For most measurements, regression analysis showed that increasing the starch concentration beyond 5 g/kg did not contribute to further significant textural changes. Torsional shear stress and strain along with Kramer shear force increased as the concentration of starch increased . The addition of ≥10 g starch/kg protein paste resulted in darker gels and gels got darker as more starch was added (R2 = .71). Results showed no benefit to increasing starch concentration in gels beyond 5 g starch/kg protein paste.Calcium‐enhanced protein recovered from black bullhead catfish was used to develop gels containing increasing amounts of potato starch (0–20 g/kg protein paste) and the effects of starch on functional, textural, and color properties were tested. Energy required to unfold protein groups was greater with the addition of 5 g starch/kg protein paste. Gels containing starch were harder, chewier, and less springy ( Fish were rinsed under running tap water, gutted, and coarsely ground twice using a meat processor  with a coarse grinding plate attachment. The coarse fish paste was transferred to lidded stainless steel trays for storage at −20°C for 12 h to prevent thermal denaturation. Chilled coarse fish paste was twice processed through the meat grinder using a fine grinder plate. The finely ground fish were individually packaged (500 g) into freezer bags , vacuum packaged , and stored at −80°C until analyses were conducted.All equipment used to handle fish were sanitized prior to fish arrival. Freshly caught  black bullhead catfish 2) to the mixture to raise to the pH to 11.0 while homogenizing using a laboratory grade, sanitized homogenizer  . Distinct layers formed with centrifugation, with lipids on the top layer and insoluble components  on the bottom. The solubilized protein in the aqueous middle layer was filtered through a cheesecloth into a glass beaker. During homogenization, pH was reduced to the protein isoelectric point of pH 5.5 with the addition of glacial l‐lactic acid . The mixture was homogenized for an additional 10 min after reaching the target pH to allow ample time for precipitation. The solution was then centrifuged at 10,000g for 15 min at 4°C and the protein fraction that formed a pellet at the bottom of the centrifuge tubes was collected and the supernatant discarded. The recovered protein was used for paste development immediately after collection.Myofibrillar fish protein was separated from the other components of the fish paste using a method called isoelectric solubilization and precipitation (ISP) that relies on pH shifts to solubilize and then precipitate the protein  chopped protein at low speed for 1 min, 20 g salt/kg protein paste was added, and the mixture was chopped for 1 min. The initial moisture of the recovered protein was confirmed by a moisture analyzer  and adjusted to 80 g/100 g by adding chilled water (4°C). Transglutaminase (5 g/kg protein paste), 3 g/kg protein paste polyphosphates  and 0, 5, 10, 15, or 20 g/kg protein paste potato starch  was added depending on the tested formula. After the final pH of the mixture was adjusted to pH 7.0–7.2  by adding lactic acid, the mixture was chopped for 3 min at high speed under vacuum (50 kPa). Thermal denaturation was prevented by maintaining the temperature of the universal food processor at 1–4°C throughout the process. The protein paste was transferred to vacuum bags and vacuum‐packed to remove air that might interfere with the texture and color analyses . A sample of protein paste was retained for differential scanning calorimetry (DSC) analysis immediately after paste development. Using a gel presser, the remaining protein paste was pressed into stainless steel tubes  and dumbbell‐shaped stainless steel torsion tubes  that were lightly sprayed with canola oil to avoid sticking of gels upon cooking. The tubes containing the pastes were stored in a refrigerator (4°C) for 24 h to allow for gel formation.2.4−1. The analysis was replicated four times per each gel sample containing different amounts of starch and the results were computed using Infinite Software . Thermograms were drawn for each sample using the mean value of four replications. Temperature onset, temperature maximum, and enthalpy required for thermal transactions were presented as mean ± standard deviation.Thermal changes in protein pastes (10–15 μg) were assessed using DSC  during which temperature was increased from 5°C to 90°C at a rate of 10°C min2.5The steel tubes containing protein pastes were removed from refrigerated storage and cooked at 90°C for 20 min in a water bath . The cooked gels were chilled in an ice bath for 15 min and then acclimated at room temperature (20–22°C for 1 h). The gels were removed from the steel tubes, cut and analyzed for expressible water content, texture, and color properties.2.6g for 15 min . The final weight was recorded. Expressible water content was calculated using the formula:Expressible water content was measured for each formula by folding 5 g of sample in two layers of Whatman No. 1 filter paper and centrifuging at 7500The test was performed in triplicates for each formula tested and the results were given as mean ± standard deviation.2.7Texture profile analysis (TPA), Kramer shear cell test, and torsional analysis were used to determine differences in texture between gel formulations. TPA describes the hardness, springiness, cohesiveness, chewiness, and resilience of gels. TPA was measured using a texture analyzer  with a compression plate attachment (70 mm). Gels were cut to 2.54 cm long with a diameter of 1.9 cm.−1 gel sample) was measured at 127 mm min−1 crosshead speed. Shear force and shear stress were calculated by dividing the peak force by the weight of each sample, and dividing the peak force by the area of the sample , respectively.Kramer shear cell test mimics the process of cutting a food sample with a knife and is another method used to measure firmness and cohesiveness of a gel sample. A texture analyzer  with a Kramer cell attachment cut through the samples with a five‐blade (3 mm thick and 70 mm wide) attachment. Peak force  twisted the samples.Texture profile analysis, Kramer shear, and torsion analyses were conducted on each gel sample formulation at least six times. Data from TPA and Kramer shear were calculated using Texture Expert software . Data from torsional analysis were calculated using Torsion Vane software (Gel Consultants).2.8L* 93.1; a* 0.3135; b* 0.3198). After collecting the data for L* , a* , and b* , whiteness was calculated using the equation:Color was measured using a colorimeter  calibrated with a standard white plate No.21333180  were assigned to significantly different values of tested gels containing different amounts of starch. Regression analysis was performed for TPA data using JMP software .ISP and formulation trials were randomized prior to beginning of the study using JMP software . One‐way analysis of variance (ANOVA) was used at a significance level of 0.05 and the mean values for each formula tested were statistically separated using Tukey's Honestly Significant Difference test. Data were presented as mean ± standard deviation. Different letters (33.1p < .05) of energy required to unfold the molecule was seen in gels containing 5 and 10 g starch/kg protein paste. Myosin tail is relatively less thermo‐susceptible than the head. Denaturation began around 26–33°C and enthalpy was increased (p < .05) with the addition of 5 g starch/kg protein paste. Peaks III and IV show that actin and sarcoplasmic protein unfolding temperatures, both at onset and maximum temperature required for denaturation, were similar between gel formulae; however, the energy required for denaturation was much higher (p < .05) for gels containing 5 g starch/kg protein paste.Thermal changes induced in protein groups in myofibrillar protein gels containing increasing amounts of starch are presented in Figure The binding effects of starch are highly dependent on the composition of the gel constituents as well as the type of starch incorporated into the protein paste. For example, amylose and amylopectin, the major components of starch, have different effects on protein gelation. Amylopectin is the main contributor to gel network formation in protein gels and increases gel strength and viscosity, whereas amylose does not show a synergistic effect in combination with protein  due to the swelling of starch granules as expected  in gels with no starch or 10 g starch/kg protein paste. The low expressible water content associated with these gels may be attributed to the calcium–starch interaction. Calcium binds to amino acids at the carboxyl side chains  than gels without starch. The addition of 5 g starch/kg protein paste starch resulted in more resilient gels (p < .05); however, cohesiveness did not change with the addition of starch (p > .05), regardless of concentration. Increased calcium content has been linked to increased endogenous TGase activity and may be reason the “no starch gels” formed a consistent network. Calcium increased thermo‐stability of the protein gels by binding to the free carboxylic groups of aspartic and glutamic acids, thus triggering cross‐linking of proteins  of the gels containing increasing amounts of starch are shown in Figure R2 = .79 and .79, respectively) from 0 to 20 g starch/kg protein paste  of gels as indicated by b* when compared to the gels containing no starch. This is consistent with previous results where starch‐containing gels were more yellow than gels with no starch  in the starch‐containing samples (p < .05) may be a result of amylose leakage from the granule which would contribute an opaque color  when starch was added to protein gels. Textural properties  did not change significantly (p > .05) with increasing amounts of starch which may be due to the interactions and competition between protein, starch, and calcium to bind water in the paste. Gels got darker, redder, and more yellow with increasing amounts of starch. Results showed no benefit in increasing starch concentration in gels beyond 5 g starch/kg protein paste.Overall, gels were harder, chewier, firmer, and more resistant to axial deformation (None declared.


