Transgenic experiments exploit integrative recombination, where a donor plasmid carrying a loxP site and DNA of interest integrate into a recipient loxP site in a target genome. Unfortunately, integrative recombination is highly inefficient because the insert is flanked by two loxP sites, which themselves become targets for Cre and lead to subsequent excision of the insert. A small number of mutations have been discovered in parts of the loxP sequence, specifically the spacer and inverted repeat segments, that increase the efficiency of integrative recombination. In this study we introduce a high-throughput in vitro assay to rapidly detect novel loxP spacer mutants and describe the sequence characteristics of successful recombinants.Cre-loxP oligonucleotides that contained a combination of inverted repeat mutations (the lox66 and lox71 mutations) and mutant spacer sequences, degenerate at 6 of the 8 positions. After in vitro Cre recombination, 3,124 recombinant clones were identified by sequencing. Included in this set were 31 unique, novel, self-recombining sequences. Using network visualization tools, we recognized 12 spacer sets with restricted promiscuity. We observed that increased guanine content at all spacer positions save for position 8 resulted in increased recombination. Interestingly, recombination between identical spacers was not preferred over non-identical spacers. We also identified a set of 16 pairs of loxP spacers that reacted at least twice with another spacer, but not themselves. Further, neither the wild-type P1 phage loxP sequence nor any of the known loxP spacer mutants appeared to be kinetically favoured by Cre recombinase.We created synthetic loxP spacer mutant screening in an unbiased manner, assuming nothing about candidate loxP sites save for the conserved 4 and 5 spacer positions. Candidate sites were free to recombine with any other sequence in the pool of all possible sites. The subset of loxP sites identified here are candidates for in vivo serial recombination as they have already demonstrated limited promiscuity with other loxP spacer and stability in the presence of Cre.This study approached  Cre ("Causes recombination") recombinase from bacteriophage P1 recognizes a specific 34 bp target sequence, termed loxP, composed of an 8 bp spacer region flanked by two identical 13 bp inverted repeats [loxP sites mediate site specific intra- or inter-strand exchange of DNA molecules catalyzed by Cre recombinase. Depending on the location and the orientation of these sites, they can invert, insert, delete or exchange fragments of DNA in prokaryotic or eukaryotic systems [loxP sites. The difficulty in accomplishing DNA insertion is that the insertion reaction results in the presence of two loxP sites in cis post-recombination, which themselves become substrates for Cre and lead to rapid excision of the inserted segment.Cre- systems -5. OrienloxP sites are available to engineer stable Cre-loxP integrative recombination. Both exploit sequence mutations in the Cre recognition sequence, either within the 8 bp spacer region or the 13-bp inverted repeats. Spacer mutants such as lox511[lox5171 [lox2272 [m2, m3, m7, and m11 [lox71, has 5 bp on the 5' end of the left inverted repeat that are changed from the wild type sequence to TACCG [lox66, has the five 3'-most bases changed to CGGTA  and the other is wild type [Two classes of variant as lox511, lox5171 and m11 -11. Inveto TACCG ,13 . Only the m2 mutant was tested and found to be functional for RMCE in bacteria and Cre-expressing eukaryotic cells (HEK-293). Other studies have evaluated the in vivo heterospecificity of the wild-type spacer versus other published loxP sequences. Siegel et al. tested heterospecific recombination between five published loxP spacer mutants and found that wild-type/FAS, wild-type/2272 and 2272/5171 pairings were incompatible while limited compatibility was found between wild-type/511 sites [lox511 spacers [lox2272 and lox5171 spacer mutants [In past experiments, novel spacer mutants were discovered by mutating a suite of bases ,7 or genoxP site . Lee and systems . In thei11 sites . However spacers . Recentl mutants . Howeverin vitro strategy to rapidly identify novel LE/RE-spacer mutants and determine their specificity.Given that spacer and inverted repeat mutants have been used together successfully, it may be possible to introduce numerous DNA segments into a given target molecule, chromosome or genome if a sufficient number of non-promiscuous LE/RE-spacer mutants can be identified. In the present study we have developed and executed a high-throughput loxP sites with six degenerate spacer nucleotides  and two central fixed spacer nucleotides (4 and 5)  contained the lox66 right arm mutant sequence  were designed that contained loxP recombination products in pUC19 vector  were sequenced in the positive orientation but only 42/768 produced high quality spacer sequences , second 8 bp spacer (104 bp), lox 66 inverted repeat mutant (111 bp), start of the pUC19 vector (133 bp) and EcoRI site (143 bp)  . The most promiscuous self-recombining sequence was GGGTAGGC which underwent 44 insertional recombination reactions. There were also a set of 16 non self-recombining spacers . The spacer GGGTAGGG deviated from the null hypothesis because it had higher promiscuity within a number of non-identical spacers rather than with itself .Each sequence read had two, usually non-identical, s Figure . Inferres Figure . Of theseviously , the remloxP spacer sequences with the greatest potential utility for genetic engineering will self-recombine and exhibit limited promiscuity. Some promiscuity is tolerable if the sites prone to interaction are used in constructs in a mutually exclusive manner. We visualized self and non-self spacer interactions as a network using Cytoscape [Traditionally candidate ytoscape  in orderloxP spacer mutant screening in an unbiased manner, assuming nothing about candidate loxP sites save for the conserved 4 and 5 spacer positions. Candidate sites were free to recombine with any other sequence in the pool of all possible sites. The present strategy also tested the general promiscuity of loxP spacer sequences. Cre-loxP spacer crosstalk was directly assayed since any two spacer sequences could interact in the Cre reaction. This revealed the promiscuity profile of each spacer sequence and identified the spacer sequences that were favoured by the Cre recombinase protein. By applying profiling algorithms typically used to analyze transcription factor binding sites [The present study approaches ng sites , we haveloxP recombination were directly challenged by the present study, the first being that Cre-loxP recombination between identical spacer sequences is favoured over recombination between non-identical spacer sequences [loxP site promiscuity is not a new observation; for example initial studies identified another loxP recombination site in the E. coli chromosome, loxB [loxP focus on pre-defined spacers there has been limited attention paid to promiscuity. Our data showed that for each spacer, self-recombination was not significantly greater than recombination with other spacers. Second, it has been assumed that the wild-type loxP spacer sequence is a favoured substrate for Cre recombinase. Although this assumption has never been explicitly stated, the majority of screens to identify novel spacers focus on point mutations to the wild-type spacer sequence. In the present study the wild-type spacer was detected in only 1 recombination reaction. This was a non-self recombination and not once did this sequence undergo self recombination . Finally, it has been assumed that directed, non-promiscuous recombination is best mediated by identical spacer mutants. In fact, none of our self-recombining spacer mutants that reacted more than once were non-promiscuous. Further, we identified 16 pairs of non-self recombining spacers; some of which recombined at levels similar to the top self-recombiners . Some sites are extremely non-reactive with other loxP sites, namely lox2272 [m3, and m7 [lox23, was detected in our self-recombining data. These results do not challenge the utility of published spacer mutants but indicate that published spacer sequences are potentially not kinetically favoured by Cre recombinase relative to other spacers. Additionally, there has been speculation in the literature that spacers at positions 2 and 7 are essential for exclusive recombination, at least between loxP mutants and the wild-type spacer [Several published spacer mutants have been described in the literature as self-recombining and are non-promiscuous  inverted repeat mutants and the other with wild-type sequence. These products are assumed to be resistant to subsequent Cre-mediated recombination. We screened all of our sequences for evidence of failure of the inverted repeat mutants to block further rounds of recombination. We found only three examples of additional recombination within our set of 3,124 successful reactions. The fact that even the most prolific spacer sequences such as GGGTAGGC did not react with the double mutants suggested that products were stable post-recombination.When using inverted repeat mutants the recombination reaction yields two different tandem loxP sites identified to date. Furthermore, previous attempts to discover novel loxP spacers have focused on mutants slightly different than, and non-reactive with, the wild-type sequence. In this work we deploy a high-throughput pipeline that rapidly identifies novel loxP spacers, regardless of sequence composition, with stability post-recombination and limited promiscuity. The set of 12 spacer sequences detected with these properties have an increased probability of successfully tolerating multiple loxP recombinations in vivo and, therefore, these sites have potential utility.There is a need for functional genomics tools that can expand the number of transgenic