The prognostic impact of surgical paraaortic staging remains unclear in patients with locally advanced cervical cancer (LACC). The objective of our study was to evaluate the survival impact of surgical staging in patients with LACC and no evidence of paraaortic lymph node (PALN) metastasis on pre-operative imaging work-up.Data of 1447 patients with cervical cancer treated between 1996 and 2016 were extracted from maintained databases of 10 French University hospitals. Patients with locally advanced disease (IB2 or more) treated by concurrent chemoradiation therapy (CRT) and no evidence of paraaortic metastasis on pre-operative imaging work-up were selected for further analysis. The Kaplan–Meier method was used to estimate the survival distribution. A Cox proportional hazards model was used to account for the influence of multiple variables.Six hundred and forty-seven patients were included, 377 (58.3%) with surgical staging and 270 (41.7%) without, with a mean follow up of 38.1 months (QI 13.0–56.0). Pathologic analysis revealed positive lymph nodes in 47 patients (12.5%). In multivariate model analysis, surgical staging remained an independent prognostic factor for DFS  and OS . The other significant parameter in multivariate analysis for both DFS and OS was treatment by intracavitary brachytherapy (OR respectively of 0.7 (0.5–1.0) and 0.6 (0.4–0.9), p < 0.05).Nodal surgical staging had an independent positive impact on survival in patients with LACC treated with CRT with no evidence of metastatic PALN on imaging.The online version of this article (10.1186/s12967-018-1703-4) contains supplementary material, which is available to authorized users. Cervical cancer is the third most common cancer in women worldwide and around half of the patients are diagnosed with locally advanced cervical cancer (LACC) . After mThe latest FIGO classification for cervical cancer do not include lymph node status  despite The debate about the most effective way to assess PALN status is ongoing. On one hand, imaging exams are non-invasive but lacks sensitivity for detecting PALN metastasis especially in cases of micrometastases . On the The objective of our study was to evaluate the survival impact of paraaortic nodal surgical staging in patients with LACC and no evidence of PALN metastasis on pre-operative imaging work-up.We conducted a retrospective study using maintained databases from 10 French institutions . These databases registered all patients diagnosed with cervical cancer at any stage between January 1996 and December 2016. The research protocol was approved by the Institutional Review Board (IRB) of the French College of Obstetrics and Gynaecology (CEROG 2016-GYN-0502).Patients with locally advanced cervical cancer (LACC) treated with CRT and no distant or para-aortic invaded nodes on pre-treatment computed tomography scanner (CT-scan) or PET-CT were selected for further analysis. LACC was defined as patients with at least stage IB2 according to the latest 2009 International Federation of Gynecology and Obstetrics (FIGO) classification. Exclusion criteria were: patients with stage IVB; patients treated by radiotherapy only; and patients with missing data for surgical staging.2 weekly base of cisplatinium ± 5FU in some centers. Some patients received intracavitary brachytherapy (15 grays) to complete pelvic conformational radiotherapy.The decision to perform surgical paraaortic staging or other complementary therapies  was center–driven. As for all aspects of patient management, the decision was made within a multidisciplinary committee and was based on both patient and tumor characteristics. During surgical para-aortic staging, all of the lymphatic tissue from the aorta was removed from the iliac bifurcation to the left renal vein. Pelvic lymphadenectomy was not routinely performed since the area is covered by traditional pelvic radiation fields. All patients were subsequently treated by CRT and received pelvic conformational radiotherapy at the total dose of 45 Grays (25 fractions) in 5 weeks with a concomitant 40 mg/mPatients with positive nodes after surgical staging were supposed to receive an extension of the radiation fields in the paraaortic region. However, some patients without surgical para-aortic staging also received paraaortic radiation therapy in the case of pelvic lymph node involvement on PET-CT. Some centers commonly performed completion surgery (hysterectomy) following RCT in patients with residual disease.Follow-up protocol included a gynecologic examination every 3 months for 2 years and then every 6 months for 2 years. Magnetic resonance imaging (MRI) or a PET-CT scan were performed when clinically indicated. Recurrences were diagnosed either on biopsy or with an imaging exam.According to previous reports, we applied the following definitions to stratify the sites of recurrence: (i) local recurrence was defined as a vaginal or central pelvic location without lymph node involvement; (ii) regional recurrence was defined as a non-central pelvic location or a peritoneal carcinomatosis and no lymph node involvement; (iii) nodal recurrence included pelvic and/or paraaortic nodal locations; (iv) distant recurrence included distant metastasis ; (v) multiple site recurrence included any combination of the locations mentioned above.The date of the end of primary treatment was used to calculate disease free survival (DFS) and OS.2 test or Fisher’s exact test for categorical variables. The Kaplan–Meier method was used to estimate the survival distribution. Comparisons of survival were made using the log rank test. A Cox proportional