P=0.007). This association did not attenuate after further adjustment for hsCRP  or excluding participants with overt CVD , diabetes , or chronic kidney disease . In summary, shorter LTL was significantly associated with an increased arterial stiffness, independent of known risk factors. This finding may shed light on the potential role of biological aging in arterial aging in American Indians.Telomere length, a marker of biological aging, has been associated with cardiovascular disease (CVD). Increased arterial stiffness, an indicator of arterial aging, predicts adverse CVD outcomes. However, the relationship between telomere length and arterial stiffness is less well studied. Here we examined the cross-sectional association between leukocyte telomere length (LTL) and arterial stiffness in 2,165 American Indians in the Strong Heart Family Study (SHFS). LTL was measured by qPCR. Arterial stiffness was assessed by stiffness index β. The association between LTL and arterial stiffness was assessed by generalized estimating equation model, adjusting for sociodemographics , study site, metabolic factors , lifestyle , and prevalent CVD. Results showed that longer LTL was significantly associated with a decreased arterial stiffness (β=-0.070,  Telomeres are repetitive DNA sequences and their associated proteins at the end of chromosomes. They protect against chromosomal degradation and prevent chromosomes from fusion, thus play a critical role in maintaining genomic stability . TelomerArterial stiffness refers to the reduced elasticity of an artery in response to pressure changes. It increases with age  and predP < 0.05). No significant difference was detected in other listed covariates across the LTL quartiles.The current analysis included a total of 2,165 American Indian participants  with a mean age of 41 ± 17 years (aged 14 to 93). The median stiffness index β was 3.24 (interquartile range: 2.36-4.58). Prevalent CVD, diabetes, and chronic kidney disease accounted for 5%, 18%, and 6% of the study population, respectively. Table P = 0.007). Further adjusting for hsCRP did not attenuate the association between LTL and log-SI , indicating that the observed association between LTL and arterial stiffness may not be confounded by inflammation. This association remained significant after excluding participants with prevalent CVD , diabetes , or chronic kidney disease . These results indicated that the observed association between LTL and arterial stiffness may not be confounded by these chronic conditions.Table Additionally adjusting for medications on hypertension, diabetes, and hypercholesterolemia did not change the association between LTL and arterial stiffness , suggestIn a large population of American Indians, we found that leukocyte telomere length was significantly and inversely associated with arterial stiffness, independent of known risk factors. This finding provides initial evidence that biological aging measured by leukocyte telomere length may play a role in arterial aging in American Indians.klotho-deficient mice as compared to their age-matched wild-type littermates /Dd) /Dd) , where P studies .2). Waist circumference was measured at the level of the umbilicus while the participant was in a supine position. Blood pressures were measured three times by trained staff using a standard mercury sphygmomanometer after the participants had been resting for at least 5 minutes and the mean of the last two measurements was used in statistical analysis. Fasting glucose, serum creatinine, and blood lipids, including total cholesterol, triglycerides, low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C), were measured by standard laboratory methods [2 [Sociodemographics  was collected by standard questionnaires. Cigarette smoking was classified as current smoking, past smoking and never smoking. Current smoking was defined as having smoked at least 100 cigarettes in the entire life, having smoked cigarettes regularly, and smoking currently. Past smoking was defined as having smoked at least 100 cigarettes in the entire life, having smoked cigarettes regularly in the past, but not smoking currently. Never smoking was defined as never smoked or having smoked fewer than 100 cigarettes in the entire life. Alcohol consumption was classified as current drinkers, former drinkers and never drinkers as previously described . Current methods . Diabete methods . Estimat methods . Chronicthods [2 . Serum hthods [2 . MedicatP-value < 0.05 was considered statistically significant. All statistical analyses were conducted using SAS statistical software .Baseline characteristics of study participants were presented according to LTL quartiles. Arterial stiffness index β was log-transformed  to improve normality. To examine the association between LTL and arterial stiffness, we constructed multivariate generalized estimating equation (GEE) model, in which log-SI was the dependent variable and LTL was the independent variable, adjusting for sociodemographics , study site, metabolic factors , lifestyle , and prevalent CVD (y/n). GEE model was used here to account for the family relatedness among participants. To examine whether and to what extent inflammation could potentially confound the association between LTL and arterial stiffness, we further adjusted for hsCRP in the GEE model described above. As prior studies reported that arterial stiffness was elevated in patients with prevalent CVD, diabetes, and chronic kidney disease , 30, 31,As previous studies reported that some medications on hypertension,  diabetes


The impact factor of a journal is one of the most discussed issues among the new editors of low-tier journals. This is probably because the impact factor is commonly used as one of the best and popular metrics of journal quality ,2. Thus,The impact factor of a journal is calculated by dividing the number of citations received by a journal within a specific year by the citable articles published in that journal in the previous two years . The forAccordingly, editorial improvement  would show up in the next year’s publications. Let’s assume that an editor has been assigned to a present journal in year A. The editor would learn the impact factor of the year (A-1) during their first months of assignment . If the editor makes any changes in the evaluation process, these would affect the publications in the year (A+1) and later. According to the impact factor formula above, editorial improvements would only affect the impact factor of the year (A+3), which is published in the year (A+4) Journal Citation Report.The primary aim of a good scientific journal should not be to have a high impact factor. Although impact factor is an important journal metric, the main goal of a journal should be to become a high-quality journal. If the scientific quality and the visibility of the articles increase, the impact factor will rise accordingly.There are four main approaches to increase the quality of a small non-profit scientific journal:How to increase the quality of a journal?• Attract quality manuscripts• Do good editorial work• Increase visibility of the journal• Trustful cooperation with owner & publisherAccording to my editorial experience at the Balkan Medical Journal as an associate editor (2004-2008) and editor-in-chief (2012-2016); asking authors no charge for submission or publication fee, open access to current issues and archive, having a user-friendly online submission system, fast and appropriate review process and publication, and reaching a large international audience/readers via popular indexes helped the journal to attract many manuscripts. The number of annual submissions to the Balkan Medical Journal has increased more than twenty-fold in the last 10 years and got over 2000. Accordingly, the number of quality manuscripts increased as well, especially those being indexed in popular indexes such as Science Citation Index Expanded (SCIE), PubMed Central (PMC), and Medline.Good editorial work mainly depends on a compatible, responsible, and motivated editorial board. The important international editor and scientific publication organizations, such as European Association of Science Editors, Council of Science Editors, World Association of Medical Editors, International Committee of Medical Journal Editors, and Committee on Publication Ethics, have defined the rules for a good editorial work. I strongly suggest that new editors read these organizations’ documents; follow their rules; attend their meetings, courses, and workshops; become a member of these organizations; and follow their discussion groups. Additionally, the editor courses given by Pippa Smart and Ana Marusic were very useful. They are not only good teachers but also good people . As a suIn addition to the major scientific literature search engines  Google is one of the most popular search engines used by academics. A journal’s visibility can be further increased by providing open access to all articles in HTML, PDF and XML format published at the journal’s website; indexing in PMC, and other popular indexes; informative e-mails sent to readers after the publication of new issues; news about the important articles; and publishing good quality review articles.The improvements made in the editorial process of the Balkan Medical Journal in recent years have increased the journal’s visibility and quality. The principal improvements include a strict review process; standardized statistical and ethical evaluation of all manuscripts before acceptance; professional English editing freely provided to all accepted papers; user friendly online submission system; publication of the journal’s archive at its web page; its open access nature; the educational activities of the journal; and indexing in PMC, resulting in easy and free access to full texts of published articles. However, the success of the journal could not be possible without the efforts of the volunteering professors that make up the editorial board and also the continuing economic support from the Trakya University.Consequently, the impact factor of the journal has increased more than ten-fold and is currently greater than 1.000 . Since AOne of my latest evaluations in 2016 revealed that the most citable articles in the Balkan Medical Journal were invited review articles and original articles followed by editorials. Case reports and other articles were cited less. Also, 81% of submissions were from Turkey, with the remaining 19% from 43 different countries. Thus, the international submissions rate should be increased. However, most of the citations to the journal’s articles were made by international authors.There are also unethical ways to increase the impact factor of a journal ,9,10, anIn conclusion, impact factors can be manipulated and have several limitations. The primary aim of a scientific journal should not be having a high impact factor but to become a high-quality journal through appropriate editorial processes and publishing high-quality, ethical articles. If the scientific quality of the articles published in a journal, as well as the visibility of the journal, are increased, the impact factor will rise.