DNA segments that can be specifically incorporated into a target sequence. For example, synthetic biology applications require a mechanism to stitch together disparate DNA molecules from various sources in an ordered, controlled manner. Serializing RMCE or insertional recombination via inverted repeats has been limited by the small number of stable, non-promiscuous loxP spacers using standard molecular biology techniques and high throughput sequencing. Using the results from 3,124 successful reactions, we provide the first profile and comprehensive analysis of sequence spacers favouring Cre-loxP recombination. Increasing the guanine content at all spacer positions save for position 8 resulted in increased recombination reactions. Interestingly, self-recombination is not preferred over non-self recombination and the collection of known loxP spacer mutants do not appear to be favoured by Cre recombinase. We have identified novel self and non-self recombining loxP spacer sites that expand the repertoire of sites available for further study and potential in vivo recombination applications. Further, given that these sites have been shown to be functional in combination with inverted repeat mutations, they should be useful for mediating direct serial insertions into a given target construct, chromosome or genome.In this study we introduce a novel, high-throughput assay to rapidly screen for successful recombination between loxP sites with six degenerate spacer nucleotides  and two central fixed spacer nucleotides  (Table lox71 left arm mutant sequence and the other (RE) contained the lox66 right arm mutant sequence [Two oligonucleotides were designed that contained sequence . These oThe amplified, double stranded LE-spacer fragment was double digested with EcoRI and HindIII, agarose gel purified, and ligated to EcoRI/HindIII linearized and agarose gel purified pUC19 vector to create a library of LE mutant sequences  and 1x  Cre Recombinase Buffer (BD Biosciences). The reaction was incubated overnight at room temperature and the desired 1.8 kbp recombination products were agarose gel purified and amplified by PCR using the RE_NotI_Forward and RE_NotI_Reverse primers  to infer the sequence of the input oligonucleotides. Successful recombinants contained serial loxP sites with one containing the lox66 and lox71 inverted repeat mutants and another with the wild-type inverted repeats. The inverted repeats flanked an 8 bp spacer region. All analysis details and scripts are provided online . A Perl script termed pair_count4.pl See  identifiline see .Spacer sequences from the sequencing reads and the inferred input sequences were stored in a MySQL database see . From thin vitro recombination and library construction work, provided direction to data analysis, and edited the manuscript. All authors approved the final manuscript.PIM conceived of the study, contributed to study design, performed bioinformatics and statistical analysis of the data and wrote the manuscript. DES contributed to study design and performed high throughput sequencing. RAH contributed to study design, performed the RH017.fasta.all.cr – Multiple FASTA file. Multiple FASTA file containing output from the sequencing reactions generated by the M13 Reverse primer.Click here for fileRH017.fasta.all.c21 – Multiple FASTA file. Multiple FASTA file containing output from the sequencing reactions generated by the M13 Forward primer.Click here for filepair_count4.pl – Perl script to extract spacer sequences from sequence reads. This file is a Perl script that detected successful recombinants from the sequencing reads. Spacer sequence was extracted and immediately deposited into a MySQL database.Click here for fileTable S1 – Table_S1_mismatch_analysis.xls – Sequence of spacers participating in recombination. This file is an Excel spreadsheet all of the loxP spacers that successfully recombined. Contains pre- and post- recombination sequence as inferred from the location of the scissile bonds and reaction mechanism (see Figure Click here for fileAnalysis_scripts.zip – zip file containing all analysis scripts. All SQL, Perl and shell scripts used in this analysis. Detailed explanation of their functionality given in the README.txt file.Click here for file


Myxococcus xanthus is a highly social prokaryote that cooperatively develops into spore-bearing, multicellular fruiting bodies in response to starvation. Here we have characterized the nature of social interactions among nine developmentally proficient strains of M. xanthus isolated from spatially distant locations. Strains were competed against one another in all possible pairwise combinations during starvation-induced development. In most pairings, at least one competitor exhibited strong antagonism toward its partner and a majority of mixes showed bidirectional antagonism that decreased total spore production, even to the point of driving whole populations to extinction. Differential response to mixing was the primary determinant of competitive superiority rather than the sporulation efficiencies of unmixed populations. In some competitive pairings, the dominant partner sporulated more efficiently in mixed populations than in clonal isolation. This finding represents a novel form of exploitation in bacteria carried out by socially competent genotypes and is the first documentation of social exploitation among natural bacterial isolates. Patterns of antagonistic superiority among these strains form a highly linear dominance hierarchy. At least some competition pairs construct chimeric, rather than segregated, fruiting bodies. The cooperative prokaryote M. xanthus has diverged into a large number of distinct social types that cooperate with clone-mates but exhibit intense antagonism toward distinct social types of the same species. Most lengthy migration events in nature may thus result in strong antagonism between migratory and resident populations, and this antagonism may have large effects on local population sizes and dynamics. Intense mutual antagonism appears to be more prevalent in this prokaryotic social species than has been observed in the eukaryotic social slime mold Dictyostelium discoideum, which also exhibits multicellular development. The finding of several cases of facultative social exploitation among these natural isolates suggests that such exploitation may occur frequently in nature in many prokaryotes with cooperative traits.Social organisms that cooperate with some members of their own species, such as close relatives, may fail to cooperate with other genotypes of the same species. Such noncooperation may take the form of outright antagonism or social exploitation.  Myxococcus xanthus reveals that some strains exploit others, with implications for the evolution of intraspecific cooperation and the generation of bacterial diversity.Experimentally competing different natural strains of the social bacterium  Escherichia coli ), 56 were negative (strains sporulated less efficiently in mixture than in isolation), whereas 16 were positive (strains sporulated more efficiently in mixture than in isolation)  and corresponded to an approximately 70-fold average decrease in sporulation efficiency in mixed competitions relative to pure-culture development.Of 72 one-way mixing effects (olation) . Thus, 2olation) , and theThe 16 positive values corresponded to competition mixes in which one strain appeared to sporulate more efficiently in mixture with a competitor than in clonal isolation and simultaneously inhibited its competitor's performance. These outcomes provide evidence of facultative social exploitation in bacteria. By “facultative,” we mean that the ability to exploit other genotypes is not derived from a genetic social defect that prevents normal cooperation under clonal conditions. Thus, the exploiters do not obligately defect from cooperation in all contexts but do so only in mixes with some genetically distinct social partners.p < 0.0001 in both cases, chi-square test). Among the 16 positive responses, 12 are made by strains A, C, and D . These are the three most dominant competitors among the nine strains examined here (see explanation of The distributions of strain representations among the 16 exploitative responses and the 16 corresponding negative responses by the inferior (exploited) partners are nonrandom, with some strains being disproportionately represented .Exploitative responses to mixing were demonstrated more robustly in two particular strain pairs that involved relatively strong positive responses in the larger matrix of competition mixes. Two competition pairings (DI and IG) with one strain showing a positive response to mixing were repeated with the dominant strain  at a lower initial frequency of 0.32 . Both D Among the 36 competition pairs initiated at a 50:50 ratio, very different mixing effect patterns were observed among all competitors against a given strain. Three examples are shown in ijW* (predicted relative sporulation), ijW , and ijC (relative mixing effect) for each of the 36 unique competition pairings. From ijC = ijW − ijW*. The relative effect of mixing on competing strains' sporulation efficiencies ij)(C and relative sporulation efficiency in mixture ij), relative to the competitors listed by row (j). Positive ijW values indicate that the column strain is superior to the row strain, whereas the only negative value indicates that the column strain (A) is inferior to the row strain (C). Values not shown in the opposite half of the matrix are simply the negative of the value shown in the reciprocal cell. For example, DBW = 4.43 (shown), whereas BDW = −4.43 (not shown). Both values mean that strain D produces approximately 27,000-fold more spores than strain B in mixed development starting at a 50:50 ratio .We examined whether the structure of competitive dominance relationships across all pairwise comparisons is strongly hierarchical . For measures of relative