hazards model including all the parameters statistically significant in univariate analysis, was used to account for the influence of multiple variables. Values of p < 0.05 were considered to denote significant differences.Databases were managed using Excel  and statistical analyses were performed using R software . Statistical analysis was based on the Student’s t test for continuous variable and the χBetween 1996 and 2016, 1447 patients were treated for cervical cancer within our institutions. Among them, 647 fulfilled the inclusion criteria and were included for analysis: of these, 377 had undergone surgical staging and 270 had not . During follow up, 140 patients died: 53 patients (14.1%) with surgical staging and 87 32.2%) without. Two hundred and two patients experienced recurrance during follow up: 102 with surgical staging and 100 without. Surgical staging was significantly associated with better DFS and OS than clinical staging (p < 0.001)  and this proportion was similar in patients with and without surgical staging. The decision of whether to apply extended radiation fields was thus not based on the results of the surgical staging. As mentioned by Pomel et al. , no studWe recognize that the retrospective nature of our study limits the generalization of our findings. However, this cohort is the largest ever reported and large multicenter cohorts are of utmost importance to accumulate evidence to resolve this long-standing controversy. Patients in our cohort had either a CT-scan or a PET-CT pre operatively despite the fact PET-CT is known a higher sensitivity for detecting lymph nodes metastases. Our choice to also include patients with CT-scan was driven by the fact that most centers do not have routinely access to PET-CT pre-operatively. Because of the retrospective nature of our study, data regarding recurrences’ management was not available. As we report the therapeutic benefit of surgical paraaortic dissection prior to initiation of concomitant radio-chemotherapy, salvage paraaortic lymph nodes removal in patients experiencing lymph nodal recurrence using minimally invasive surgery could be a valid therapeutic approach as recently suggested by Gallotta et al. .A commonly used argument against surgical staging is the subsequent delay in starting concomitant chemo-radiotherapy. We were not able to evaluate this parameter but in light of our results, with increased survival rates in patients with surgical staging, it is safe to think that this hypothesis can be ruled out. This is all the more true when taking into consideration that others have reported no significant delays in starting chemoradiation therapy in these patients . FinallyThe complex interactions between the different variables determining prognosis, have delayed initiation of a randomized controlled trial to answer the issue. The LILACS study by Frumovitz et al.  should bWe found surgical staging had a therapeutic value in women with node metastases not detected on pre-operative imaging, with significant improvement in DFS and OS achieved by tailoring radiation therapy plans or modifying planned therapy, and identifying patients with peritoneal spread. This benefit could vary from one patient to another due to the numerous therapeutic factors involved in improving survival, as well as from one center to another as experience in laparoscopic staging is a determining factor to limit associated morbidity. Further studies should help select patients that will benefit the most from surgical staging.Additional file 1: Figure S1. Kaplan–Meier curve for overall survival in patients with surgical staging stratified by final pathological analysis of paraaortic lymph nodes. In black: patients without lymph nodes metastases. In red: patients with paraaortic lymph nodes metastases.


We investigated the regulation of chemical signals of house mice living in seminatural social conditions. We found that male mice more than doubled the excretion of major urinary proteins (MUPs) after they acquired a territory and become socially dominant. MUPs bind and stabilize the release of volatile pheromone ligands, and some MUPs exhibit pheromonal properties themselves. We conducted olfactory assays and found that female mice were more attracted to the scent of dominant than subordinate males when they were in estrus. Yet, when male status was controlled, females were not attracted to urine with high MUP concentration, despite being comparable to levels of dominant males. To determine which compounds influence female attraction, we conducted additional analyses and found that dominant males differentially upregulated the excretion of particular MUPs, including the pheromone MUP20 (darcin), and a volatile pheromone that influences female reproductive physiology and behavior. Our findings show that once male house mice become territorial and socially dominant, they upregulate the amount and types of excreted MUPs, which increases the intensities of volatiles and the attractiveness of their urinary scent to sexually receptive females. Mus musculus) excrete large quantities of major urinary proteins (MUPs) that bind and transport hydrophobic ligands, including several volatile pheromones3. Upon excretion, MUPs slow down the release of volatiles from scent marks8, which may prolong their attraction and influence on conspecifics. MUPs are often suggested to show high individual diversity and thereby mediate individual and kin recognition10. They are encoded by 21 paralogous loci, but MUP genes are highly homologous12 and no individual variation has been detected in wild