Vibrio cholerae O1 and occasionally O139. A growing body of evidence indicates that immune responses targeting the O-specific polysaccharide (OSP) of V. cholerae are involved in mediating protection against cholera. We therefore assessed whether antibody responses against OSP occur after vaccination with live attenuated oral cholera vaccine CVD 103-HgR, and whether such responses correlate with protection against cholera.Cholera is an acute voluminous dehydrating diarrheal disease caused by toxigenic strains of 8 colony-forming units (CFU) of oral cholera vaccine CVD 103-HgR and then orally challenged with approximately 1 × 105 CFU of wild-type V. cholerae O1 El Tor Inaba strain N16961, either 10 or 90 days post-vaccination.We assessed adult North American volunteers (n = 46) who were vaccinated with 5 × 10P = 0.002; r = −0.36, P = 0.01; respectively), and none of 27 vaccinees who developed a ≥1.5 fold increase in any antibody isotype targeting OSP on day 10 following vaccination compared to baseline developed moderate or severe cholera following experimental challenge, while 5 of 19 who did not develop such anti-OSP responses did (P = 0.01).Vaccination was associated with induction of significant serum IgM and IgA anti-OSP and vibriocidal antibody responses within 10 days of vaccination. There was significant correlation between anti-OSP and vibriocidal antibody responses. IgM and IgA anti-OSP responses on day 10 following vaccination were associated with lower post-challenge stool volume  of V. cholerae lipopolysaccharide (LPS). Despite this, no previous work has directly assessed correlation of OSP-immune responses and protection against cholera. In this study, we assessed adult North American volunteer’s antibody responses targeting OSP after vaccination with live attenuated oral cholera vaccine CVD 103-HgR, and we assessed correlation of protection against cholera with such antibody responses. Oral vaccination was associated with the induction of significant IgM and IgA responses against OSP, and these responses correlated with vibriocidal responses. There was significant negative correlation of OSP responses and post-challenge stool volume, and none of the volunteers who developed an anti-OSP antibody responses of any isotype of ≥1.5 fold developed moderate or severe cholera following experimental challenge. In summary, vaccination with live attenuated oral cholera vaccine CVD 103-HgR induces antibodies that target V. cholerae OSP, and these responses highly correlate with protection against cholera.Cholera is a severe watery diarrheal disease, caused by pathogenic strains of  Vibrio cholerae can be characterized into over 200 serogroups [V. cholerae serogroups O1 and O139 are the causes of epidemic cholera, a severe dehydrating illness of humans [V. cholerae O1 and O139 are noninvasive intestinal pathogens that express cholera toxin within the intestine of infected humans. Cholera toxin (CT) is an ADP-ribosylating protein that binds to intestinal epithelial cells, resulting in increased intracellular cAMP, and subsequent chloride, sodium, and water excretion by intestinal epithelial cells, resulting in the secretory diarrhea characteristic of cholera [V. cholerae O1 are Ogawa and Inaba and serospecificity is mediated by the O-specific polysaccharide (OSP) component of lipopolysaccharide (LPS) [V. cholerae OSP, suggesting that OSP is the prime antigenic target assessed by the vibriocidal assay [rogroups . V. chol cholera . The mec cholera –7. Protede (LPS) , 8. Currde (LPS) . The vibal assay .V. cholerae O1 classical Inaba strain 569B [V. cholerae O1 organisms [V. cholerae O1 OSP occur after vaccination with CVD 103-HgR, and whether such responses to vaccination correlate with protection against cholera following subsequent experimental challenge with wild type V. cholerae O1 organisms.A live attenuated oral cholera vaccine, CVD 103-HgR  was recently FDA-approved in the United States. CVD 103-HgR is an engineered attenuated derivative of rganisms . We werehttp://clinicaltrials.gov/show/NCT01895855) that was approved by the Institutional Review Boards of the University of Maryland, Baltimore; Cincinnati Children’s Hospital Medical Center, Ohio; and University of Vermont Burlington, Vermont. Written informed consent was obtained from participating healthy adults 18–45 years of age.This study involved analysis of anonymized samples from a previously reported clinical trial; this current use was approved by the Partners-Massachusetts General Hospital Institutional Review Board, Boston, Massachusetts. The serum samples were collected in a previously reported clinical trial (8 colony-forming units (CFU) of CVD 103-HgR, and then orally challenged on either day 10 (n = 26) or day 90 (n = 20) with 1 × 105 CFU of wild type V. cholerae O1 El Tor Inaba N16961, as previously described [The clinical study during which samples were collected has been previously described . We limiFor analyses using diarrhea following challenge as an efficacy endpoint, diarrhea was defined as the passage of ≥2 loose stools (grade 3–5) over a 48-hour period ≥200 mL on volume or a single loose stool ≥300 mL, as previously described . ModeratV. cholerae OSP purified from PIC018  and PIC158 (Ogawa El Tor), as previously described [Stored serum specimens were tested to measure IgM, IgA and IgG immune responses against escribed , 13, 14.2O2 (Sigma), and determined the optical density at 405 nm with a Vmax microplate kinetic reader . Plates were read for 5 min at 30s intervals, and the maximum slope for an optical density change of 0.2U was reported as millioptical density units per minute (mOD/min). We normalized ELISA units (EU) by calculating the ratio of the optical density of the test sample to that of a standard of convalescent-phase pooled sera (prepared from previously infected cholera patients) included on each plate.We measured OSP-specific IgM, IgA and IgG responses in stored serum using standard enzyme-linked immunosorbent assay (ELISA) protocols, as described previously , 16. In P values were two-tailed, with a value of ≤0.05 considered the threshold for statistical significance. We performed analyses using GraphPad Prism, version 5.01 .We analyzed differences in immune responses between groups using Mann-Whitney U tests. We used Fisher’s exact tests to assess differences by responder frequency. We used Spearman’s test to assess correlations of anti-OSP and vibriocidal antibody responses with cumulative diarrheal volume, as well as to assess correlations between immune responses. All Characteristics of study participants are described in V. cholerae O1 Inaba OSP within 7 days of oral vaccination with CVD 103-HgR, and these values remained elevated until at least day 28 following vaccination. The peak effect was seen at 10 days after vaccination. In the absence of wild type experimental challenge, anti-OSP Inaba IgM mean responses decreased to baseline by day 90 following vaccination. Elevated IgA anti-OSP Inaba mean increases were also present within 10 days following vaccination, and decreased to baseline by day 28. Anti-Ogawa IgM and IgA mean antibody increases were also induced following vaccination with CVD 103-HgR, although these responses were generally of lower magnitude and shorter duration than responses targeting Inaba OSP, consistent with the fact that CVD 103-HgR is an Inaba-based vaccine. Mean increases in anti-OSP IgG were not detected following vaccination.To assess anti-OSP responses following vaccination alone, we analyzed anti-OSP antibody responses in blood samples from both day 10 and day 90 challenge cohorts up to their respective days of challenge . We founWe found no significant boosting of IgM or IgA anti-OSP mean responses following challenge, when analyzing immune responses in the cohort of volunteers vaccinated with CVD 103-HgR challenged 10 days after vaccination . In compWe did not detect a difference in mean antibody responses targeting OSP following vaccination when considering cohorts by blood group O characterization .V. cholerae , for the purposes of assessing whether anti-OSP responses correlated with protection against cholera, we combined day 10 and day 90 challenge cohorts, and assessed outcome by post-vaccination day 10 immune responses. We found that IgM anti-OSP Inaba responses on day 10 following vaccination with CVD 103-HgR correlated with protection against moderate or severe diarrhea following subsequent experimental challenge  and IgA (P = 0.05)  . InteresV. cholerae O1 challenge and post-vaccination day 10 anti-Inaba OSP IgM responses  and anti-Inaba OSP IgA responses  , 5 of 22 , and 5 of 38 , who did not develop such anti-OSP responses did develop moderate or severe diarrhea. Fold increase of anti-OSP values was also associated with protection against all degrees of diarrhea  following wild type challenge. Six of 27 vaccinees who developed a ≥1.5 fold increase in any antibody isotype  targeting OSP on day 10 following vaccination compared to baseline developed mild diarrhea; none developed moderate or severe diarrhea following experimental challenge, while 11 of 19 who did not develop such anti-OSP responses did develop diarrhea  , but no P = 0.01) (P < 0.0001) (P = 0.03), and there was no correlation of IgG OSP responses and vibriocidal responses   Figs. Se 0.0001) . Serum Iesponses .V. cholerae O-specific polysaccharide (OSP), and that these responses correlate with protection against moderate or severe diarrhea following experimental challenge with wild type V. cholerae O1. Antibodies targeting OSP induced by vaccination with CVD 103-HgR were largely of the IgM and IgA isotypes. Since V. cholerae is a noninvasive mucosal pathogen of the human intestine, IgM and IgA immune responses would be predicted to be the most pertinent isotypes involved in mechanistically mediating protection against cholera, since both IgM and IgA are actively secreted into the intestinal lumen by intestinal epithelial cells [In this study, we found that recipients of live attenuated oral cholera vaccine CVD 103-HgR develop prominent immune responses targeting al cells –19.V. cholerae OSP is currently unknown, as is the duration of protection afforded by vaccination.Induction of IgA antibody is associated with oral and mucosal vaccination, especially in response to protein antigens that are antigenically processed by T cells , 21. OSPV. cholerae OSP highly correlated with vibriocidal responses in vaccine recipients. This would be expected, since we have previously shown that the vibriocidal response can largely be adsorbed away by V. cholerae OSP [V. cholerae. The vibriocidal assay assesses complement-dependent mediated bacterial cell lysis in an in vitro assay [V. cholerae OSP. Indeed, in our current analysis, we show that immune responses targeting V. cholerae OSP correlate with protection against experimental challenge with wild-type V. cholerae. This association was most clear when considering IgM responses targeting V. cholerae OSP, but also approached statistical significance (P = 0.06) when considering IgA responses targeting V. cholerae OSP. There was no association of mean IgG anti-OSP responses and protection against cholera. IgG is not actively transported into the intestinal lumen in appreciable quantities in the presence of an intact epithelium, and IgG responses would not be predicted to play a major role in mechanistically mediating protection against V. cholerae. The few vaccine recipients who did develop IgG responses targeting OSP in this study, also developed IgM and IgA anti-OSP responses.In our study, IgM and, less so, IgA antibody responses targeting erae OSP . It is mro assay , 26. Bacro assay , 28. Mucro assay –29. Our V. cholerae OSP or vibriocidal responses when vaccine recipients were experimentally challenged with wild-type V. cholerae just 10 days after vaccination. We did, however, find significant boosting of anti-V. cholerae immune responses and vibriocidal responses following experimental challenge with wild-type V. cholerae when 90 days had elapsed since vaccination. A possible explanation for this observation is that mucosal immune responses 10 days following vaccination may have been so prominent that there was minimal if any immunological and antigenic processing of V. cholerae by host immune cells; specifically, that intestinal IgM and IgA anti-OSP antibody levels may have been so prominent within 10 days of vaccination and bound with such high avidity to intra-luminal wild-type V. cholerae organisms that not only was clinical protection present, but V. cholerae organisms were also not presented to human