mixing effect ijW. The remaining strains, B, I, F, E, H, and G, exhibit five, four, three, two, one, and zero cases of superiority, respectively. The ijW values among the three pairings of A, D, and C reflect the sole nontransitive triad  of performance relationships in each respective hierarchy. The top four competitors were all isolated from North America (as opposed to Eurasia), but such a ranking distribution has a 9.2% likelihood of occurring by chance .Strains A, D, and C shared the highest rank dominance (superiority over seven competitors each) and are ordered by decreasing average D. discoideum [K representing the degree of linearity on a scale from zero  to one (perfect linearity). With our group size of nine individuals, dominance hierarchies with 16 or fewer nontransitive triads exhibit significant linearity. Our hierarchy had only one such triad, representing extremely strong and significant linearity , meaning that this set of strains shows many distinct fitness ranks (seven of nine possible) rather than few.Kendall's technique  was usedscoideum . The nul, fruiting bodies were transferred to two nutrient agar plate types (ten to each type for AC and 40 to each type for BI), with each plate type containing distinct antibiotic corresponding to the competitors' resistance states. Fruiting bodies were then scored for subsequent growth after 5 d.Distinct antibiotic-resistance states of mixed competitors also allowed us to examine whether competitors ever mix within fruiting bodies or rather segregate during development after forced mixing in liquid. For two pairings (AC and BI) in which both competitors sporulated at relatively high levels in the mixed populations, we tested whether only one competitor could be found within individual fruiting bodies or rather both. For each pairp < 0.0001, binomial test).If competitors always segregate into separate fruiting bodies, only half of the total number of transferred fruiting bodies should germinate, regardless of the fruiting-body type ratio, because each fruiting body will germinate on only one of the two selective agar types. In the AC competition, all ten fruiting bodies germinated on whichever selective media they were placed, indicating that A and C form chimeric populations within fruiting bodies and likely do so in all fruiting bodies. The probability of all 20 fruiting bodies germinating under the total segregation hypothesis is extremely low  all germinated, whereas only three of the 40 fruiting bodies under selection for strain I (novobiocin plates) germinated. This result might reflect either total segregation of the competitors into distinct fruiting bodies (with I fruiting bodies in the minority) or segregation of B in most fruiting bodies with a small percentage of chimeras in which both B and I are present. Under the total segregation model, our novobiocin result gives the estimate that approximately 7.5% of fruiting bodies are of type I, which leads to the prediction that only 92.5% (37) of fruiting bodies will grow on rifampin agar. The total segregation hypothesis is unlikely to be correct, however, because the probability of all 40 fruiting bodies growing on rifampin when only 37 are expected is only 4.4% . In the quantitative spore production assays, strain B sporulated approximately 28-fold more efficiently than strain I, averaged over all fruiting bodies and replicates. However, strain B was found in only 12.3-fold more fruiting bodies than strain I, suggesting that even if the total segregation hypothesis were correct, strain I should sporulate less efficiently within its respective fruiting bodies than strain B.M. xanthus under environmental conditions in which social cooperation is crucial to fitness and survival. This antagonism negatively affected social productivity in almost all cases, with several pairings of antagonistic competitors reducing total spore production by greater than 90%. Most individual responses to mixing were strongly negative, but some strains exploited others by performing better in mixture than in isolation. Overall fitness ranks among the isolates were strongly hierarchical, with the three best strains forming a dominant triad (with nontransitive fitness ranks within the triad) and the remaining six exhibiting a perfect hierarchy.Intraspecific antagonism among relatively asocial bacteria is well known ,18–20, bM. xanthus as a species is not composed of a small number of cooperative units. Rather, this species has diverged into a large number of distinct social types that cooperate with clone-mates and perhaps very close relatives but exhibit intense antagonism toward distinct social types of the same species. This leads to the prediction that most long-distance transports of soil particles will result in strong antagonism between migratory and resident populations of M. xanthus. Our results show that such antagonism often causes large reductions in the total spore production of chimeric populations and can even cause whole populations to become extinct  segregated into separate fruiting bodies after forced mixing and that the M. virescens isolate inhibited sporulation of the M. xanthus isolate [M. xanthus are capable of forming chimeric fruiting bodies, suggesting that such chimerism might also occur under natural conditions, even if rarely. Such within-species chimerism has also been observed in D. discoideum fruiting bodies [Myxococcus genotypes may actually mix in the wild is under further investigation.A previous  isolate . In contg bodies . It has g bodies . Under sMyxococcus and other genera. Questions of future interest emerge from comparison of the M. virescens- vs.-M. xanthus study with our results, including whether genotypes of distinct species classifications ever form fruiting chimeras and whether qualitative antagonism patterns during interspecific competitions are consistent or highly variable across multiple isolates from each species.Until recently, myxobacterial classification was based exclusively on characterization of morphological traits, including the size, shape, and color of fruiting bodies and growing swarms. A recent comparison of a 16S rRNA-based phylogeny of the myxobacteria with traditional classification showed that the two approaches yielded largely consistent results across the monophyletic group . At the E. coli have evolved intense forms of intraspecific antagonism [E. coli, the best known form of intraspecific antagonism is mediated by the production of colicin proteins from plasmid-borne genes that can be readily exchanged among genotypes via conjugative plasmid transfer [M. xanthus hosts self-replicating plasmids [M. xanthus has no known mechanism of conjugative DNA transfer between cells. Thus, both the evolutionary and biochemical mechanisms of antagonism are likely to be fundamentally different between these species.Relatively asocial bacterial species such as tagonism . Our restransfer . In contplasmids , and M. M. xanthus carries genes for the production of a variety of secondary metabolites not present in most bacteria that may serve as anticompetitor agents [At the biochemical level, r agents . InitialM. xanthus development among spatially isolated genotypes suggests that local diversity generated by migration of previously isolated genotypes will, on average, be short lived. Previous studies have shown that nontransitive fitness relationships among three or more competitors in structured habitats can serve to maintain biological diversity [M. xanthus, and it will be of interest to determine whether nontransitivity is detected more frequently among close neighbors in the soil than was observed here among isolates from distant origins.Regardless of its underlying biochemical mechanisms, the hierarchical antagonism observed during iversity ,20,28. IAlthough the average effect of competitive encounters here is strongly negative, 44% of competitive pairs had one partner that showed enhanced sporulation efficiency in response to mixing see  and 4. APrevious reports of bacterial social exploitation have involved genotypes modified or evolved in laboratory environments, whereas this study demonstrates intraspecific social exploitation among distinct natural bacterial isolates. The superiority of exploitative clones over their competitors in mixture observed here is not due to major differences in pure-culture performance. Rather, it is accomplished by the formidable competitive ability of some dominant genotypes to both inhibit  a competitor's sporulation ability and simultaneously convert a higher percentage of cells into spores specifically due to the presence of the inferior competitor.M. xanthus based on morphological criteria  were used in this work. DK801 , DK816 , DK836 , and DK897  were obtained as clonal stocks from D. Kaiser; clones of Mxx15 , Mxx23 , Mxx41 , Mxx104 , and Mxx144  were purified from culture isolates provided by H. Reichenbach.Nine clonal isolates previously classified as mariner transposon carried by the suicide plasmid pMycoMar [M. xanthus lab strain DK1622.Three criteria were used in strain selection: (i) robust fruiting body formation and high sporulation efficiency, (ii) ability to obtain strain variants resistant to novobiocin, rifampin, or kanamycin, and (iii) zero or small effect of antibiotic resistance on sporulation efficiency. Strains were selected for use prior to collection of any data about their competitive ability against other strains. Strains A, B, D, E, and F were marked with spontaneous mutations conferring resistance to rifampin, while strains C, D, G, and I were likewise marked with spontaneous mutations to novobiocin resistance. Kanamycin resistance was conferred to strains C and E by chromosomal integration (via electroporation) of the plasmid pREG1727  into thepMycoMar . PositioijW competition matrix . Factoring in marker effects to predict the outcome of hypothetical competitions between unmarked strains did not