populations13. Interestingly, MUP excretion is sexually dimorphic15, under endocrine control18, and dynamically regulated19 depending upon health22, nutritional status23, and social interactions25. Our aims here were to test whether house mice regulate the excretion of MUPs or volatile pheromone ligands depending upon their social status, and whether such regulation influences the attractiveness of their odor to potential mates24.House mice , and especially if males are socially ‘dominant’38. Several volatile odor compounds (VOCs) have been identified as sexual pheromones , including α- and β- farnesene39, 2-sec-Butyl-4, 5-dihydrothiazole (SBT), 3, 4-dehydro-exo-brevicomin (DHB), and 6-hydroxy-6-methyl-3-heptanone (HMH40), though not in all strains41. Exposure to a combination of male pheromones  induces female olfactory preferences for these compounds40 and accelerates vaginal opening42. ‘Dominant’ males excrete higher quantities of MUPs45 and volatile pheromone ligands (α- and β- farnesene and SBT46) than defeated ‘subordinates’. These findings might help explain how females discriminate winners from losers35 and how dominant, territorial males achieve greater reproductive success than subordinates49.Male house mice are territorial and much evidence indicates that intra- and inter-sexual selection are mediated by chemosensory signals. Dominant territorial males scent mark their territories, and males deposit more scent-marks after winning an agonistic encounter compared to losers (winners are commonly labeled as ‘dominants’ and losers as ‘subordinates’)Mus laboratorius50), but their ecological relevance should not be assumed52 . Second, previous studies have mainly used the outcome of short-term, dyadic agonistic interactions as proxies for social status, and winners and losers are labeled as ‘dominants’ and ‘subordinates’, respectfully. These assays are also used to study social defeat57, which results in behavioral58 and physiological changes57, including detrimental health effects59. However, they do not necessarily provide a valid proxy for assessing a male’s ability to obtain a territory nor subsequent changes in behavior and physiology60 of dominants or subordinates under more natural social conditions. Indeed, a recent study on laboratory mice indicates that winning dyadic agonistic assays did not predict social dominance in more complex social environments45. Third, it is unclear whether MUP excretion is regulated according to social status, or vice versa, as previously suggested44 and it is also unclear whether winners upregulate or losers down-regulate MUPs and volatile pheromones. Longitudinal studies are needed to examine pheromone excretion in social interactions and with controls for comparison. Finally, the regulation of MUPs and volatiles have mainly been investigated independently, with few exceptions61 and studies are needed to examine how MUP regulation influences odor, as well as the excretion of volatiles.These studies have provided valuable insights into chemosensory-mediated sexual selection, however, several questions still need to be addressed. First, studies on chemical communication in mice are primarily conducted with laboratory strains (Mus musculus musculus) to address the following aims: (1) We tested whether MUP production provides a reliable indicator of social status in seminatural conditions over 12 weeks, and we also analyzed protein excretion from controls kept in standard housing conditions during the same time. (2) We conducted olfactory assays to test whether female mice discriminate the scent of dominant versus subordinate males32 and whether variation in male urinary protein concentration influences female preferences when male social status was controlled. (3) We conducted in-depth proteomic analyses using SWATH  to measure the regulation of specific MUP proteoforms, and gas chromatography- mass spectrometry (GC-MS) to analyze variation in specific volatile pheromones. We expected dominant males to upregulate, or subordinates to down-regulate  protein excretion, or both. To determine whether regulation is temporary or long-lasting, we continued measuring MUP excretion after social interactions were terminated. We expected females to have lower levels of protein excretion than males, and to regulate protein excretion depending upon their social status, as previously suggested62. We expected that females would discriminate and prefer the urine of dominant over that of subordinate males, and that they would prefer the scent of males with high MUP concentration when social status is controlled (as observed in rats63). Finally, we expected that mice would show differential regulation of particular MUP proteoforms43, volatile pheromones39, or both, depending upon their social status.We conducted a study on social behavior and regulation of MUPs and other pheromones in wild-derived (F1 from wild-caught) house mice , Z = 3.70, p < 0.01; Supplementary Table F1,245 = 0.3, p = 0.60; Supplementary Table Males that became socially dominant (DOM) in the seminatural enclosures upregulated urinary protein excretion, as expected, whereas subordinates (SUB) and controls (CTRL) showed no significant changes during this time Fig. . Linear F4,288 = 3.5, p < 0.01; Supplementary Table post hoc analyses revealed a significant increase in urinary protein excretion in subordinate females during the enclosure phase compared to levels before  with dominant and subordinate mice increasing excretion during the enclosure phase compared to prior levels . Female mice differed in total urinary protein excretion during the