immune cells in Peyer’s patches or regional lymph nodes at an appreciable level. However, when experimental challenge was delayed for 90 days after vaccination, we did indeed find boosting of IgM and IgA responses targeting OSP, and new development of IgG responses targeting V. cholerae OSP. These observations might suggest that mucosal immune responses were then less prominent at this 90 day mark, and that this decrease allowed additional immunological processing and handling of V. cholerae organisms. Such a scenario could result in the observed rapid boosting of the IgM and IgA anti-OSP responses following the 90 day challenge to values that were actually higher than following primary immunization. This de facto boosting also appears to have led to isotype switching of the antibody response with development of IgG isotype antibodies targeting OSP. Whether there was antibody maturation and increased affinity afforded by this boosting was not assessed in this study. These observations agree with a prior analysis performed in North American volunteers who received a previous version of CVD 103-HgR and were then challenged with wild type V. cholerae O1 either 7, 30, or 180 days after vaccination [Interestingly, we found no boosting of immune responses targeting cholera gravis [V. cholerae OSP by A/B/O blood group. As such, CVD 103-HgR appears to be equivalently immunogenic with regard both to V. cholerae OSP as well as vibriocidal responses irrelevant of this major blood group category. These data suggest that the association of blood group and severity may not be directly due to differences in the ability to mount anti-OSP antibody responses.Although the mechanism has not been clearly elucidated, humans with blood group O are at increased risk of severe cholera, especially in its severest form: a gravis , 32. In V. cholerae Ogawa OSP, as well as Inaba OSP. V. cholerae can be classified into over 200 serogroups with V. cholerae O1 and O139 being able to cause epidemic cholera. V. cholerae O1 itself can be classified into a number of serotypes including Inaba and Ogawa, with the difference being the presence of a 2-O-methyl group in the non-reducing terminal sugar of the Ogawa OSP, which is absent from Inaba OSP [V. cholerae O1 Inaba provide more protection against Ogawa than previous Ogawa infection provides against Inaba [V. cholerae O1 serotype organism. We found that vaccination with CVD 103-HgR induced immune responses against both Inaba and Ogawa V. cholerae OSP, with responses being most prominent to the Inaba serotype. Our data suggest that CVD 103-HgR would provide protection against both Inaba and Ogawa serotype O1 organisms.We also assessed the ability of CVD 103-HgR to induce anti-OSP responses against naba OSP . Anti-Inst Inaba , 33, 34.V. cholerae OSP, nor did we assess antibody affinity or avidity. We also did not assess duration of immune responses via long-lived plasma cells or memory B cell responses targeting V. cholerae OSP. This volunteer study also did not include children, who may respond differently to vaccination with CVD 103-HgR, especially to T cell-independent antigens such as OSP, although the induction of IgM responses to OSP as occurred in adults in this study would presumably still occur. Our study is only one of correlations, and is not able to assess the possible mechanism of protection that may be afforded by antibodies targeting V. cholerae OSP. Despite these limitations, our analysis shows that CVD 103-HgR induces immune responses targeting V. cholerae OSP; these responses are likely largely mucosal in nature; mucosal immune responses appear to be very high level soon after vaccination; and boosting of anti-OSP immune responses can occur following repetitive exposure to V. cholerae. We also found that blood group did not appear to affect the ability to mount an anti-OSP immune response, and that immune responses induced by CVD 103-HgR included antibodies targeting both Inaba and Ogawa serotype V. cholerae. Most importantly, we found that anti-OSP immune responses correlated with protection against diarrhea following wild-type experimental challenge, especially moderate or severe diarrhea. These data further support the growing body of evidence that immune responses targeting V. cholerae OSP may be a critical component of providing protection against cholera. Establishing the mechanism of this protection is warranted.Our study has a number of limitations. We did not directly assess induction of mucosal immune responses targeting S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 FigV. cholerae O1 Inaba and Ogawa OSP 10 days after oral vaccination with CVD 103-HgR by O blood group status of vaccine recipient. X axis indicates the time points of samples while Y-axis denotes OSP specific antibody responses. Each single dot indicates an individual OSP antibody value, horizontal bars indicate the geometric mean (GM), and error bars indicate 95% confidence intervals. Responders were defined as having an increase in kinetic ELISA units ≥ 1.5-fold on day 10 post-vaccination compared with day 0 pre-vaccination.Serum IgM, IgA and IgG antibody responses targeting (PDF)Click here for additional data file.S2 FigV. cholerae O1 Inaba and Ogawa OSP 10 days after oral vaccination with CVD 103-HgR and subsequent development of mild (<3L), moderate (≥3L) or severe (≥5L) diarrhea versus no diarrhea following wild type V. cholerae O1 Inaba experimental infection 10 or 90 days after vaccination. X axis indicates the time points of samples while Y-axis denotes OSP specific antibody responses. Each single dot indicates an individual OSP antibody value, horizontal bars indicate the geometric mean (GM), and error bars indicate 95% confidence intervals. Responders were defined as having an increase in kinetic ELISA units ≥ 1.5-fold on day 10 post-vaccination compared with day 0 pre-vaccination. Ina indicates Inaba and Oga indicates Ogawa.Serum IgM, IgA and IgG antibody responses targeting (PDF)Click here for additional data file.S3 FigV. cholerae O1 Inaba and Ogawa OSP 10 days after oral vaccination with CVD 103-HgR and subsequent development of moderate (≥3 − <5L) or severe (≥5L) diarrhea versus no or mild (<3L) diarrhea following wild type V. cholerae O1 Inaba experimental infection 10 or 90 days after vaccination. X axis indicates the time points of samples while Y-axis denotes OSP-specific antibody responses. Each single dot indicates an individual OSP antibody value, horizontal bars indicate the geometric mean (GM), and error bars indicate 95% confidence intervals. Responders were defined as having an increase in kinetic ELISA units ≥ 1.5-fold on day 10 post-vaccination compared with day 0 pre-vaccination. Ina indicates Inaba and Oga indicates Ogawa.Serum IgM, IgA and IgG antibody responses targeting (PDF)Click here for additional data file.S4 FigV. cholerae challenge. Day 0 is pre-vaccination. In total, 46 vaccinees are included until day 10, then 20 vaccinees until day 90. Each single dot indicates an individual vibriocidal antibody titer, horizontal bars indicate the geometric mean (GM) and error bars indicate 95% confidence intervals (CI). P values represent significant differences of the mean between groups. Asterisks denote significance between responder and non-responder frequency at every time point with baseline (*** P < 0.0001). Responders were defined as having a ≥4-fold increase in vibriocidal titer compared to the baseline (day 0).Figure only contains results for samples collected prior to wild type experimental (PDF)Click here for additional data file.S5 FigEach single dot represents an individual vibriocidal antibody titer, horizontal bars represent the geometric mean (GM) and error bars represent 95% confidence intervals (CI). Responders were defined as having a ≥ 4-fold increase in vibriocidal value on day 10 post-vaccination compared with day 0 pre-vaccination.(PDF)Click here for additional data file.S6 FigV. cholerae O1 Inaba and Ogawa at different time points post-vaccination in recipients (N = 26) of oral cholera vaccine CVD 103-HgR who were then experimentally challenged with wild type V. cholerae O1 Inaba N16961 10 days after vaccination. Day 0 is pre-vaccination. Other dates are timed from vaccination. Each single dot denotes an individual vibriocidal antibody titer, horizontal bars denote the geometric mean (GM) and error bars denote 95% confidence intervals (CI). P values represent significant differences of the mean between groups. Asterisks denote significance between responder and non-responder frequency of every time point compared with baseline (*** P < 0.0001). Responders were defined as having a ≥4-fold increase in vibriocidal titer compared to the baseline (day 0).Serum vibriocidal antibody responses to (PDF)Click here for additional data file.S7 FigV. cholerae O1 Inaba and Ogawa at different time points post-vaccination in recipients (N = 20) of oral cholera vaccine CVD 103-HgR who were then experimentally challenged with wild type V. cholerae O1 Inaba N16961 90 days after vaccination. Day 0 is pre-vaccination. Other dates are timed from vaccination. Each single dot represents an individual vibriocidal antibody titer, horizontal bars represent the geometric mean (GM) and error bars represent 95% confidence intervals (CI). P values represent significant differences of the mean between groups. Asterisks denote significance between responder and non-responder frequency of every time point compared with baseline (*** P < 0.0001 and ** P < 0.01). Responders were defined as having a ≥4-fold increase in vibriocidal titer compared to the baseline (day 0).Serum vibriocidal antibody responses to (PDF)Click here for additional data file.S8 FigV. cholerae O1 Inaba and Ogawa 10 days post-vaccination and subsequent development of moderate (≥3L) or severe (≥5L) diarrhea versus no diarrhea following wild type V. cholerae O1 Inaba experimental infection 10 or 90 days after vaccination. Each single dot indicates an individual vibriocidal antibody titer, horizontal bars indicate the geometric mean (GM) and error bars indicate 95% confidence intervals (CI). Responders were defined as having ≥4-fold increase in vibriocidal value (day 10 post-vaccination compared to day 0 pre-vaccination). Asterisks denote significant difference (** P < 0.01) of responder frequency between no diarrhea versus moderate/severe diarrhea group in Fisher’s exact test.Serum vibriocidal antibody responses to (PDF)Click here for additional data file.S9 FigV. cholerae O1 Inaba and Ogawa 10 days post-vaccination and subsequent development of mild (<3L), moderate (≥3 − <5L) or severe (≥5L) diarrhea versus no diarrhea following wild type V. cholerae O1 Inaba experimental infection 10 or 90 days after vaccination. Each single dot denotes an individual vibriocidal antibody titer, horizontal bars denote the geometric mean (GM) and error bars denote 95% confidence intervals (CI). Responders were defined as having ≥4-fold increase in vibriocidal value (day 10 post-vaccination compared to day 0 pre-vaccination). Asterisks denote significant difference  of responder frequency between no diarrhea versus mild/moderate/severe diarrhea group in Fisher’s exact test.Serum vibriocidal antibody responses to (PDF)Click here for additional data file.S10 FigV. cholerae O1 Inaba and Ogawa 10 days post-vaccination and subsequent development of mild (<3 L), moderate (≥3L) or severe (≥5L) diarrhea versus no diarrhea following wild type V. cholerae O1 Inaba experimental infection 10 or 90 days after vaccination. Each single dot represents an individual vibriocidal antibody titer, horizontal bars represent the geometric mean (GM) and error bars represent 95% confidence intervals (CI). Responders were defined as having ≥4-fold increase in vibriocidal value (day 10 post-vaccination compared to day 0 pre-vaccination). Asterisks indicates significant difference (** P < 0.01) of responder frequency between no/mild diarrhea versus moderate/severe diarrhea group in Fisher’s exact test.Serum vibriocidal antibody responses to (PDF)Click here for additional data file.S11 FigDashed horizontal lines mark moderate (3L) or severe (5L) diarrheal and dashed vertical line denotes 4-fold vibriocidal change value (day 10 post vaccination compared to day 0 pre-vaccination).(PDF)Click here for additional data file.S12 FigDashed horizontal lines mark moderate (3L) or severe (5L) diarrheal and dashed vertical line denotes 4-fold vibriocidal change value (day 10 post vaccination compared to day 0 pre-vaccination).(PDF)Click here for additional data file.S13 Fig(PDF)Click here for additional data file.