alter the structure of the n matrix .9 cells/ml unless specified otherwise. For pure-culture assays, 100 μl of pure-culture resuspension was dispensed in the center of TPM plates (1.5% agar) for starvation. For paired competition assays, pure resuspended cultures from paired competitors were mixed at the appropriate ratio (50:50 in the complete matrix of pairwise mixes or 32:68 in a later experiment), and the total mixed volume (100 μl) was dispensed on the center of TPM plates. Thus, the total initial cell number and density were the same in competitive mixtures and clonal treatments. Developmental plates were incubated for 5 d, after which the cell populations were harvested with a scalpel blade, transferred into 1 ml of double-distilled H2O, and heated at 50 °C for 2 h to select for viable spores. Samples were then sonicated by microtip and diluted into CTT soft agar (0.5% agar) with the appropriate antibiotic treatment . In cases of mixed competitions where no colonies grew at our lowest dilution factor (101), a value of ten spores/assay was entered for data analysis, providing conservatively low estimates of fitness inferiority in these cases.All agar plates were incubated at 32 °C and 90% rH, and all liquid cultures were grown at 32 °C, 300 rpm. Strains were grown on CTT agar medium  from ultIn each competition mixture, the paired strains had distinct antibiotic-resistance marker states, allowing quantification of each competitor's spore production. In most cases, both competitors bore resistance to distinct antibiotics, allowing for direct mixing of one resistant representative from each strain. For two pairings (AB and GI), however, only variants with the same resistance marker were obtained. In these two cases, the resistant variants of both strains were mixed with the original antibiotic-sensitive clone of the respective competitor and their sporulation efficiencies in mixture were measured independently.In the complete competition matrix, each of all possible pairwise competitions were performed in at least three temporally independent blocks, with two replicates of each particular competition performed within each block. Assays of pure-culture sporulation efficiency for all competitors were performed simultaneously with the respective competition assays, also with 2-fold replication within each block. Because variation across blocks was larger than within blocks, we conservatively used within-block means of relevant values as data points for statistical analysis.(D) of strain i in pure culture is the frequency of cells surviving development as viable spores:The untransformed sporulation efficiency iN represents the viable population size of i before starvation (t0) and after 5 d of starvation (t5). The efficiency of i in mixed competition with strain j is similarly given aswhere i and j on the sporulation efficiency of strain i is given asThe effect of mixing strains iC(j) indicates that strain i sporulates more efficiently in the presence of strain j than in clonal isolation, whereas a negative value indicates that mixing with j negatively affects the efficiency of i. The relative mixing effect of strains i and j is given asThus, a positive value of i andj during development assumes that there are no effects of mixing on sporulation efficiency and is defined asThe predicted relative sporulation efficiency (log-transformed) of strains The actual relative sporulation efficiency (log-transformed) of two strains is defined as the log difference of the strains' actual sporulation efficiencies in mixed competition with each other:ij) value of each strain (across each strain's eight pairings) was used as the statistical unit to estimate the significance of the overall one-way effect of mixing.To estimate the significance of the negative overall mean of http://www.ncbi.nlm.nih.gov/gquery/gquery.fcgi) accession number for DK1622 is M34114.The NCBI Entrez (


ENG gene causes type I hereditary hemorrhagic telangiectasia (HHT1), an autosomal dominant disorder. Compared to normal population, HHT1 patients have a higher prevalence of AVM in multiple organs including the brain. Vessels in bAVM are fragile and tend to rupture, causing hemorrhagic stroke. High prevalence of pulmonary AVM in HHT1 patients are associated with a higher incidence of paradoxical embolism in the cerebral circulation causing ischemic brain injury. Therefore, HHT1 patients are at risk for both hemorrhagic and ischemic stroke. This review summarizes the possible mechanism of ENG in the pathogenesis of cerebrovascular diseases in experimental animal models and in patients.Endoglin  is a transforming growth factor β (TGFβ) associated receptor and is required for both vasculogenesis and angiogenesis. Angiogenesis is important in the development of cerebral vasculature and in the pathogenesis of cerebral vascular diseases. ENG is an essential component of the endothelial nitric oxide synthase activation complex. Animal studies showed that ENG deficiency impairs stroke recovery. ENG deficiency also impairs the regulation of vascular tone, which contributes to the pathogenesis of brain arteriovenous malformation (bAVM) and vasospasm. In human, functional haploinsufficiency of  ENG, or CD105) is located on chromosome 9q34.11. It is a type III transforming growth factor β (TGFβ) receptor interacting with TGFβRI  and/or TGFβRII . In the endothelium, ENG interacts with the activin receptor-like kinase 1 (ALK1 or ACVRL1), a type 1 TGFβR. ENG binds with TGFβ1 and TGFβ3 with high affinity in the presence of other TGFRs but not with TGFβ2–4. ENG also binds to activin-A, bone morphogenetic protein 2 (BMP2) and BMP7. Protein studies suggested that ENG plays an important role in modulating the TGFβ signaling pathway.In human, endoglin gene , also known as Osler-Rendu-Weber Syndrome. HHT is an autosomal dominant disease. The clinical features of HHT patients are telangiectases in mucocutaneous membrane and arteriovenous malformation (AVM) in multiple organs, including the skin, liver, lung, intestine and brain. AVMs are abnormal vessels that shunt blood directly from arteries to veins. Brain AVM (bAVM) tends to rupture, which can cause life-threatening intracranial hemorrhage and hemorrhagic stroke. Hemorrhage from bAVM can also cause long-term disability. Elevated levels of angiogenic factors including vascular endothelial growth factor (VEGF) were found in sporadic bAVM patients, 27. High levels of VEGF are also associated with increase of blood-brain barrier (BBB) permeability and bAVM hemorrhage–29. Similarly, HHT patients that have a higher incidence of AVMs in multiple organs also have an increased level of plasma VEGF. All of these evidence suggest that angiogenesis is involved in the pathogenesis of bAVM.Mutations in the Since ENG plays an important role in the angiogenesis, in this review, we summarize the influences of ENG on endothelial function and the angiogenesis, as well as how ENG-deficiency contributes to the pathogenesis of cerebrovascular diseases, including ischemic stroke and intra-cranial hemorrhage, as well as cerebrovascular malformation, stenosis and occlusion.Eng gene knockout mice were generated, 31. Homozygous deletion of Eng gene in mice causes embryonic death by E10.5–11.5, 31. The endothelial cells derived from ENG deficient human embryonic stem cells failed to organize effectively into tubular structures in vitro. VEGF induced vascular network was also reduced in the metatarsal bone of Eng heterozygous knockout (Eng−/−) mouse embryo. Consistently, depletion or inhibition of ENG gene in human endothelial cells mitigated VEGF-induced angiogenesis. These findings suggest that ENG is required for the differentiation and sprouting of endothelial tubes, which are important processes of angiogenesis.To study the functional role of ENG in development, , 32, 33. The recruitment of vascular smooth muscle cells and pericytes to newly formed vascular network is impaired in Eng deficient mouse embryos.ENG also mediates endothelial-mesenchymal communication during angiogenesis. To understand bAVM pathogenesis and to develop therapeutic strategies, many Eng deficient mouse models were generated. Using these animal models, we are able to elucidate bAVM pathogenesis and test new therapies.As mentioned in previous sections, ENG deficiency is associated with HHT1, a familial disease that has bAVM as one of its major phenotypes. Brain AVM contains abnormal vessels, that are prone to rupture, causing intracranial hemorrhage and hemorrhagic stroke. In addition, patients with ENG deficiency (HHT1) have a higher incidence of pulmonary AVM (PAVM), which is associated with a high incidence of paradoxical embolism in the cerebral circulation and ischemic brain injuryEng gene in mouse causes embryonic lethality, 31, mice with heterozygous deletion of Eng (Eng+/−) are used to study the pathogenesis of HHT patients. Eng+/− mice exhibit many phenotypes that resemble those of HHT1 patients, including mucocutaneous telangiectases, external bleeding, and AVMs in the liver, lung, brain and gastrointestine. Enlarged cerebrovascular structure was found in some Eng+/− mice with evidence of hemorrhage. However, penetrance of bAVM in Eng+/− mice is very low, only 7%, suggesting that heterozygous Eng deletion alone is not sufficient to cause bAVM formation. In addition, the differences of the penetration of HHT phenotypes in 129/Ola and C57BL/6 Eng+/− mice suggests that modifier genes are contributing to the severity and heterogeneity of AVMs in HHT patients.Since homozygous deletion of , 27, 30. The intensity of VEGF staining is also correlated with microvessel density in nasal mucosa from HHT patients. Together, abnormally high level of VEGF appears to be a fundamental part of AVM pathophysiology, 37–39. Based on these studies, we overexpressed VEGF in the mouse brain in conjunction with Eng deletion to generate bAVM models. In adult Eng+/− mice, intra-brain