enclosure phase with dominant and subordinate females excreting more total urinary protein than caged controls  urinary protein excretion in males showed a significant interaction of male social status and enclosure phase . Contrary to our expectations, females showed no initial preferences for male urine with high versus low protein concentration  stimulus , but not relative intensity of MUP protein excreted , and separately for MUP1, MUP2, MUP3, MUP5, MUP17, and MUP20  but not MUP4 . Dominant and subordinate males did not differ in the extent of fold change in relative intensity or protein amount of urinary proteins  for both intact and denatured urine , but not females . SPLS-DA and univariate tests showed that caged control males’ urine released significantly more SBT than urine of enclosure males . Third, body mass had no influence on male urinary protein excretion, indicating that MUP excretion is not an index of size. Neither body mass nor age correlated with social status, unlike some rodent studies68, though such correlations may be a consequence of social rank influencing access to food, rather than vice versa70. Our results align with other studies that find no relationship between body mass and social status or urinary protein excretion45, though the correlation between size and social rank may occur in Mus musculus domesticus, and not Mus musculus musculus71. Fourth, MUP upregulation by territorial males was relatively stable over the 12 weeks of living in seminatural conditions . Future studies on chemosensory sex recognition73 should therefore consider the possibility that sex differences might be inflated in the laboratory.Females did not regulate protein excretion according to social status, contrary to a previous suggestion74. Therefore, behaviors associated with social dominance, such as winning fights and scent-marking, may upregulate MUP expression by triggering pulsatile hormonal secretions. Unfortunately, previous studies have focused on physiological and behavioral changes following social defeat rather than winning75, whereas our findings cannot be explained by subordinates losing fights26, having poorer health, nutrition, or condition23. Regardless of the physiological mechanism, there are several possible adaptive benefits to upregulating the amount or type of MUPs excreted for dominant males, which include (1) enhancing female olfactory attraction; (2) prolonging the release of VOCs from a male’s scent marks76, which may extend their apparent freshness and enhance pheromonal effects on female physiology3; (3) increasing the aversiveness of a male’s territory to rival males32; (4) regulating energy expenditure and metabolism77; and (5) facilitating toxic waste excretion80. These hypotheses are not mutually exclusive, and we investigated the first hypothesis.The mechanisms controlling the regulation of MUP excretion and their adaptive functions are unclear. Urinary MUPs are transcribed in the liver and expression is under complex endocrine control, involving several hormones and pulsatile secretion34. Female olfactory preferences were expressed only during estrus, suggesting a mating function. The lack of preference by diestrous females can be explained by cyclical silencing of vomeronasal sensory neurons, which makes female mice “blind” to certain male pheromones during diestrus and restores discrimination during estrus81. Female preferences may have been mediated by MUPs, VOCs, or both . We presented females with samples of male urine collected from the same individual that differed in protein concentration – at levels comparable to the differences between dominant and subordinate males – and surprisingly, females showed no preference for high protein concentration. This result means that differences in the amount of urinary protein concentration per se are not sufficient to explain female olfactory preferences for dominant males . Therefore, we conducted proteomic and GC-MS analyses to test for differential regulation of particular MUP proteoforms and changes in known pheromones.Our olfactory experiments showed that estrous female mice were more attracted to the scent of dominant than subordinate males, which is consistent with previous studies using dyadic agonistic assays as proxies for social status25 to determine whether urinary protein regulation was consistent for all MUP proteoforms, or whether only specific MUPs were regulated depending on social status. We did not observe any difference between dominant and subordinate mice in relative spectra intensities of seven identifiable proteoforms . However, differences between dominant and subordinate males became evident when we compared within-individual fold changes of MUP proteoform production. Dominant males significantly increased the excretion of four MUP proteoforms  in social conditions, whereas subordinate mice showed no significant fold changes. These results indicate that dominant male house mice show differential upregulation of specific MUP proteoforms.We applied high-resolution mass spectrometry83. Furthermore, MUP20 is predominately expressed in male urine , it is a female attractant66, and it mediates spatial learning85 by stimulating neurogenesis86. MUP20 also stimulates male-male aggression83 and maternal aggression from lactating females87. Studies are needed to determine whether honest signaling is enforced by punishment of dishonest signalers . Our findings support previous studies of Mus musculus domesticus showing increased MUP20 production in relation to social status after a three-day44 and a 17-day enclosure experience45 in