We send our condolences to her friends, family and colleagues.All of us at Biology Open were very sad to learn of the death of Patricia Keely, one of our longest-serving Editors. Patricia joined the team upon the launch of The Company of Biologists' newest journal in 2011, demonstrating her strong support for not-for-profit scientific publishing by taking a chance on a fledgling journal that had no previous track record. She brought her expertise to bear on many articles over the years, dedicating a considerable amount of time and effort and exercising her judgement with great wisdom. Although her illness meant that she was unable to attend our most recent Editor meeting, I was in contact with her during her treatment and she remained remarkably upbeat and enthusiastic about potential new treatments, despite her deep understanding of how serious the situation was and how poor her prognosis. Reading the many tributes to her has only impressed upon us the considerable legacy she leaves (


The formation and isomerisation of nido-[C2B10H12]2– species is investigated through DFT calculations, which reveal novel basket and inverted nido intermediates and unusual inverconversion pathways, including basket collapse and pivoting triangles and diamonds. nido-[C2B10]2– species are key intermediates in the polyhedral expansion from 12- to 13-vertex carboranes and metallacarboranes, and the isomer adopted by these nido intermediates dictates the isomeric form of the 13-vertex product. Upon reduction and metallation of para-carborane up to five MC2B10 metallacarboranes can be produced , the structures of which imply the intermediacy of 1,7-, 3,7-, 4,7-, 7,9- and 7,10-isomers of the nido-[C2B10]2– species. In this paper we use density functional theory (DFT) calculations to characterise the reduction of closo-C2B10H12 carboranes and the subsequent isomerisations of the nido-[C2B10H12]2– dianions. Upon reduction para-carborane initially opens to 2–  and 2–  and isomerisation pathways connecting 1,7 to 7,9, 4,7 to 7,10 and 1,7 to 3,7 have been characterised. For ortho- and meta-carborane the experimental reduction produces 7,9 in both cases and computed pathways for both processes are also defined; with ortho-carborane rearrangement occurs via7,8, whereas with meta-carborane 7,9 is formed directly. The 7,9 isomer is the global minimum nido-structure. The characterisation of these isomerisation processes uncovers intermediates that adopt new structural motifs that we term basket and inverted nido. Basket intermediates feature a two-vertex basket handle bridging the remaining 10 vertices; inverted nido intermediates are related to known nido species, in that they have 5- and 6-membered belts, but where the latter, rather than the former, is capped, leaving a 5-membered open face. These new intermediates exhibit similar stability to the nido species, which is attributed to their relation to the 13-vertex docosahedron through the removal of 5-connected vertices. Isomerisation pathways starting from nido geometries are most often initiated by destabilisation of the cluster through a DSD process causing the 3-connected C7 vertex to move into a 4-connected site and a neighbouring B vertex to become 3-connected. The ensuing rearrangement of the cluster involves processes such as the pivoting of a 4-vertex diamond about its long diagonal, the pivoting of two 3-vertex triangles about a shared vertex and DSD processes. These processes are all ultimately driven by the preference for carbon to occupy low-connected vertices on the open 6-membered face of the resulting nido species.Dianionic  A key synthetic route that relies upon this is the polyhedral expansion method, whereby 2-electron reduction of a closo precursor  results in the formation of a nido fragment, which can then be capitated with a {BR} or {M} fragment. Application of this method led to both the first 13-vertex metallacarborane2 and the first 13-vertex carborane.3 In 2007 we showed that the polyhedral expansion of a single carborane precursor, , with {M} = {Ru(p-cymene)} led to the formation of five isomeric supraicosahedral metallacarboranes of the form RuC2B10  suggested the first nido-fragments formed upon reduction were 2–  and 2– . At the time, the further isomerisation of these species to the remaining nido species was not considered, however computing these directly provided relative energies, H  of +29.5, +24.0, +22.0 and +1.4 kcal mol–1 for 1,7, 3,7, 4,7 and 7,10 respectively, all relative to 7,9 at 0.0 kcal mol–1. In contrast, related experimental studies adopting ortho- or meta-carborane precursors show that 7,9 is the only nido species formed.7As predicted by Wade's Rules, RuC2B10 .4 This icloso (hetero)boranes has been the subject of continued investigation. Starting from ortho-carborane, the conversion to meta-carborane at 450 °C and then to para-carborane at 700 °C has been known for over 50 years and indeed was the route to their first syntheses.10 The processes involved in their rearrangement have been studied for many years, both theoretically16 and experimentally through labelling studies.19 In 1966, Lipscomb13 introduced the diamond-square-diamond (DSD) mechanism (16 suggested the triangular face rotation (TFR) mechanism, which can also be described as three concerted DSD processes  level. Multi-step DSD-derived processes were found to dominate, however two TFR-type pathways were also located. High symmetry processes involving multiple simultaneous DSDs  were discounted due to their unfeasibly high activation energies. Later studies also demonstrate an energetic preference for low symmetry processes.14 Wales also ruled out the closo-nido-closo rearrangement pathway, which requires opening of the cage to a high energy pseudo-nido intermediate.The thermal rearrangement of 12-vertex echanism , while irocesses . The DSD11 showed, through density functional theory (DFT) calculations, that a single step TFR process was favoured in the ortho- to meta-carborane isomerisation, while a two-step DSD pathway was preferred for isomerisation from meta- to para-carborane. Brown and McKee had discounted a two-step pathway from ortho- to meta-carborane, due to a high initial barrier between ortho-carborane and the intermediate involved. We later showed that the equivalent intermediate was formed upon the oxidation of 7,9 and characterised a lower energy process to form ortho-carborane, in agreement with experiment.12 Most recently, Sugden and co-workers14 investigated both of these isomerisation pathways through ab initio molecular dynamics (DFT-MD) adopting the PBE functional.More recently, Brown and McKeeet al.20 computed 7,9 directly, through HF calculations, for comparison with plausible protonated [nido-C2B10H13]– structures. Later, Hermansson and co-workers21 showed in a study of the electron affinities of carboranes  that sequential addition of two electrons to meta-carborane resulted in 7,9, while geometries produced from reduction of ortho- and para-carborane showed only minor distortions and did not resemble nido fragments. More recently, 12-vertex nido carboranes and (bis)carboranes have featured in our investigations of the aforementioned oxidation of 7,9 to ortho-carborane,12 the room-temperature C–C activation of an arene at a 13-vertex metallacarborane,22 co-production of isomeric 13-vertex cobaltacarboranes from polyhedral expansion of a tethered carborane precursor23 and in the rational design of derivatives of 7,9 stabilised towards aerial oxidation.24In stark contrast, few computational studies have involved reduced 12-vertex carboranes, despite the isomeric form of the reduced species ultimately dictating the isomer of the supraicosahedral product. McKee para-carborane and formation of 1,7 and 4,7, revealing pathways interconnecting all five nido fragments inferred experimentally . In order to model the reduction of para-carborane, first the neutral geometry was optimised, then two electrons were added and the system was re-optimised. This resulted in an initial reduced minimum, Int(A)  features 4-membered (B3–B8–B4–C1) and 5-membered (B3–C1–B5–B6–B2) open faces and is related to the nido geometry, 1,7, by a DSD process in the C1–B3–B8–B4 face. This was characterised by decreasing the C1···B8 distance, giving TS . Through this process, C1, which is 3-connected in Int(A), becomes 2-connected in the transition state and returns to 3-connected in 1,7, giving a barrier of 19.0 kcal mol–1. Alternatively, movement of B3 across the 5-membered face towards B6 in Int(A) gives Int, through a lower barrier of 4.4 kcal mol–1. Int is topologically equivalent to Int(A) and features 4-membered C1–B3–B2–B6 and 5-membered C1–B3–B7–B8–B4 faces. We refer to such a topology as a basket intermediate (discussed in detail later) where here C1 and B3 vertices bridge the remaining 10 vertices in a way that resembles a basket handle. From Int decreasing the B3···B6 distance causes the 4-membered face to close viaTS2  in what is effectively a barrierless process. Thereafter, a DSD process occurs in the C1–B3–B6–B5 diamond, breaking the C1–B6 connection and forming the B3–B5 connection, allowing C1 to become 3-connected and furnishing the nido geometry, 4,7. These energy profiles suggest that processes decreasing the number of connections to carbon vertices and increasing the number of connections to boron vertices are favoured. The lower barrier to formation of 4,7 than of 1,7 also suggests that processes involving movement of boron are easier than those involving movement of carbon. This is supported by electronegativity arguments; the radial orbitals of the carbon vertices, being more contracted than those of boron, do not allow stabilisation of higher-connected sites or longer connections. The movement of vertices from the initial para-carborane geometry to give Int(A) can be rationalised by visualisation of the LUMO of para-carborane .DFT calculations were performed at the BP86/6-31G** level using Gaussian 03 and we report zero-point corrected electronic energies, arborane . This