injection of an adeno-associated viral vector expressing VEGF (AAV-VEGF) significantly increased the penetrance of cerebrovascular abnormality. Almost all-adult Eng+/− mice that received intra-brain injection of AAV-VEGF showed cerebrovascular abnormality. However, unlike HHT1 patients, the vascular abnormality in Eng+/− mice is at the capillary level.Based on clinical studies, we and others found that VEGF levels are increased in the plasma of HHT patients and in surgically resected sporadic human bAVM specimens. Since the accumulation of bone marrow-derived macrophage in VEGF-induced brain angiogenic regions peaks earlier than the increase of vessel density, macrophages likely play a role in angiogenesis.Bone marrow-derived cells can infiltrate into the brain angiogenic region. We found that macrophages are the major bone marrow-derived cells recruited to the brain angiogenic fociEng+/− mice, the influence of bone marrow derived cells in the development of bAVM has been studied. Transplantation of Eng+/− bone marrow to wild type mice induced vascular dysplasia in the brain angiogenic regions, while transplantation of wild type bone marrow to Eng+/− mice reduced the severity of vascular dysplasia in the brain angiogenic foci of Eng+/− mice. These data suggested that Eng gene mutation in bone marrow cells cause vascular dysplasia. Importantly, these data suggested that transplantation of normal bone marrow cells to bAVM patients could be a therapeutic option.Using Eng+/− mice by overexpression of VEGF, arteriovenous shunts were not detected in these mice. Studies have shown that a combination of homozygous Eng inactivation and additional stimulations are needed for robust bAVM formation. Genetic studies also indicated that mutations of Eng modifier genes contribute to AVM formation, 44.Although we were able to induce vascular dysplasia in the brain of Eng deletion, Allinson et al. generated an Eng-floxed (Eng2f/2f) mouse line that have the Eng gene exons 5–6 flanked by loxP sites. When Cre recombinase is present, the DNA sequence between the loxPsites will be deleted. To test whether homozygous Eng gene deletion plus angiogenic stimulation can initiate bAVM formation, an adeno virus expressing Cre recombinase (Ad-Cre) and AAV-VEGF were co-injected into the brain of Eng2f/2f mice, 46 to induce brain focal Eng gene deletion and angiogenesis. Eng2f/2f mice with focal Eng gene deletion and angiogenic stimulation developed vascular dysplasia beyond the capillary level around the AAV-VEGF injection site eight weeks after the vector injection. Robust bAVM have also developed in the AAV-VEGF induced brain angiogenic region in mice subjected to global Eng deletion at the age of 8 weeks old. The bAVM phenotype in these mice highly resembled the phenotype of human bAVM. Furthermore, Eng-null endothelial cells were found in the dysplastic vessels in the bAVM lesion. Our studies are consistent with the studies on skin AVM development, and support the notion that an injury (angiogenic stimulation) is needed to induce bAVM.To avoid embryonic death caused by homozygous Eng-deficient bAVM mouse models have been used to analyze the function of macrophages during bAVM pathogenesis. Although Eng deficiency has been shown to impair monocyte migration into injured tissue–50, an increased number of bone barrow-derived macrophages and activated residential microglia was found in the bAVM lesion in mouse and human. Compared with normal macrophages, Eng-deficient macrophages show slower but more persistent infiltration into the brain angiogenic regions. Delayed clearance of macrophages and persistent inflammation could exaggerate abnormal vascular phenotypes in bAVM.Eng gene in adult mice, several cell-specific cre transgenic mouse lines have been used to induction of Eng deletion in specific cell-types. For example, the promoter of SM22α (smooth muscle actin) is used express cre in smooth muscle specifically. Although SM22α is predominantly expressed in smooth muscle cells in normal mice, Cre expression driven by the SM22α promoter in this transgenic mouse line was also found in other cell types, including endothelial cells, 53. SM22α Cre; Eng2f/2f mice have Eng gene deleted in the SM22α expressing cells during the embryonic developmental stage. We found 90% of SM22α Cre; Eng2f/2f mice have spontaneously developed bAVM by 5 weeks of age and 50% of them died by 6 weeks of age. BAVM lesions varied in size and location in these mice. In addition to bAVMs, some of SM22α Cre; Eng2f/2f mice also developed spinal and intestinal AVMs. Because AVM develops in this mouse line spontaneously without exogenous VEGF stimulation, this model is an ideal model for testing new therapeutic strategies.In addition to conditional knockout of Eng gene not only expresses in endothelial cells, 6, but also expresses in activated monocytes/macrophages, mesenchymal cells, fibroblasts, and smooth muscle cells, 10. Using transgenes that express cre specific cell-types, the Eng gene was conditionally deleted in different cell types in adult mice to determine which cell type is most crucial for AVM development, 55. In SclCreER; Eng2f/2f mice, which have Eng deleted in endothelial cells only, AVM formed in the skin around the ear wound and back wound, 55. We found that bAVM develops in the brain angiogenic region in Pdgfb-iCreER; Eng2f/2f mice that have Eng gene deletion specifically in endothelial cells. Myh11Cre ER-mediated Eng deletion in smooth muscle cells in adult mice did not cause AVM formation in the wound area of the skin. Furthermore, LysMCre; Eng2f/2f mice, which have Eng deleted in macrophages, did not develop AVM in any organ and in the brain angiogenic regions. These studies indicate that Eng deletion in endothelial cells is essential for AVM formation in the brain and other organs, 54.As mentioned above, Eng-deficient bAVM mouse models were valuable resources to test new therapies for the treatment of bAVM. Current treatments for bAVM are mostly invasive and associated with high morbidities and mortalities. Since high VEGF level is involved in the pathogenesis of bAVM, we have tested the feasibility of use soluble FMS-like tyrosine kinase 1 (sFLT1) gene therapy to treat bAVM. Soluble FLT1 is an alternative transcript of FLT1 (or VEGFR1) containing only the extracellular domains of the receptor. Soluble FLT1 binds VEGF with high affinity in tissue, reduces VEGF signaling through its membrane-bound receptors, and thus inhibits VEGF-induced angiogenesis. Systemic delivery of AAV9-sFLT1 into a bAVM mouse model that has Eng gene deleted globally reduced abnormal vessels in the bAVM region. Intravenous delivery of AAV9-sFLT1 to SM22α Cre; Eng2f/2f mice that have spontaneously developed bAVMs reduced mortality and bAVM penetrance. This study demonstrated that mouse models are important tools to test new therapies., 22, 58, which ameliorates endothelial cells apoptosis regardless of the presence or absence of TGFβ. During hypoxia stress, TGFβ induces apoptosis of endothelial cells, 61, but reduces the death of neurons and vascular smooth muscle cells. Therefore, ENG is likely to antagonize the inhibitory effects of TGFβ1 on human vascular endothelial cells, 63 and protect endothelial cells against apoptosis via TGFβ signaling or other independent pathways.Hypoxia induces the expression of ENG in human and mouse brain microvascular endothelial cells.Under hypoxia conditions, ENG expression increases in many cell-types, such as, human microvascular endothelial cells-1 (HMEC-1) and monocytic U-937 cell. It is likely that hypoxia regulates ENG expression through crosstalk of several signaling pathwaysENG gene. Therefore, ENG expression can be induced by hypoxia through hypoxia-inducible factor-1 (HIF-1). A subsequent study suggested that hypoxic induction of Eng expression in bEnd.3  cells was activated through ERK-p38 MAPK and JNK pathway, instead of HIF-1. In addition, Smad3 was reported to interact with HIF proteins to induce the overexpression of ENG. Although these studies implicated links among multiple factors, further studies are required to better elucidate the exact transcriptional regulation of ENG expression under hypoxia conditions.The transcriptional regulation of ENG expression under hypoxia condition was studied by a reporter assay using HeLa cells, and by the electrophoretic mobility shift assay (EMSA) using human umbilical cord vein endothelial cells (HUVECs). These assays confirmed the presence of a hypoxia response element (HRE) in the enhancer region of . However, it was not clear at that time whether the angiogenesis was beneficial. In acute ischemic stroke patients, there is a robust mobilization of immature hematopoietic cells, colony-forming cells and long-term culture initiating cells. It has been suggested that the degree of immature hematopoietic cell mobilization is directly correlated with the recovery of neurological function, 67. An increase of ENG positive micro-particles including exosomes and shedding vesicles, which are small vesicles released by specific cells , were detected in patients’ sera collected 3 days after stroke compared to that of healthy people. Certain types of ENG positive micro-particles increased further in stroke patients with severe disability. The ENG positive micro-particles decreased gradually after the initial increases. The number of these circulating ENG positive micro-particles was positively correlated with the stroke severity, even after adjusting for other demographic and clinical variables, such as hypotension and other stroke comorbidities. Similarly, ENG positive circulating micro-particles released from endothelial cells were also increased in patients with acute ischemic stroke. The increase of ENG positive cells was positively associated with the severity of neurological function at hospital admission, larger brain lesion volume and unfavorable functional outcome at hospital discharge. The