highly male-biased populations. Taken together, these studies suggest that upregulation of specific MUP proteoforms provides a reliable indicator of dominant male social status, which may deter challenges from subordinate rivals, as well as attract potential mates.Upregulating particular MUP proteoforms may function as a mechanism for dominant males to elicit specific information or responses from conspecifics. Adding recombinant MUP3 to male urine, for example, promotes aggression from male conspecifics80, and we expected that elevating MUP excretion would increase the intensities of volatile pheromones and other ligands in urine. Indeed, we found a strong, positive correlation between urinary protein concentration and intensity of VOCs, and denaturing the proteins increased the overall ion intensities of all VOCs detected and analyzed , which showed the highest relative intensity in dominant males. HMH is an androgen-dependent volatile compound previously found to accelerate female puberty in some laboratory strains65. HMH intensities are higher in males than in females, and females prefer HMH when mixed with DHB and SBT, but not HMH alone42. Thus, elevated HMH levels in dominant males might function to attract adult females and accelerate sexual maturity in peripubertal females. Elevated HMH levels might explain female preferences for dominant males’ scent in the olfactory discrimination assay. We compared the intensities of other male pheromones, including 3,4-dehydro-exo-brevicomin (DHB), 2-sec-butyl-4,5-dihydrothiazole (SBT) and β-farnesene, and found no significant differences between dominant and subordinate males. The intensity of SBT was higher in caged controls than enclosure males, supporting results that compared isolated versus group-living mice in laboratory conditions89. These findings raise additional caveats about studying chemosignals in standard laboratory conditions. We found no evidence for female-specific VOCs , and VOC profiles of dominant and subordinate females were almost indistinguishable. It is surprising that we did not detect more compounds that differ between dominant and subordinate males, but MUPs show extremely strong binding affinity for male pheromones, and so some ligands remain bound, even after extensive purification90. Thus, it is possible that GC-MS will fail to detect actual differences in the amounts of these bound ligands.MUPs control the binding of volatile male pheromones, but there are surprisingly few studies on the regulation of MUPs on volatiles – and none in natural or seminatural conditions. Our previous analyses verified the presence of male pheromones in our enclosure mice49, our findings suggest that MUP excretion provides a reliable indicator of genetic quality to potential mates.Male mice increased the excretion of major urinary proteins (MUPs) once they acquired a territory and became socially dominant, which may function to attract females. Estrous females were more attracted to the scent of dominant than subordinate males, however, they were not attracted to the scent of urine with elevated MUP concentration when male social status was controlled. Dominant males also differentially upregulated specific MUPs, including (MUP20) and a volatile pheromone (HMH), a MUP-ligand that may influence female behavior and reproduction. Future studies are needed to examine the proximate mechanisms through which dominant males regulate these pheromones and how they influence mating success. Since social status of male house mice is influenced by phenotypic and genetic quality Mus musculus musculus) trapped at seven locations within a 300 m radius in Vienna, Austria  and crossed between trapping sites to avoid inbreeding. F1 mice were weaned at 21 ± 1 d, separated from siblings at the age of 35 ± 1 d and individually housed in standard mouse cages  containing wooden bedding , a cardboard roll, cotton nestlets© , and a plastic nest box . Food (Altromin rodent diet 1324) and water were provided ad libitum and temperature was maintained at 22 ± 2 °C. Mice were kept on a 12:12 h light:dark cycle with red lights on at 1500. The authors use these descriptions of housing, diet, and light:dark cycle to define standard housing conditions. At weaning, all animals received an ear-punch for individual identification and to obtain tissue for DNA extraction. The Ethical and Animal Welfare Commission at the University of Veterinary Medicine Vienna approved the experimental protocols (Permit No. 02/08/97/2013). We confirm that all experiments and animal handling were performed according to the ethical standards and guidelines outlined by the Ethical and Animal Welfare Commission.Experimental animals were F1 offspring of wild-caught house mice  or caged control group . Mice in the enclosure group were assigned to one of eight replicate populations, and each population had eight mice . Males within populations were matched for body mass (range = 0.6 g). To test whether initial urinary protein excretion predicted males’ subsequent social status, we also assigned males depending on their relative urinary protein production , and each population contained one high, two intermediate, and one low urinary protein producer (see Supplementary Table ad libitum and temperature was maintained at 22 ± 2 °C. Mice were kept on a 12:12 h light:dark cycle with white light off (and red lights on) at 1500. The caged controls were litter-mates of mice in the enclosure group, and were housed under standard colony conditions during the experiment.F1 mice were assigned to either 49. Mice obtaining a dominance index ≥80% within