fe2.1,7 and 4,7 were considered as starting points towards formation of the remaining nido species, 3,7, 7,9 and 7,10. Initially, we considered the possibility of TFR processes linking nido isomers  were unsuccessful from either 1,7 or 4,7. Several atoms had to be fixed in position in order to prevent non-targetted rearrangement of the cluster. This suggested that the TFR process, though relevant to closo carborane isomerisation, was higher in energy than other processes available to the more flexible nido species. It was noted that the lowest energy vibrational mode of all nido species computed involves rotation of the 6-membered face above the 5-membered 2–3–4–5–6 belt, where the largest displacement is seen in the 7-position. Taking the lead from the mode-following approach of Wales,15 we used the transition state (TS) optimisation option in Gaussian 03,25 which follows the lowest energy vibrational mode to a saddle-point on the PES, thus allowing low energy transition states to be sought a priori, direct from selected minima. The present C2B10H12 clusters  lend themselves to mode-following since the lowest energy vibrational mode always involves displacement of cluster vertices and so is productive towards cluster rearrangement. Mode-following was therefore always attempted in the first instance for any transition state search . From 1,7 and 4,7 this revealed surprising and contrasting isomerisation processes, which see rearrangement of 1,7 to 7,9 in a single step and 4,7 to 7,10 in a multi-step process.The geometries of  isomers ; in 1,7,2.11,7 provided a transition state, TS  is formed through a DSD process in the C7–B12–B6–B2 diamond and exhibits a 3-connected boron vertex, B12, which protrudes from the 6-membered open face by 0.81 Å w.r.t. the C7–B8–B9–B10–B11 least-squares mean plane . Visualisation of the single imaginary vibrational mode of TS sees movement of B12 relative to the open face, where the B12···B9 distance is 3.01 Å at the transition state geometry. Characterisation of TSvia IRC calculations revealed a remarkable and unanticipated process in which the cluster inverts in one step from 1,7, which has a CB5 6-membered open face, to 7,9, with a C2B4 open face. 1,7 to allow vertex movement to be followed; two snap-shots, SS1 and SS2, are shown to further aid in visualising the process and a movie is provided in the ESI.TS involves pivoting about the long C7···B11 diagonal of the C7–B6–B11–B12 diamond. SS1 illustrates the midpoint of this process. The pivoting continues, opening the C1–B2–C7–B6–B11–B5 face and closing the C7–B8–B9–B10–B11–B12 face of the starting structure. At SS2 the original open face has closed to give a geometry resembling a mirror image of TS, but with both C-vertices now on the open face. Finally, a barrierless DSD process in the C7–B6–B12–B8 diamond moves C7 into the 3-connected site to give 7,9.Mode-following from 2.24,7 to 7,10 was found to be a multistep process   again gives a low isomerisation barrier of 11.0 kcal mol–1. TS1 is related to 4,7 by a single DSD process in the C7–B8–B3–B2 diamond, resulting in a geometry topologically equivalent to TS, exhibiting a 3-connected B8 vertex protruding from the open face by 0.56 Å. Further distortion of the cluster has also occurred, where the B3–C4 connection has lengthened from 1.67 Å to 2.01 Å, producing an open B1–B3–B9–C4 diamond. The imaginary mode associated with TS1 displays rotation of the B3–B8–B9 and C4–B9–B10 triangles, giving a hinging motion about the shared B9 vertex. Towards Int1 , this motion further elongates the B3–C4 connection to 2.57 Å, shortens the B9–B1 distance to 2.46 Å and sees formation of an incipient B8–B10 connection of 2.04 Å, giving a 5-membered C7–B8–B10–B11–B12 face. Mode-following from Int1 gives TS2  in which B8 has moved above the open face and the B8–B3 and B8–B9 connections are lengthened, but not broken, from 1.80 Å and 1.68 Å respectively in Int1 to 1.90 Å and 1.74 Å respectively in TS2. Similarly, the long B1···B3 distance is lengthened further from 1.99 Å in Int1 to 2.11 Å in the transition state. From TS2 to Int2 the movement of B8 across the larger face is continued, leading to formation of connections B8–B12 and B8–B11 in Int2 while breaking the B3–B8 and B9–B8 connections. C2 symmetric Int2  is a further example of a basket intermediate with two equivalent 5-membered CB4 faces, where the B3–B9 edge comprises the basket handle and the C vertices adopt bridgehead positions, 4 and 7. It is also relatively thermodynamically stable, having a lower energy than the starting nido species, 4,7. However, it is kinetically unstable due to the low barrier to its subsequent rearrangement. Rearrangement of Int2 to give a nido fragment is equivalent to that found for Int(A). Mode-following gives TS3 , in which the B3–B2 connection is lengthened from 1.84 Å to 2.25 Å at the transition state and the B9–B8 connection shortens to 2.02 Å, giving a structure that closely resembles TS and TS2. In visualising the IRC calculations from TS3 B3 is initially seen to remain 3-connected, however a DSD process in the B3–B8–B12–C7 diamond gives a 3-connected C7. Overall this transformation sees 4,7 rearrange to 7,10 where the former C4 vertex now adopts the C10 position and the B3–B8–B9 triangle is effectively transferred across the open face of the cluster in order for this to be accomplished. The process is exothermic with ΔH = –20.6 kcal mol–1.In contrast, the isomerisation of  process  which wa2.3nido fragment, 3,7, lies between 1,7 and 4,7 in energy  we focussed on the reverse process, from 3,7 to 1,7. In this case, however, the lowest mode led to a facile, degenerate, two-step DSD process that links equivalent forms of 3,7 , that was comparable in energy to 1,7 and 3,7. Int2 is topologically equivalent to Int1. Therefore a process analogous to that linking 4,7 to Int1, where two triangle pivot about a shared vertex, was sought through STQN calculations. This resulted in location of TS3, through which the C1–B3–B2 and B2–B6–C7 triangles  to 1,7. Int2 features one 5-membered face including C1. By analogy now with TS, it was envisaged that a process pivoting the B12–B5–B10–B11 diamond about its long B12···B10 diagonal would provide the 1,7 geometry. Such a process, located by decreasing the C1···B5 distance with the B5–B11 distance frozen, gave TS2 . A snap-shot geometry, SS is given in 5–B10–B11–B12 diamond pivots about the long B10···B12 diagonal. This gives (in the reverse direction) Int1 . Mode-following from Int1 gave TS1 directly, emphasising the ability of this technique to give the lowest energy transition state associated with a minimum, i.e. here locating the lower TS1 at ΔH = +1.6 kcal mol–1 rather than the slightly higher TS2 at ΔH = +2.1 kcal mol–1 w.r.t. Int1. The process linking 1,7 to Int1 involves a double-DSD step, where the first DSD (in the C7–B2–B6–B12 diamond) has already occurred at TS1 and the second furnishes Int1. The overall isomerisation process forming 3,7 from 1,7 has a barrier of 17.6 kcal mol–1, corresponding to TS2, and is exothermic by ΔH = –5.5 kcal mol.The final remaining target 2.4nido species targeted. However, for completeness, it is desirable to connect all species to 7,9, the global minimum. 1,7 connects to 7,9 directly, whereas 3,7 connects to 7,9via1,7 and 4,7 connects to 7,9via7,10 (see Discussion section). The remaining isomerisation, from 7,10 to 7,9, is discussed below and shown in 7,10, mode-following results in a degenerate process where the 3-connected C7 becomes 4-connected and B8 and B9  become 3-connected. This is similar to the initial movement of vertices seen for the formation of 3,7 from 1,7, however here it does not lead to an isomerisation process. A linear transit was therefore adopted, increasing the C7–B2 distance, to cause a DSD process in the C7–B8–B2–B12 diamond. This gave TS1  which leads to a basket intermediate, Int , where C7–B11 forms the basket handle. Int is related to 7,9 through a DSD process in the C7–B11–B6–B12 diamond, which was characterised through location of TS2 . The isomerisation from 7,10 to 7,9 therefore involves two DSD processes, through a basket intermediate, with an overall barrier of 26.3 kcal mol–1.At this stage isomerisation processes have been characterised that rationalise the formation of all five 3.ortho- and meta-carborane each lead to 7,9.7 Hermansson et al.21 showed in a study of carborane electron affinities that, at the HF level of theory, sequential addition of two electrons to meta-carborane produced a 7,9nido geometry, whereas ortho-carborane was only slightly distorted. Through the present DFT calculations, we have now characterised the rearrangement processes undergone by both of these species following 2e reduction, ultimately giving 7,9, which can be rationalised by relating them to the processes seen above. By analogy to the computational treatment of the reduction of para-carborane, 2e were added to the optimised geometry of ortho-carborane and the structure re-optimised as a dianion. This gave Int(B)  is another example of a basket intermediate, where here the geometry is C2 symmetric and the C1 and C2 vertices form the basket handle. The C1–C2 connection in Int(B) is shortened to 1.52 Å w.r.t. 1.64 Å in ortho-carborane, indicative of single bond character. From Int(B), a basket collapse process was characterised through TS  and breaking of the C1–C2 connection to give 7,9 in a single step. However, this process exhibits a high overall barrier of 34.5 kcal mol–1, consistent with it being dominated by the breaking of a C–C connection with single bond character. An alternative basket collapse process was characterised through mode-following   was located, and provided a basket intermediate, Int1. From here, C1–C2 bond breaking proceeds through TS2 at over 10 kcal mol–1 lower than TS , giving a barrier of 29.9 kcal mol–1 from 7,8. This process leads to Int2 at H = +2.8 kcal mol–1, which is identical to Int. Therefore the basket collapse process described above to give 7,9 is repeated in this pathway; here