increased level of circulating ENG positive micro-particles after acute stroke may have been caused by either increased circulating cells as a self-repair response to stroke or a sign of increased apoptosis of circulating cells in response to hypoxic conditions.Previous studies revealed that ENG was highly expressed in the penumbra region of human stroke lesion, where an increase of angiogenesis was found. ENG overexpression also protects against TGFβ-induced apoptosis of endothelial cells, 59. Reduction of vascular cell-apoptosis after hypoxia improves blood supply to ischemic tissue, 71. Increase of ENG expression in endothelial cells could also be hazardous, because BBB permeability was increased in some of the capillaries that express high level of ENG, which was accompanied with mononuclear cell infiltration in the surrounding brain tissues. These findings suggests that pronounced ENG overexpression might impair vessel wall integrity. Alternatively, lack of ENG expression may indicate severe vessel damage. ENG and TGFβ are involved in the pathogenesis of post-ischemic brain injury in human. Abnormal ENG and TGFβfunction might lead to long-term neurological deterioration or cognitive disturbance after acute ischemic stroke, 73. Homeostasis of ENG expression is crucial for maintaining normal angiogenesis, vascular remodeling and reduction of stoke injury.The role of ENG in stroke injury is complex, and is influenced by the local microenvironment. Constitutive expression of ENG enhances the TGFβ signaling and promotes new vessel wall remodeling. Nutrient supply supporting neuron survival is carried through blood. Higher microvessel density in the peri-infarct region is associated with lower morbidity and mortality. Hypoxia-induced angiogenesis increases blood flow and oxygen delivery to ischemic tissues, which contributes to the recovery after stroke.The survival of neurons in peri-infarcted regions is associated with the extent of patient recovery after stroke. The micro-vessel density remains higher in the infarct area compared with the corresponding contralateral side three months after stroke. Increased vessel density restores cerebral blood flow, salvages ischemic tissue, enhances neuronal survival and improves functional recovery of stroke survivors.Angiogenesis occurs in human brain after stroke. Through examining human postmortem brain samples with ischemic infarcts caused by occlusive vascular diseases, capillary networks with regular connection and micro-vessels were found in the brain samples of patients who died within one week after stroke, and the neo-vasculature was in filled with blood in the brain samples collected from patients that died 2–3 weeks after stroke and is elevated in inflammatory tissue and healing wound. In patients, somepro-angiogenic genes, including Tie-2, matrix metalloproteinase-2 (MMP-2), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), hepatocyte growth factor-α (HGF-α) and monocyte chemoattractant protein-1 (MCP-1), were upregulated in ENG expressing micro-vessels in stroke affected tissue. These key angiogenic elements play important roles in endothelial cell migration, differentiation and tube-formation, as well as vessel stabilization and stem cell homing into the region of angiogenesis and revascularization.ENG is expressed in proliferating vascular endothelial cellsEng deficient mice (Eng+/− mice), the functional performance after stroke was poorer than wild type animal both in the acute phase and the sub-chronic stage (one month after stroke), suggesting that there is an association between delayed functional recovery and Eng deficiency. The infarct volume and atrophic volume are larger in Eng+/− mice. The density of micro-vessels within the infarct and peri-infarct region are lower in Eng+/− mice than wild type mice, 80. In vitro study showed that Eng+/− endothelial cells express a lower level of VEGF compared to that of wild type endothelial cells. Eng+/− macrophages express lower levels of VEGF receptor 1 (VEGFR1) and 2 (VEGFR2) at the baseline and lower level of VEGFR2 after VEGF stimulation than wild type macrophages. Although Eng+/− macrophages and wild type macrophages express similar levels of MMP9 at the baseline, unlike in wild type macrophages, the expression of MMP9 did not increase in Eng+/− macrophages after VEGF treatment. In the brain of Eng+/− mice, VEGF-induced upregulation of VEGFR2 expression was also impaired. Together, these data suggest a reduced angiogenic response in the absence of normal Eng function may be responsible for the impairment of tissue repair in Eng deficient mice after experimental stroke.In Eng deficiency is associated with impairment of macrophage recruitment and clearance in the peri-infarct area during stroke recovery. Eng expression was upregulated during the transition from monocyte to macrophage. Eng deficiency in endothelial cell reduced adhesion and transmigration of leukocytes in response to ischemic injury. Recruitment of monocytes to the infarcted tissue and subsequent vessel formation was severely impaired in HHT1 patients (who have ENG haploinsufficiency) suggesting that ENG deficiency impairs monocyte adhesion and migration. In the acute phase (3 days) of stroke, Eng deficient mice had fewer macrophages in the peri-infarct region. However, at 60 days after stroke, a time that is considered as recovery stage, there was an increase number of macrophage in the peri-infarct region of Eng deficient animals, suggesting a delayed homing and clearance of over-activated macrophage. However, the roles of post-ischemic inflammation might be bidirectional. The inflammatory response after ischemic stroke could contribute to a secondary brain injury, because the influx of inflammatory cells amplifies brain cell death. On the other hand, inflammation also facilitated the clearance of damaged tissues and promoted tissue repair. Therefore, the consequences of impaired macrophage in homing and clearance in the stroke tissue require further studied.In addition, our study suggested that Eng+/− mice had severer brain injury than wild type mice since the first day of experimental stroke, which could not be explained by impaired tissue repair. As discussed in above, hypoxia induce endothelial Eng expression, which prevents hypoxia-induced apoptosis of endothelial cells. Therefore, vascular damage in Eng+/− mice could be more severe than in wild type mice after ischemic injury. In addition, Eng haploinsufficiency has been shown to be associated with reduced production of nitric oxide and increased production of superoxide under eNOS induction. Nitric oxide produced by endothelial cell induces vascular relaxation. Bioavailability of nitric oxide is lower in Eng+/− mice than in wild type mice. Enhancing superoxide production in Eng deficient mice reduces vascular relaxation, and increases vessel damage and oxidative stress, all of which increases brain injury during the acute stage of ischemic stroke.Interestingly, Since ENG plays an important role in angiogenesis and lack of ENG dampens angiogenesis, therapeutic stimulation of ENG could promote angiogenesis, vascular remodeling and improve stroke recovery, as well as reduce morbidity and mortality of stroke patients.ENG gene, which contains only the extracellular domain of the full-length ENG. Soluble ENG enters the circulation in various conditions that related to the endothelial injury, activation, inflammation and senescence. Our group showed that sENG level is increased in the surgical resected human bAVMs. We have also shown that co-injection of an adenoviral vector expressing sENG with AAV-VEGF into mouse brains caused capillary dysplasia. It is still unclear how overexpression of sENG causes cerebrovascular malformation. One of the possibilities is that circulating sENG acts as a decoy inhibiting the effect of ENG on the endothelium, leading to vascular malformation during angiogenesis.Soluble ENG (sENG) is an alternative transcript of Eng+/− mice have a lower level of NO metabolites (nitrites) in the plasma and in the urine than that of wild type mice, suggesting that the NO level is lower in Eng deficient animals. The hypotensive and vasodilatory response induced by endothelium-dependent vasodilators was less intensive in Eng+/− mice than wild type mice. However, the difference of this vasodilation effect between Eng+/− mice and wild type mice disappeared after NO synthesis was inhibited. These findings suggested that the NO level or the subsequent vessel response to NO is reduced in Eng+/− mice. However, after eliminating the endogenous NO, the vasodilatory effect induced by exogenous NO donor (nitroprusside) was similar in Eng+/− and wild type mice. The peripheral progenitor cells of HHT patients expresses lower level of eNOS  mRNA. Endothelial NOS produces NO in response to humoral and mechanical stimuli. However, resistance arteries in Eng+/− mice displayed an eNOS-dependent impairment in the myogenic response  despite of a reduced eNOS level. Eng deficient endothelial cells had uncoupled eNOS, which produce less NO but more superoxide. Taken together, these studies indicate a role of Eng in the regulation of vascular tone.Nitric oxide (NO) is a potent vascular smooth muscle relaxant, which is synthesized by the vascular endothelium. . The potential role of ENG on the production of NO suggests that ENG might be associated with vasospasm after SAH. In patients with SAH, the level of sENG increased in the cerebrospinal fluid (CSF) and decreased in the serum, 95. In the subgroup with cerebral infarction due to post-SAH vasospasm, the level of sENG was higher in the CSF and lower in the serum than the patients who did not have post-SAH cerebral infarction, 95. The level of sENG during the first two weeks of SAH might be a predictive factor for the long-term outcome, such as, 6 months after SAH. Similar to sENG, the ENG positive endothelial micro-particles were