an enclosure compartment were considered to be dominant; otherwise they were considered subordinate. Adult survival was monitored daily and offspring born in the enclosures were removed upon discovery.To assess social status, behavioral observations were recorded three to five days per week for 30 min/d between 1500 and 1700 (peak activity) for 12 weeks . Before release into the enclosures, males received unique fur cuts, allowing easy identification under red light; females were identified by unique ear punches. Observers recorded interactions , the location of the interaction, and the mice involved. A dominance index for each mouse was calculated as (number of aggressive + investigatory interactions)/2) and then hung inside the metabolic cage. Upon excreting >70 µL of urine, mice were either returned to their home cage (cage controls) or released back into the enclosure (during the enclosure phase). Only 2% (17/700) of urine collections provided an insufficient volume during the 60-min sampling period, in which case mice were returned to their cage or enclosure without sampling. Urine and feces were transferred to separate Eppendorf tubes or glass vials , immediately frozen, and stored at −80 °C. As part of our experimental design, storage times differed depending on collection date, but handling was the same for each sample to avoid possible biases. Previous studies used a subset of these samples to compare the effects of housing  on urinary volatiles80 and proteins25.For monitoring urinary protein and pheromone production, we collected six urine samples from each mouse over 20 weeks at four week intervals . The Y-maze apparatus consisted of clear PVC tubes (5 cm Ø) and a PVC connector  were unrelated, wild-derived mice . Subjects were housed with a sister in our animal facility, and they were unfamiliar to urine stimulus donors. Estrous cycling was stimulated by introducing male bedding into the home cage of test mice and daily vaginal smears were performed to determine estrus ; (2) adult male versus adult female urine ; (3) dominant males versus subordinates ; and (4) high versus low urinary protein concentration  collected on two different days; 3:1 high:low PC ratio, see below).We compared the following stimuli in our olfactory discrimination experiments: (1) adult male urine versus water  was measured in triplicates using a standard Bradford assay92 on a 96-well microplate. Triplet values occurred within ±10% range, otherwise the analysis was repeated. We used total urinary protein excretion and adjusted with creatinine concentration to calculate urinary protein excretion (protein:creatinine ratio or PC ratio), as this value is expected to account for renal activity and urine dilution . We confirmed that MUPs comprise most (85 ± 7%) of the total protein in the urine of these mice in a preliminary study25. We provide results for PC ratio, total urinary protein , and creatinine.Total urinary protein excretion (mg ml25. Relative quantification was conducted using two different databases containing (1) all reviewed proteins from Swissprot, and (2) 9 reviewed MUP proteins from Uniprot. In total, 42 different MUP sequences were found in Uniprot, though many of these were not distinguishable using a bottom up approach due to signal peptide cleavage at protein maturation. Thus, we employed a conservative approach by including only reviewed MUPs which have been identified independently by at least two different working groups.Urine samples of 12 dominant and 5 subordinate males were obtained during the enclosure phase and pooled for each individual for protein analysis. As previously described, protein identification and quantification was conducted using a label-free proteomics technique known as SWATH80. Compounds were identified using mass spectral library comparison (NIST08) combined with manual interpretation. Exact peak alignment was difficult during the first 5 min of each run, which resulted in a wide peak detection window and increased chances of aligning separate peaks into one. Thus, the dataset suffered from poor peak alignment and difficulties with identifying compounds due to co-elution. After leaving out the first 5 min of each run, we were able to use a significantly smaller peak detection window, which improved dataset quality and enabled reliable peak identification. However, information on the first 5 min of each run (and differentiating compounds therein) is missing from this dataset.Urine samples of 25 males  and 8 caged controls) and 15 females  and 5 caged controls) were obtained during the enclosure phase and pooled for each individual. As previously reported, 15 µL of intact or denatured urine were analyzed using gas chromatography coupled with mass spectrometry (GC-MS)93) and the assumptions of the methods used were first verified.Statistical analyses were performed using the R statistical package  to examine the relationship between urinary protein excretion  and social status, we compared three ‘social status’ groups: dominants, subordinates, and caged controls in standard housing conditions. PC ratio and creatinine values were natural log transformed to achieve a normal distribution. Social status, enclosure phase , and the interaction of social status * enclosure phase were used as fixed factors; age and body mass were covariates in each model. The random factor was composed of individuals nested in population. We used the varIdent function to account for heteroscedasticity in the social status factor and fit the LME model using the maximum likelihood method. The anova.lme function produced F