forming the C1–B7 connection through a DSD process in the C1–B11–B7–B6 diamond of the Int2basket. The nido-7,8 isomer is implicated experimentally in the synthesis if 4,1,2-MC2B10 species from ortho-carborane, where exopolyhedral hydrocarbyl or silyl tethers connecting the C-vertices ensure the C positions remain adjacent.27 With the removable silyl tether metallation with M = {CoCp} leads to the concurrent formation of the expected 4,1,2-MC2B10 species, but also the 4,1,6-MC2B10 isomer, indicating that isomerisation of the nido-7,8 fragment to the 7,9 form is possible.23 The initial strengthening of the C1–C2 connection in Int(B)cf. ortho-carborane contrasts with the 2e addition to 1,2-Ph2-1,2-closo-C2B10H10 , where the C1–C2 connection breaks due to a σ-C–C antibonding component in the LUMO orbital of the neutral species.24 In the LUMO of ortho-carborane  and along the B4–B5 (and B7–B11) connections. Upon visualising the optimisation an initial lengthening of connections with antibonding interactions was noted (see ESIo-B)SS shows the C1–C2 connection initially lengthens from 1.64 Å in ortho-carborane to ca. 1.7 Å. In addition, the distances from the C vertices to B3 and B6 and the B4–B5 and B7–B11 connections are also lengthened at o-B)SS(. As the optimisation continues the C2v symmetry is reduced to C2 by reformation of the C2–B3 (1.55 Å), C1–B5 (1.71 Å) and C1–C2 (1.52 Å) connections in Int(B).The experimental reductions of arborane  a π-antimeta-carborane led directly to the location of 7,9 SS1SS2SS2   and indecloso-C2B10 carboranes with metal fragments produces a range of MC2B10 species which imply the intermediacy of 1,7-, 3,7-, 4,7-, 7,9- and 7,10-isomers of the nido-[C2B10]2– species. Here we have used DFT calculations to characterise the isomerisation pathways that link these various nido isomers. Our study has revealed several unusual new intermediates and their unforeseen rearrangement pathways which are categorised and rationalised below.Polyhedral expansion of para-carborane, 1,7 and 4,7 are formed as the initial nido species  and therefore 3,7 likely isomerises to 7,9via 1,7. Similarly, a direct pathway from 4,7 to 7,9 was not found and so formation of 7,9 from 4,7 is thought to proceed through 7,10 . An additional nido species, 7,8, was found to be formed following 2e addition to ortho-carborane and isomerises to 7,9 through a barrier of 29.9 kcal mol–1. 2e reduction of meta-carborane leads directly to 7,9. Degenerate pathways, where the start and end points of a rearrangement are the same nido species, were characterised for 7,9 , 7,10 , two examples for 3,7  and 7,8 .Following the addition of two electrons to the optimised geometry of  species . Thereafbasket intermediates, which are often energetically comparable to conventional nido fragments, were located along several of the characterised pathways. In a basket intermediate, two vertices form a basket handle bridging the remaining 10 vertices, with examples located in this study including Int(A), Int, Int, Int1 and Int2  and Int2 and Int(B) (with C2 symmetry with the CH vertices in the bridgehead positions and basket handle positions respectively). As shown in C1-basket may be produced through removal of one of the 5-connected vertices 6–9 (red), with lengthening of the 1–4 distance to produce the requisite 4- and 5-membered faces. The C2-basket intermediates are related to the relevant C1-basket by a DSD in the 1–2–5–9 diamond and lengthening of the 3–4 distance. Two additional key intermediates, Int1 and Int2, we refer to as inverted nido geometries, due to the 6-membered belt of vertices, rather than the 5-membered belt, being capped by a single vertex. The inverted nido motif is derived from the docosahedron by removal of a 5-connected vertex . A classical nido geometry is produced through removal of a 6-connected vertex .28 In order to test the validity of this empirical observation, the idealised [B13H13]2– docosahedron was computed, the appropriate vertices removed and the structure re-optimised as [B12H12]4– fragments. The nido and inverted nido were located as minima .The structural types discussed above tend to access specific rearrangement processes. From re found : a commonido-carboranes, [C2B10H12]2–, have been characterised through DFT calculations. para-Carborane rearranges to 1,7 and 4,7 as the first nido species, where experimentally, 1,7, 3,7, 4,7, 7,9 and 7,10 are produced. Isomerisation processes have been characterised connecting 1,7 to 7,9, 4,7 to 7,10, 1,7 to 3,7 and 7,10 to 7,9, thus rationalising the experimental formation of these species and showing how they are interconnected. Reduction of ortho-carborane gives 7,8 as the first nido species which subsequently isomerises to the expected 7,9, while meta-carborane rearranges directly to 7,9. The initial movements of vertices away from closo geometries, following the addition of two electrons, is related to the LUMO of the neutral species.The rearrangements of dianionic 12-vertex a priori mode-following calculations, a series of common intermediate topologies as well as the unexpectedly complex processes by which they interconnect have been uncovered and rationalised. Basket intermediates (e.g.Int(A), Int, Int2, Int and Int(B)) are characterised by a two-vertex basket handle bridging the remaining 10 vertices; inverted nido intermediates  and Int2) exhibit a 5-membered belt and a 6-membered belt capped by the remaining vertex. The geometries of these new intermediates, like nido species themselves, are related to the 13-vertex docosahedron by removal of a single vertex. The pathways through which carborane dianions isomerise, driven by the thermodynamic preference for low-connected C vertices, are most often initiated by movement of the 3-connected C7 vertex, common to all nido species, into a 4-connected position through a DSD step, forcing a B vertex into a destabilised 3-connected site and leading to rearrangement of the cluster. Isomerisation continues through processes such as the pivoting of a 4-vertex diamond about its long diagonal. This can directly lead to a nido geometry or produce a basket or inverted nido intermediate. Basket intermediates can undergo a basket collapse process, characterised by DSD steps, giving rise to a nido geometry, while inverted nido intermediates convert to nido geometries through the pivoting of two 3-vertex triangles about a shared vertex.In the characterisation of these isomerisation processes, where possible through 30 and 6-31G** basis sets31 for B, C and H atoms. Zero-point corrected energies, H, are reported in kcal mol–1 relative to 7,9. Analytical frequency calculations were used to confirm geometries as minima  or transition states . Transition states were further characterised through IRC calculations.33 Mode-following calculations used the ‘OPT = TS’ option in Gaussian along with the GDIIS algorithm, where convergence constraints were set to ‘verytight’ in order to force the optimisation to move away from a formally minimum energy starting geometry, itself optimised with default convergence constraints. Synchronous Transit-Guided Quasi-Newton (STQN) calculations were run with the ‘QST2’ option (two intermediate geometries given as input) and the structure generated used as input in a transition state optimisation.Calculations were performed using Gaussian 03, Revision D.01 employing the BP86 functional


Three members of type-II receptor protein tyrosine phosphatases  are known as presynaptic organizers. Synaptic adhesion-like molecules (SALMs) have recently emerged as a family of postsynaptic organizers. Although all five SALM isoforms can bind to the type-IIa RPTPs, only SALM3 and SALM5 reportedly have synaptogenic activities depending on their binding. Here, we report the crystal structures of apo-SALM5, and PTPδ–SALM2 and PTPδ–SALM5 complexes. The leucine-rich repeat (LRR) domains of SALMs interact with the second immunoglobulin-like (Ig) domain of PTPδ, whereas the Ig domains of SALMs interact with both the second and third Ig domains of PTPδ. Unexpectedly, the structures exhibit the LRR-mediated 2:2 complex. Our synaptogenic co-culture assay using site-directed SALM5 mutants demonstrates that presynaptic differentiation induced by PTPδ–SALM5 requires the dimeric property of SALM5.Synapse formation is triggered by  trans-synaptic interactions. Here the authors give molecular insights into synaptic differentiation by determining the structures of the synaptic adhesion-like molecules SALM2 and SALM5 bound to the presynaptic organizer PTPδ.Synaptic organizers are cell adhesion molecules that facilitate synapse formation through  Trans-synaptic interactions between pre- and postsynaptic organizers can induce synapse formation6. Type-IIa receptor protein tyrosine phosphatases (RPTPs) and neurexins are the two major presynaptic organizers7. In mammals, the type-IIa RPTPs have three members, PTPδ, PTPσ, and LAR. These members possess a large extracellular domain (ECD) consisting of three immunoglobulin-like (Ig) domains and four to eight fibronectin type-III (Fn) domains, followed by a single transmembrane helix and a cytoplasmic domain harboring two protein tyrosine phosphatase (PTP) domains 9, netrin-G ligand-3 (NGL-3)11, neurotrophin receptor tyrosine kinase C (TrkC)12, and Slit- and Trk-like family proteins (Slitrks)14. The binding of the type-IIa RPTPs with IL-1RAcP, IL1RAPL1, and Slitrks is controlled by the two short peptide inserts derived from alternative splicing of the type-IIa RPTP genes at the sites corresponding to a loop within Ig2  and the junction between Ig2 and Ig3 , intellectual disability, or schizophreniains Fig. : the fir20. SALM family proteins have five isoforms (SALM1–SALM5). Their ECDs share the same domain organization consisting of a leucine-rich repeat (LRR) domain, an Ig domain and an Fn domain with 35% amino-acid sequence identity  family proteins have recently emerged as a family of postsynaptic organizers that bind to the type-IIa RPTPsity Fig. . In cont24. The number of excitatory synapses in