increased in SAH patients with vasospasm.Cerebral vasospasm is one of the most common complications of subarachnoid hemorrhage (SAH) and is associated with high morbidity and mortality. NO is found to be an important mediator of vasospasm. Several studies suggest that sENG is a naturally occurring antagonist of TGFβ. In contrast to the lower level of sENG, the level of TGFβ1 in the serum was higher in patients with vasospasm after SAH than those without vasospasm. Moreover, sENG interferes the binding between TGFβ1 and its receptors. TGFβ1 has been suggested to be involved in eNOS activation. Therefore, the reduced sENG levels in patients with post-SAH vasospasm might reflect an impaired production of vasorelaxant factors, such as NO. However, there is no direct evidence supporting the cause-and-effect relationship between vasospasm and sENG. Further studies of post-SAH vasospasm in Eng-deficient mice might be helpful in exploring the association of sENG and vasorelaxation.Soluble ENG are present in both healthy people and patients with pathological conditions (such as preeclampsia and SAH), 95. In patients with Doppler sonographic vasospasm, the serum level of sENG was similar to those without vasospasm. However, the serum level of sENG was reduced in patients with cerebral infarction due to severe vasospasm and hydrocephalus, suggesting that the sENG level in the serum might beserved as a biomarker for cerebral ischemia subsequent to vasospasm. Cerebral hypo-perfusion or hypoxia could induce increases of focal expression of ENG and might contribute to the increase of sENG in the CSF of patients with vasospasm. Both extravasation of sENG from blood or intrathecal production of sENG could cause the increase of sENG in the CSF and decrease of sENG in the plasma. Further studies are needed to ravel the origin of sENG during post-SAH vasospasm.Interestingly, the changes of sENG in the cerebrospinal fluid (CSF) and the serum of patients with SAH and vasospasm are oppositeAlthough it is not clear how ENG-positive micro-particles and sENG increased in patients with post-SAH vasospasm, the results of these studies indicated that, the circulating sENG is a promising biomarker for cerebral vasospasm after SAH.. The expression of ENG is very low in normal human arteries and is restricted to the endothelial cells of adventitial microvessels. In contrast, higher ENG expression is present in the advanced atherosclerotic plaque of human patients. The site of ENG expression are slightly difference between atherosclerotic plaques in carotid arteries and aorta. In aortic atherosclerotic plaque, ENG is predominantly expressed in smooth muscle cells. However, in carotid plaque, ENG is expressed in endothelial cells of neo-vessels within the lipid core and plaque shoulders. ENG expression is higher in carotid plaque containing higher levels of collagen and lessintra-plaque thrombi, which are characteristics of stable plaques. These evidence indicate that ENG may promote the formation of intra-plaque neo-vessels and collagen and reduce the vessel leakage and hemorrhage. However, ENG expression in the neo-vessels of carotid atherosclerosis has also been found to be positively correlated with the advanced grade of plaques. The distinct ENG expression patterns in different types of plaques suggest that ENG might play different roles in the course of atherogenesis progression.Carotid atherosclerotic stenosis is a major cause of ischemic stroke. As mentioned in earlier sections, ENG is expressed mainly in endothelial cells, smooth muscle cells and macrophages, which are the three major cells involved in the pathogenesis of atherosclerosis. Therefore, Eng may serve as a biomarker for evaluating the therapeutic effect of drugs in treating atherosclerosis. More studies are needed to elucidate the role of ENG in the pathogenesis of atherosclerosis.Atorvastatin is a drug to treat carotid atherosclerotic plague. In a mouse model of atherosclerosis, atorvastatin treatment decreased the level of Eng in the serum and increased Eng expression in the plaque. Middle cerebral artery (MCA) of MMD patients had thicker intimal walls than control vessels collected from aneurysm patients, indicating intimal hyperplasia in MMD. The expression of ENG and HIF-1 are increased in the intima of MMD patients. In addition, TGFβ3 expression was also detected, which was predominantly in the endothelium and was co-localized with HIF-1 and ENG. Although the study did not find an association between cerebral blood perfusion and ENG expression, the low spatial resolution method used to evaluate the cerebral blood flow of the entire MCA territory might not be accurate enough to detect the real perfusion through the MCA branches that were used to measure the ENG expression. The increased expression of ENG and HIF-1 in MMD is consistent with the increased expression of ENG under hypoxia condition. Therefore, ENG may play roles in the pathogenesis of cerebrovascular stenosis or occlusion.Moyamoya disease (MMD) is a rare, progressive cerebrovascular disorder caused by blocked arteries at the base of the brain in an area called the basal ganglia. MMD is one of the major causes of stroke in children and adults characterized by progressive stenosis or occlusion of terminal portion of internal carotid arteries and development of fragile collateral vessels. Resveratrol is a natural component of a number of fruits, including grapes, blueberries and raspberries. The skin of red grapes is used to extract resveratrol. In vitro, resveratrol reduces sENG secretion and pro-inflammatory factors of cultured endothelial cells. Therefore, it might be a promising non-specific inhibitor of sENG. A proper level of ENG expression might be crucial for maintaining normal angiogenesis and vascular remodeling in the brain. However, there is no report of direct regulation of ENG for treating cerebrovascular disease to date. In vitro study showed that statins could increase sENG secretion from endothelial cells; and in vivo administration of stain increased Eng expression in the carotid plaque of a mouse model. Statins are a group of medications that has been used to treat patients with carotid artery atherosclerosis and other ischemic cerebrovascular diseases. More studies are needed to test whether ENG can be used as a target for developing new therapies for the treatment of cerebrovascular diseases.Since ENG has been implicated in the pathogenesis of various cerebrovascular diseases , modulatIn summary, ENG plays a critical role in angiogenesis, vascular development and regulation of vascular tone. ENG deficiency is associated with the development of AVM in HHT patients, exacerbates stoke injury and impairs stroke recovery. ENG might be a potential biomarker for vasospasm after SAH and cerebrovascular stenosis. Therefore, experimental or therapeutic modulating of ENG expression are useful ingeneration of disease models in animals to study disease pathogenesis and in development of novel therapies to treat cerebrovascular diseases. The exact function of ENG in cerebrovascular diseases remains to be revealing.


Neurocognitive impairments are a core and enduring feature of psychosis that continue to persist despite pharmacological interventions. Neurocognitive interventions have emerged as a supplementary treatment option to improve cognition in early psychosis patients. Recently, focus has shifted to using social cognitive training (SCT) as evidence suggests that targeting social cognition may lead to improvements not only in cognition but also in real-world functioning . This improvement is thought to be mediated by restoration of functional brain activity in patients undergoing neurocognitive interventions, especially associated with medial prefrontal cortex . In this study, we report our interim findings of the effects of a 10-hour SCT on cognition and resting-state functional connectivity (rsFC). Our hypothesis was that training would improve cognition and normalize functional connectivity.https://www.brainhq.com/), while another naturalistic arm (n=18) received treatment as usual (TAU). Both treatment arms were assessed on a battery of neurocognitive tests and underwent a multimodal imaging protocol, including a 10 min resting-state fMRI, at two timepoints . Seed-based voxel-wise rsFC was performed and individual-level rsFC correlation maps were calculated between the bilateral medial prefrontal cortex (mPFC) and the whole brain.In this randomized-controlled study, one recent onset psychosis (ROP) patient arm (n=18) underwent a 6-week (10-hour) computerized SCT , processing speed (p<0.05) and resilience to both immediate and delayed memory decline over 6 weeks (p<0.05), as compared to TAU. Comparison of FC between the two measurement time points, suggested increased FC between mPFC and left inferior temporal gyrus (ITG), as well as increased FC between mPFC and left somatosensory area in ROP patients that underwent SCT relative to TAU.We have shown improvements in processing speed, verbal memory, and spatial working memory that agree with previous studies using computerized cognitive interventions. Moreover, the improvement in the spatial working memory domain was significant in the most demanding test condition with 10 elements - indicative of benefits from the fine-tuning of higher-level executive functions. The neuroimaging results also suggested that the improvements may have been mediated by the improvement of FC in regions typically associated with social cognition and facial recognition . These results are in line with recent studies investigating not only the feasibility of SCT as an intervention, but also the effects on cognition and underlying neural alterations resulting from intensive computerized neurocognitive interventions . Future studies using machine learning methods will be necessary to determine functional biomarkers in order to personalize SCT at the individual-level.