and p values for testing significance of model variables. Pairwise comparison post hoc tests were performed for the interaction social status * enclosure phase in each model using the glht function with Tukey correction for multiple testing (package multcomp95).We used linear mixed effects models  with sex, housing, and the sex * housing interaction as fixed effects on the dependent variables of PC ratio, total urinary protein, and creatinine excretion measured during the enclosure phase (three separate models). Mouse ID and population were included as random effects. An inverse link function was used to account for the inverse Gaussian distribution of the urinary protein variables. Wald chi-square tests97 were used to determine significance of the model effects . Associations between body mass parameters  and social status of enclosure males were examined using Mann-Whitney U tests. Due to pregnancy effects, females were excluded from mean mass and change in mass comparisons.Sex differences were examined using generalized linear mixed models  were used to measure frequency, duration, and latency for each zone  to normalize model residuals). Estrous state of the subjects was included as a fixed factor; subject ID and male stimulus donors’ ID were included as random factors. Models were fitted by residual maximum likelihood and the anova function produced F and p values. Benjamini-Hochberg method (p.adjust function) was used to control multiple hypothesis tests for false discovery rate.When normality of the variables was not rejected (Shapiro-Wilks test), One-Sample T-tests (μ = 0) were used for the positive control, sex, and concentration discrimination experiments. Wilcoxon Signed Rank Tests were used for non-normal data to examine a deviation from a zero median. Fisher Exact test (1:1 null expectation) was used to determine a significant bias in initial preference. To account for the paired design for the social status discrimination experiment, McNemar test was used to compare initial preferences, and linear mixed-effects (LME) models /Xbefore and compared between social status groups as described below. Additionally, fold change in protein amount and relative intensity of MUPs was compared to null expectations using One-Sample T-tests with Benjamini-Hochberg correction. To investigate changes in concentration of individual MUPs from introducing singly housed males into a social setting (enclosures), we ran a multivariate general linear model with individual MUP protein concentrations  as continuous variables and social status , enclosure phase (before vs during), and the interaction of social status * enclosure phase as factors. Age and body mass were used as covariates in the model. The random factor was composed of individuals nested in population. To investigate whether concentration of individual MUPs before introduction into enclosures were predictive of male social status, we used Mann-Whitney U tests with Bonferroni correction.Analyses were carried out on the relative intensity and protein amount of 7 MUPs and 115 non-MUP proteins detected using SWATH. Relative intensity for each compound was calculated by dividing the peak intensity of each compound by the total chromatogram intensity. Protein amount of each compound was calculated by multiplying the relative intensity of each compound by the total amount of protein in the sample (determined by Bradford assay). To investigate the effects of social experience on urinary protein excretion, we calculated the difference in protein amount and relative intensity from XAnalyses were also carried out on the relative intensity of MUP-bound volatiles obtained by GC-MS. We ran an exploratory data analysis  on a dataset containing peak ion intensities (N = 962) after leaving out the first 5 min of each run. Second, we took a targeted approach by analyzing manually collected peak intensities of 34 known urinary volatiles for each sample  based on pairwise Bray–Curtis similarity values calculated from the log(x + 1)- transformed data of protein amount or relative intensity. NMDS allows visualization of high-dimensional chemical similarity by placing each individual in a 2D scatter plot such that points closer together represent individuals with relatively high chemical similarity. The grouping structure of individuals according to their social status was analyzed using nonparametric ANOSIM, a permutation test to evaluate differences between two or more groups without assumptions about data distribution or homoscedasticity. A significant test result indicates that the chemical similarity within the groups is significantly greater than the chemical similarity between the groups. These analyses were implemented using the 99. Intrinsic variable selection was performed to filter out relevant variables, and the data was projected onto uncorrelated latent variables. These linear combinations of the relevant variables were then used in linear discriminant analysis to predict class membership. The package spls100 provides an implementation of SPLS-DA, namely the function splsda. The optimal model parameters were searched for with the function cv.splsda and the number of latent variables  and the sparsity parameter  were selected with 5-fold cross validation.To identify compounds that differentiate between social status groups, we used Sparse Partial Least Squares Discriminant Analysis (SPLS-DA) with subsequent univariate test. SPLS-DA is a classification method appropriate for data with a high number of potentially correlated variables and needs no preliminary variable filteringWe ran 16 classification models to identify urinary proteins that differentiated the social status groups based on the following variables: (1) relative intensity; (2) protein amount; (3) difference (during - before enclosure) in relative intensity; (4) difference in protein amount; (5) fold change in relative intensity; and (6) fold change in protein amount. Additionally, for intact and denatured urine samples, 12 classification models were estimated to obtain volatile urinary compounds that differentiated between the social status groups and housing conditions (see Supplementary Table Supplementary Information