the hippocampal CA1 region is obviously reduced in SALM3-null mice18. Knockdown of SALM5 reduces the number of excitatory and inhibitory synapses24. SALM5 has been reported to be associated with ASD, intellectual disability, and schizophrenia in humans28.Previous studies have shown the differences in the functional roles of SALM1–SALM5 in neuronal development. Although all isoforms can bind to the type-IIa RPTPs, only SALM3 and SALM5 are capable of inducing excitatory and inhibitory presynaptic differentiation in contacting axonstrans-synaptic adhesions mediated by SALMs and the type-IIa RPTPs in neuronal development, their underlying structural mechanisms remain elusive. In this study, we present the crystal structures of apo-SALM5 and the PTPδ–SALM2 and PTPδ–SALM5 complexes. Together with structure-based mutagenesis, in vitro binding analysis with surface-plasmon resonance (SPR) spectroscopy and synaptogenic co-culture assay, we reveal the structural basis of trans-synaptic interactions between the SALMs and type-IIa RPTPs.Despite the functional importance of We determined the crystal structures of apo-SALM5 (LRR–Ig), and PTPδ Ig1–Ig3)–SALM2 (LRR–Ig) and PTPδ (Ig1–Fn1)–SALM5 (LRR–Ig) complexes at 3.08, 3.16, and 4.18 Å resolutions, respectively  in PTPδ Ig2 was observed in either the PTPδ–SALM2 or PTPδ–SALM5 complex, suggesting that meA is completely disordered and not involved in the binding of PTPδ to SALM2 or SALM5. Consistently, our SPR analysis showed that the meA-containing and meA-lacking PTPδ isoforms  bind to SALM5 with similar affinities  resides in the junction between Ig2 and Ig3 of PTPδ , whereas Asn158 hydrogen bonds with Gln134*  is 26–96 times lower than those to other type-IIa RPTP-binding postsynaptic organizers 30. Moreover, the dimerization of SALM1–SALM3 potentially promotes the clustering of the proteins at the postsynaptic density through the interaction of their PDZ-binding motifs with PSD-95.Our structures revealed the 2:2 binding mode of PTPδ and SALMs. SALMs form a dimer, which bridges two PTPδ monomers. Although the interaction between PTPδ and SALMs is independent of the dimerization of SALMs, the dimerization of SALM5 is required for its synaptogenic activity. The dimerization of SALM5 may be necessary to reinforce the binding to the type-IIa RPTPs in neurons, because the affinity of PTPδ to SALM5  for in vitro binding studies have been unsuccessful in our hands.The preference of SALMs for the splice variants of PTPδ was previously studied by cell-surface-binding assay, which suggested that the preference is different among SALMs29. The crystal structures revealed that IL1RAPL1 and TrkC compete with proteoglycans for interaction with the type-IIa RPTPs30. Therefore, IL1RAPL1 and TrkC need to remove HSPGs from the type-IIa RPTPs to shift from the axon guidance state to the synapse formation state. In contrast, the SALM5-binding surface of PTPδ does not overlap with the proteoglycan-binding site, suggesting that SALM5 can bind to the type-IIa RPTPs regardless of its binding to HSPGs. This may be important for SALM5 to function in neurons, since the affinity of SALM5 to the type-IIa RPTPs is lower than those of other postsynaptic organizers.The Ig1 domain of the type-IIa RPTPs contains a proteoglycan-binding site. Heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans promote and inhibit axon extension, respectively, through their interactions with the type-IIa RPTPs30  and mouse SALM2 LRR–Ig (residues 32–390) are PCR amplified from a human cDNA library  and a mouse cDNA library  and cloned into the pEBMulti-Neo vector with the N-terminal Igκ signal sequence and C-terminal His6 tag. The cDNA encoding the entire ECD of human SALM5 lacking the signal sequence was cloned into pEB6-Igκ-Fc4 to yield pEB6-Igκ-SALM5-Fc. The mutants of PTPδ Ig1–Fn1 (A9B+)-His6, SALM5 LRR–Ig-His6 and SALM5 ECD-Fc were produced by PCR-based site-directed mutagenesis. All proteins were transiently expressed in Expi293F cells (Thermo Fisher Scientific). The secreted proteins in the culture media were purified by Ni-NTA resin (Qiagen). The culture media containing the secreted proteins were loaded onto Ni-NTA resin equilibrated with 20 mM Tris-Cl (pH 7.4) containing 200 mM NaCl. The resin was then washed with 20 mM Tris-Cl (pH 7.4) containing 200 mM NaCl and 20 mM imidazole. The bound proteins were eluted with 20 mM Tris-Cl (pH 7.4) containing 200 mM NaCl and 250 mM imidazole. The purified PTPδ proteins were dialyzed against 20 mM Tris-Cl (pH 7.4) containing 150 mM NaCl. The SALM proteins were further purified by a Superdex200 10/300 size-exclusion column  with 20 mM Tris-Cl (pH 7.4) containing 250 mM NaCl.The cDNAs encoding mouse PTPδ Ig1–Ig3 (residues 28–325) and PTPδ Ig1–Fn1  were cloned into the pEBMulti-Neo vector  with the N-terminal signal sequence derived from pHLsec vector2SO4 and 0.1 M ADA (pH 6.5). The crystals of apo-SALM5 and the PTPδ–SALM2 and PTPδ–SALM5 complexes were cryoprotected by supplementation of the reservoir solutions with the final concentrations of 30% ethylene glycol, 35% PEG400 and 35% xylitol, respectively, and then flash-frozen in liquid N2.For crystallization of apo-SALM5, SALM5 LRR–Ig was concentrated to 100 µM using an Amicon Ultra-4 filter (Millipore) and crystallized using the sitting drop vapor diffusion method at 20 °C by mixing 0.5 µL of the protein solution and 0.5 µL of the reservoir solution containing 10% PEG3350 and 0.2 M ammonium tartrate. For the crystallization of the PTPδ–SALM2 complex, SALM2 LRR–Ig was mixed with PTPδ Ig1–Ig3 at the final concentration of 57 µM each and co-crystallized using the sitting drop vapor diffusion method at 20 °C by mixing 0.5 µL of the complex solution and 0.5 µL of the reservoir solution containing 7% PEG20000 and 0.1 M MES (pH 6.0). For the crystallization of the PTPδ–SALM5 complex, SALM5 LRR–Ig was mixed with PTPδ Ig1–Fn1 at the final concentration of 70 µM each and crystallized using the sitting drop vapor diffusion method at 20 °C by mixing 0.5 µL of the complex solution and 0.5 µL of the reservoir solution containing 7% PEG4000, 0.1 M NaCl, 0.1 M Li35 and CCP4 program suite36. All structures were solved by the molecular replacement method using Molrep37 or Phaser38. The LRR structure of NGL-3 (PDB 3ZYO)39 was used as the search model for the structure determination of apo-SALM5. The apo-SALM5 structure and the previously determined PTPδ structure (PDB 4YFC and 4YFE)15 were used as the search models for the structure determination of the PTPδ–SALM2 and PTPδ–SALM5 complexes. Model building and autocorrection/refinement were carried out using the programs Coot40 and Phenix41, respectively. Data collection and refinement statistics are shown in Table The diffraction data set of apo-SALM5 was collected at 100 K at beamline BL-1A of Photon Factory . The diffraction data sets of the PTPδ–SALM2 and PTPδ–SALM5 complexes were collected at 100 K at beamline BL41XU of SPring-8 . The data sets were processed with HKL2000SPR measurements were carried out using Biacore T200  at 25 °C in 10 mM Hepes-Na (pH 7.5) containing 200 mM NaCl and 0.05% Tween20. SALM5 proteins were immobilized on a CM5 sensor chip by the amino-coupling method. PTPδ proteins were injected as analytes with adequate concentration series (0.1–260 μM) depending on the affinities. The sensorgrams are shown in Supplementary Figs. −1 and applied onto an ENrich SEC 650 (10 × 300 mm) column (Bio-Rad) in 20 mM Tris-HCl (pH 7.4) buffer containing 250 mM NaCl. The MALS data were collected by a DAWN HELEOS 8 + detector (Wyatt Technology) with an RF-20A UV detector (Shimadzu) and analyzed by the program ASTRA (Wyatt Technology).SALM proteins were concentrated to 0.5 g L2. The cells were plated at a density of 2.5 × 105 cells per cm2 on glass cover slips (Matsunami glass) coated with poly-L-lysine (Sigma) and mouse laminin (Life Technologies). The cells were cultured in the Neurobasal A supplemented with 5% FCS, 2% B-27 supplement, 100 U mL−1 penicillin, 100 µg mL−1 streptomycin and 0.5 mM L-glutamine for 24 h, and then in the same medium without FCS. Expression vectors for mutated forms of human SALM5-Fc were generated by PCR-based mutagenesis using pEB6-Igκ-SALM5-Fc as a template. Fc and mutated forms of SALM5-Fc in FreeStyle 293-F cell culture media were bound to Protein A-conjugated magnetic particles . Beads coupled with Fc or Fc-fusion proteins were added to cortical neurons on days in vitro 11. After 24 h, cultures were fixed for immunostaining with mouse anti-Bassoon antibody  followed by Alexa555-conjugated donkey anti-mouse IgG  and FITC-conjugated donkey anti-human IgG .Primary cerebral cortical cultures were prepared from mice at postnatal day 0. The cerebral cortices were treated with 1% trypsin and 0.1% DNase I in phosphate-buffered saline (PBS) for 5 min at room temperature. The cells were washed three times with Neurobasal A medium (Life Technologies) with 2% B-27 supplement (Life Technologies) and 5% fetal calf serum (Life Technologies) and dissociated by passing through a fire-polished Pasteur pipette in PBS containing 0.05% DNase I, 0.03% trypsin inhibitor and 2 mM MgCln = 28–33 beads).For the quantitative measurements of Bassoon immunofluorescent signals, four or five optical images from two independent beads-neuron co-cultures were obtained using a confocal microscope . Bassoon signal intensities on the beads were measured as the fluorescence mean density within a 7 μm diameter circle enclosing a coated bead. The fluorescent mean densities of the surrounding regions within a 14-μm diameter circle were then measured and subtracted as background signals. Statistical significance of difference was evaluated by one-way ANOVA followed by post hoc Tukey’s test (The coordinates and structure factors of apo-SALM5 and the PTPδ–SALM2 and PTPδ–SALM5 complexes have been deposited in the Protein Data Bank under the accession codes 5XWS, 5XWU, and 5XWT, respectively. Primer sequences used in this study are listed in Supplementary Data Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1