Glioblastoma is the most aggressive cancer of the brain. The dismal prognosis is largely attributed to the heterogeneous nature of the tumor, which in addition to intrinsic molecular and genetic changes is also influenced by the microenvironmental niche in which the glioma cells reside. The cancer stem cells (CSCs) hypothesis suggests that all cancers arise from CSCs that possess the ability to self-renew and initiate tumor formation. CSCs reside in specialized niches where interaction with the microenvironment regulates their stem cell behavior. The reciprocal interaction between glioma stem cells (GSCs) and cells from the microenvironment, such as endothelial cells, immune cells, and other parenchymal cells, may also promote angiogenesis, invasion, proliferation, and stemness of the GSCs and be likely to have an underappreciated role in their responsiveness to therapy. This crosstalk may also promote molecular transition of GSCs. Hence the inherent plasticity of GSCs can be seen as an adaptive response, changing according to the signaling cue from the niche. Given the association of GSCs with tumor recurrence and treatment sensitivity, understanding this bidirectional crosstalk between GSCs and its niche may provide a framework to identify more effective therapeutic targets and improve treatment outcome. Glioblastoma (GBM), World Health Organization (WHO) grade IV glioma, is the most aggressive primary brain tumor in adults and accounts for over 50% of the tumors of the brain . CurrentTwo hypotheses have been proposed on the origin of such tumor heterogeneity. Clonal evolution hypothesis suggests that most cancers arise from a single altered cell which facilitates tumor initiation and progression. As the tumor progresses, accumulated genomic instability results in the appearance of new genetic variants. Those variants with selective growth advantage expand to become the predominant subpopulation in the tumor. The presence of multiple subpopulations in a tumor thus supports the theory of tumor heterogeneity. On the other hand, cancer stem cells (CSCs) hypothesis suggests that intratumor heterogeneity arises from CSCs that possess the ability to self-renew and initiate tumor formation. CSCs give rise to phenotypically diverse cancer cells and reside in specialized niches where interaction with the microenvironment regulates their stem cell behavior. This behavior suggests a possible linkage between therapy outcome and genomic composition of the tumor. Recent experimental evidence supports the concept of CSCs plasticity and the ability of non-CSCs to dedifferentiate into CSCs . This coAdvances in genomic sequencing and transcriptomic profiling reveal the existence of multiple molecular subtypes, namely, proneural, neural, classical, and mesenchymal, within a tumor, highlighting the heterogeneous nature of GBM , 16. EacThe existence of different molecular subtypes within a tumor  and at sIt is now established that an epithelial-mesenchymal-like transition (EMT), termed as proneural-mesenchymal transition (PMT), exists in GBM , 35, 37.Heterogeneity among tumor cells not only arises within a single tumor as a result of molecular and genetic changes but also is affected by different microenvironments within different regions of the tumor –42. GBM β (TGF-β) and platelet derived growth factor (PDGF), which in turn increase expression of stemness-related genes such as SRY-box-2 (Sox-2), oligodendrocyte lineage transcription factor-2 (Olig-2), Bmi-1, and CD133 in GSCs  pyridine derivatives reduced mesenchymal tumor cells growth and inhibited xenograft growth in glioma bearing mouse brains [γ-aminobutyric acid (GABA), which is involved in the production of neurotransmitters. Conversely, mesenchymal-like GSCs lack NAA and GABA but have high levels of high mobile lipids indicative of an astroglial-like metabolism [In GBM, IDH-1 wildtype proneural subclasses are characterized by having high glutamate level . On the e brains , 141. Ustabolism , thus detabolism . Along ttabolism . Unlike tabolism . How theSeveral oncogene driven pathways converge to drive changes in metabolic programming. A key to understanding the therapeutic significance of metabolic changes in the tumor is to integrate information obtained through omics profiling via genomics, epigenetics, and transcriptomics as well as metabolomics of different stages of tumor, so as to decipher critical enzymatic players at a systems level for possible therapeutic targets.Vascular abnormalities in GBM can result in disruption of the blood-brain barrier (BBB). BBB is composed of astrocytes, endothelial cells, and pericytes that tightly regulate the transfer of molecules between the blood and the brain. Intact BBB ensures that the brain is immune-privilege . Disruptαvβ3 [β, stromal-derived factor-1 (SDF-1), and NO, maintain and promote GSCs [β plays dual role in the tumor microenvironment. On the one hand, TGF-β released from TAMs induces matrix-metalloproteinase-2 (MMP-2) and MMP-9 expression from GBM to augment GC invasion [β released from GSCs actively suppresses M1 macrophages, inhibits phagocytosis, and induces polarization of microglial and macrophages into the immunosuppressive M2 phenotype and thus enhances the capacity of TAMs to inhibit T cell proliferation, thereby promoting tumor progression [In the tumor microenvironment, tumor associated macrophages (TAMs) are commonly found in the vicinity of GSCs and correlate with the density of GSCs perhaps owing to the higher level of chemoattractants such as VEGF , 154. Thαvβ3 . Conversote GSCs –164. TGFinvasion , 165–167gression .γ (IFN-γ) production, suggesting that soluble factors secreted by GSCs exerted an immunosuppressive phenotype in MDSCs [Regs [MDSCs are a heterogeneous population of immature myeloid progenitors that mediate immune suppression and support glioma growth, invasion, vascularization, and expansion of regulatory T cells via various molecules. It is believed that MDSCs interact with gliomas and GSCs ; howeverin MDSCs . Using ain MDSCs . Arginasin MDSCs . The groCs [Regs . SimilarCs [Regs . TogetheIt is interesting to note that each molecular subtype has different frequency and types of immune infiltrates. Higher frequency of TAMs was detected in mesenchymal subtypes in comparison to other GBM subtypes . In factα (TNF-α), which in turn activates transcription factor nuclear factor-κB (NF-κB) to drive mesenchymal transition [β (CEBP-β), CD44 and NF-κB activation portends poor survival in GBM patients [Triggering PMT in tumor cells in the perivascular niche not only induces stem cells phenotypes but also maintains the stemness of cancer cells. One of the molecules that is crucial in PMT is osteopontin, which is secreted by immune cells under inflammatory conditions and promotes GSCs phenotype by activating CD44 . Higher ansition . Anotheransition . Silenciansition . Togethepatients . Importapatients , 35, 176It remains unclear what factors are responsible for mediating the interaction between GSCs, ECs, and TAMs. Given the complexity of the glioma inflammatory niche, elucidating the molecular mechanism involved in various interactions is critical to address several key questions: do TAMs acquire dissimilar function when interacting with different microenvironment generated by the various molecular subclasses? Does combining TAMs targeted therapy with standard treatment regimen give better treatment response than GSCs-targeted therapy, irrespective of the molecular subclasses? More importantly, is subtype-specific therapy essential for personalized medicine? These are some of the questions that remain to be addressed; TAMs remain a promising target for the design of therapeutic intervention.The inherent plasticity of GSCs thus suggests that its response to environmental cue is an adaptive response. Given the correlation of GSCs with tumor recurrence and response to treatment, further understating of GSCs biology and its surrounding niche may provide a framework to identify more effective treatment interventions .Targeting the tumor microenvironment represents a promising approach to prevent tumor progression. However, several key questions remain unanswered. Although different molecular subtypes exhibit varying degree of hypoxia and immune cell infiltration, it is still not clear whether there are differences in the composition of the distinct tumor niches. Patient stratification for molecular therapies using tissues obtained from single surgical site may not present accurate information, as evidenced by the significant differences between samples obtained from different regions from the same tumor mass. Given that the tumor cells are genetically and epigenetically diverse, it is conceivable that the interaction between the tumor cells and the microenvironment is niche type specific to better accommodate the needs of the GSC. This heterogeneity in the niche will affect tumor cells response to therapies. Furthermore, the tumor microenvironment is dynamic. Changes in oxygenation, which in turn affects hypoxic conditions and metabolic states, neovascularization, and tumor invasiveness, will alter receptivity of the niches to accommodate more aggressive GBM growth. On the other hand, therapies may convert a tumor niche into a benign type or even eliminate it. For example, radiation therapy may convert the perivascular niche into hypoxic niche that promotes mesenchymal tumor growth. Conversely, short-term anti-VEGF therapy may convert a hypoxic niche into the perivascular niche that normalizes the vasculature and hence facilitates drug delivery. However, it is difficult to target specific niches for therapy; hence deciphering common shared pathways among various niches is the most efficient approach in designing therapeutic strategies. Thus, a more structured modulation of the glioma microenvironmental niche may complement the conventional treatments to achieve more effective control of the malignancy of GSC-driven GBM.