, HTC, MCV, and MCH, respectively. It can be concluded that ovine RBC subpopulations, based on their morphometric parameters, can be obtained by using computer-assisted image analysis of RBC morphometry and multivariate statistical methods, including principal component and cluster analysis. RBC morphometry, including classification into subpopulations, could serve as a basis for future possibilities in the diagnostic interpretation of anaemic syndromes in veterinary medicine, especially in normocytic, macrocytic, and microcytic anaemias in sheep. The Lika pramenka breed is the most numerous autochthonous population of sheep in Croatia . In receUntil recently, morphometric studies of RBCs were essentially based on linear measures of RBC size. Use of an ocular micrometer and a lens micrometer (micrometric slide) is the only valid and recognised method of measuring the size of RBCs . MorphomRBC senescence has so far been investigated through the isolation of RBC populations of different mean cell ages. Most of the research has been conducted on RBCs separated on the basis of differences in cell density or volume/size ,14. Of tData concerning the morphometric size and shape parameters of sheep RBCs obtained using computer-assisted image analysis have not been investigated. In addition, to the best of our knowledge, there are no data in recent literature on any analyses of ovine RBC subpopulations with respect to morphometric size and shape parameters, both approaches using computer-assisted image analysis of RBC morphometry and multivariate statistical methods, including discriminant and cluster analyses. The hypothesis of this research is based on the assumption that there might be a connection between RBC morphometric and haematological parameters, as well as differences in the distribution of RBC subpopulations in categorised groups according to the obtained haematological parameters’ values in Lika pramenka sheep. Therefore, the aims of this study are to determine the values of RBC haematological and morphometric size and shape parameters, to form RBC groups according to the obtained values of haematological parameters, to determine the differences in morphometric parameters between the formed RBC groups and to determine RBC subpopulations and their respective proportions in the formed groups.All procedures used in this research were in compliance with the European guidelines for the care and use of animals in research (Directive 2010/63/EC) and approved by the Committee for Ethics in Veterinary Science, Faculty of Veterinary Medicine, University of Zagreb, Croatia  and Veterinary and Food Safety Directorate, Ministry of Agriculture, Republic of Croatia .“Innovative functional lamb meat products” financed by the Croatian Science Foundation (IP-2016-06-3685). It was conducted on a sheep farm of Lika pramenka breed, in the ownership of GEA-COM Ltd., situated in Crkvina, Krnjak area, Croatia. The farm is situated at an altitude of 215 m above sea level and is composed of 200 sheep and 20 rams of varying age.The animals are kept extensively, grazing in the field in the warmer periods of the year, and in a barn during winter, when they are fed with hay from the surrounding pastures. Independently of the season, the sheep are also fed with pelleted complete feed .® Tube, K2EDTA). The samples were then stored at +4 °C and processed in the laboratory on the same day, approximately 5 h after the collection.The research comprised 36 randomly chosen sheep, 2 to 5 years old, with an average body weight of 37.58 ± 2.66 kg. The sheep were clinically examined during May, outside of the reproductive season, and their blood was drawn for further haematological analyses. Blood was collected by venepuncture of the jugular vein, using vacuum tubes with EDTA. Before the venepuncture, hair surrounding the puncture site was clipped off and skin was disinfected according to the usual veterinary aseptic procedure. After the venepuncture and collection of 5 mL of blood in vacuum tubes, 500 µL of blood was separated in micro test tubes . Haematological analyses were performed on a fully automated counter made for in vitro use, the Abacus Junior Vet haematology analyser . All analyses were repeated two times, with a maximal error of <4%. Automated sampling was performed with authorised reagents  in four phases. Among standard haematological parameters, those regarding RBCs were chosen for further analyses: haemoglobin (HGB), haematocrit (HTC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), and red blood cell distribution width (RDW). Furthermore, a smear was prepared from each sample and stained using Pappenheim’s panoptic staining method with the May–Grünwald and Giemsa stains ,18.2), outline (μm), convex, minimal and maximal radius (µm), length and breadth (μm). Some of the RBC morphometric size measures were used to calculate RBC shape parameters according to the following formulas: ellipticity = length/breadth; elongation = [(length−breadth)/(length + breadth)]; solidity = area/convex; roundness = (4 × area)/[π × (major axis)2]; form factor = [4π × area/outline2]; contour index = outline/√area.RBC morphometry was conducted using a computer-assisted program package for image analysis, SFORM . The system consists of a high-resolution colour camera  that digitalises and transfers images from an Olympus BX 41  light microscope, following observation under the magnification of 1000×, onto a personal computer. Morphometric analysis was conducted on the blood smears stained according to Pappenheim’s method. Staining makes RBCs more visible and gives them contrast, which enables automated measurements with the SFORM software. A total of 36 stained blood smears were analysed, with approximately 110 RBCs per smear. Only RBCs that did not overlap with other cells or debris were measured and analysed (n = 4017). The borders of the RBCs were marked automatically using the marking option in the SFORM program, with manual corrections using a computer mouse . The folThe results were statistically processed using the SAS 9.4 software package . Descriptive statistics was made using PROC MEANS and PROC FREQ modules. The normal distribution of data was assessed with the PROC TRANSREG module. In the case of non-normality and heteroscedasticity of variances, the transformation of variables was performed by log or exponential transformation prior to analysis using a Box–Cox transformation.Dependent variables were analysed by multivariate variance analysis (MANOVA) based on the Wilks’ lambda criterion using the GLM procedure. A multiple comparison test of the least-square means with Tukey’s correction was performed to compare each group. Groups were formed by categorizing continuous variables of the analysed values of haematological parameters . Those parameters were categorized into two groups: those with lower values or values below the physiological range (Groups 1) and those with higher or values above the physiological range (Groups 2), bearing in mind that the groups have approximately the same number of samples each. Age as a variable was removed since it had no effect on the tested haematological or morphometric RBC parameters.p < 0.05.To analyse the RBC subpopulations, principal component and cluster analyses were carried out in several steps. The principal component analysis was performed using the PROC FACTOR module in order to obtain eigenvalues of morphometric variables using Kaiser’s criterion (λ ≥ 1) and to establish the number of the main components for the next analysis. The number of clusters in K-means cluster analysis was determined using the HPCLUS procedure, which selects the best k value (number of clusters or subpopulations) using the aligned box criterion value. To obtain well-separated clusters with non-random initialisation, the FASTCLUS procedure was used. To specify the mean absolute deviation criterion and to prevent outliers from distorting the results, the FASTCLUS procedure was conducted using the LEAST and STRICT options. The last cluster analysis was done with the same procedure (PROC FASTCLUS) to assign outliers and tails to clusters using zero iterations. Although cluster analysis had determined that the best number of clusters was four, one cluster was incorporated into the nearest cluster due to the low number of RBCs that formed it. To assess the differences in the distribution of RBC subpopulations between the groups, the FREQ procedure was carried out using the Hi-squared test. The level of statistical significance was set at Descriptive statistical analyses of the data on the morphometric size and shape parameters of RBCs in Lika pramenka sheep are shown in Morphometric parameters of ovine RBCs in the categorized groups according to the values of the haematological parameters.The mean and standard error values of the RBC morphometric size and shape parameters of groups formed by the categorisation of the haematological parameter values in Lika pramenka sheep are presented in Using principal component analysis prior to the clustering of RBC morphometric parameters, three characteristic components (factors) were obtained with eigenvalues greater than 1 (λ ≥ 1), which account for the 93.8% cumulative variance of the morphometric variables . The firBy using cluster analysis, three precisely defined RBC clusters or subpopulations were obtained based on RBC morphometric size and shape parameters . The firBy testing the distribution of RBC subpopulations according to the morphometric variables of RBC size and shape, a significant difference was observed between the groups formed by categorization of haematological parameters values . Significantly higher proportions of ES 2 and ES 3 subpopulations, as well as a significantly lover proportion of ES 1 subpopulation, were established in HGB 2, HCT 2, MCV 2 and MCH 2 groups compared to HGB 1, HTC 1, MCV 1 and MCH 1 groups, respectively .It was found that the proportions of ES 1 and ES 3 subpopulations were significantly lower while ES 2 proportion was significantly higher in the MCHC 2 group than in the MCHC 1 group. In addition, the RDW 2 group had a significantly lower proportion of ES 2 and ES 3 subpopulations and a significantly higher proportion of ES 1 subpopulation in comparison to the RDW 1 group.Detection of changes in RBC morphology is clinically important in haematology and medicine , especiaDetection of RBC morphology is routinely performed by subjective microscopic evaluation, which is difficult and highly dependent on the operator’s expertise. Therefore, it is necessary to implement an automated methodology to analyse red blood cell shape changes to support and improve the operator’s capability and to expedite measurements ,22. Inte2) does not match the values reported by Adili et al. [2), obtained from both ewes and rams, were almost equal, which is surprising.Data concerning RBC morphometric size and shape parameters in the Lika pramenka sheep breed, in general, have not been investigated, and only a few RBC morphometric size parameters  in sheep have been determined using an ocular micrometer and a lens micrometer. Therefore, according to the above-mentioned results of this study, most of the investigated morphometric parameters values cannot be compared. The morphometric study of RBCs seems to show that the diameter, circumference and area of RBCs have a significant influence on the determination of the species of domestic animals . So, oviIn this research, the differences in the mean values of RBC morphometric size and shape parameters were determined between groups formed by categorization of haematological parameters values  in Lika pramenka sheep. In addition, significantly higher values of RBC area, outline, convex, minimal radius, maximal radius, length, and breadth, were established in the groups with higher vs. the groups with lower values of HGB, HCT, MCV, MCH, MCHC, respectively. The obtained results of RBC morphometric size parameters determined in this study are consistent with those of Ravi Sarma , who staIt has long been known that RBCs comprise various subpopulations, which can, among others, be separated through Percoll density gradients . HoweverThis is, to the best of our knowledge, the first report on the morphometric parameters of sheep RBCs that includes differentiation into subpopulations and focuses on clinically healthy sheep outside of the reproductive season. Morphometric parameters of the size and shape of RBCs in Lika pramenka sheep appear to be the most representative to mark changes in the normal morphometry of RBCs. Furthermore, it can be concluded that ovine RBC subpopulations based on morphometric size and shape parameters can be identified using computer-assisted image analysis of RBC morphometry and multivariate statistical methods, including principal component and cluster analysis. However, data on RBC subpopulations are scarce or non-existent. Therefore, it would be of great interest in the future to compare RBC subpopulations among different sheep breeds or animals with different (patho) physiological status . RBC morphometry could prevent any potential confusion in studying anaemic syndromes, i.e., it could serve as a basis for diagnostic interpretation of anaemic syndromes in veterinary medicine, especially concerning normocytic, macrocytic, and microcytic anaemias in sheep. In conclusion, RBC morphometry may have important applications and become a part of standard procedures for diagnostic interpretation and treatment of haematological disorders in which the detection of ovine RBCs shape changes is crucial.