The advent of multiple exciton harvesting schemes and prolonging exciton lifetimes to improve performance attributes of solar cells based on conjugated organic materials presents some interesting challenges that must be overcome in order to realize the full potential of these strategies. This is especially important for applications involving multi-chromophoric conjugated polymers where interactions between multiple spin-forbidden triplet excitons can be significant and are mediated by chain conformation. We use single molecule spectroscopic techniques to investigate interactions between multiple triplet excitons and emissive singlets by monitoring time-dependent fluorescence quenching on time scales commensurate with the triplet lifetime. Structurally related conjugated polymers differing by heteroatom substitution were targeted and we use a stochastic photodynamic model to numerically simulate the evolution of multi-exciton populations following photoexcitation. Single chains of poly(3-hexylthiophene) (P3HT) exhibit longer-lived triplet dynamics and larger steady-state triplet occupancies compared to those of poly(3-hexylselenophene) (P3HS), which has a larger reported triplet yield. Triplet populations evolve and relax much faster in P3HS which only becomes evident when considering all kinetic factors governing exciton population dynamics. Overall, we uncover new guidelines for effectively managing multi-exciton populations and interactions in conjugated polymers and improving their light harvesting efficiency. There is now widespread interest for mitigating performance losses by generating multiple excitons per photon absorbed or extending exciton lifetimes5. Singlet fission–the generation of two triplet excitons from one singlet exciton–and heavy atom substitution to increase triplet character, respectively, have attracted the most attention11, but applications involving conjugated polymers are limited13. In fact, mechanistic studies of singlet fission have concentrated on crystalline solids and small molecule arrays with well-defined chromophore orientations. Furthermore, efforts to increase triplet exciton character and tune triplet interactions in polymers may be complicated by contributions from large vibrational displacements along high frequency modes that modulate spin-orbit coupling strength14.Conjugated organic polymers have demonstrated promise in solar cell applications but extreme heterogeneity and efficient intrinsic loss mechanisms15. For example, the longer lifetimes of triplets creates complex photophysical scenarios due to the presence of multiple excitonic states of different spin on many chromophore segments that interact over a broad range of time scales . Interestingly, previous pulse radiolysis studies found that isolated conjugated polymer chains can support a large number of triplets (~30) simultaneously16. While much of the current focus has emphasized elucidating triplet formation mechanisms on ultrafast time scales, relatively little is known about how populations of multiple triplet excitons evolve on longer time scales .Perhaps the most significant obstacle for effectively utilizing multi-exciton generation and harvesting strategies can be traced to the multi-chromophoric nature of polymers  and variable inter-chromophore coupling due to conformational heterogeneity17. However, by dispersing polymers into inert glassy hosts, intermolecular interactions and packing heterogeneity are negated in addition to spatially confining excitonic kinetics and interactions to a single polymer chain (SPC). Single molecule spectroscopy can then be used to interrogate triplet population dynamics and interactions with emissive singlets by monitoring fluorescence quenching on nanosecond to millisecond time scales21. However, much of the earlier investigations of excitonic processes at the SPC level focused on energy transfer within the singlet manifold where funneling between chromophore sites typically dominates responses26. This regime is most prevalent when yields of spin-forbidden triplet excitons are small (<10%) although the presence of even one triplet can have significant consequences at the SPC level29. Triplet interactions with emissive singlets are commonly inferred from intermittency behavior of SPC fluorescence intensity transients in the form of flickering  on millisecond time scales30 or blinking behavior due to sensitization of reactive oxygen species occurring on time scales of seconds31. More specialized single molecule spectroscopic tools have proven effective for exposing triplet interactions on faster time scales, such as, fluorescence correlation spectroscopy32 and excitation intensity modulation spectroscopy33. While these approaches can access population dynamics of excitonic configurations, kinetic models describing discrete excitonic interactions are much more complicated when multiple triplets are involved. For this reason, it is generally assumed that rates describing multi-excitonic triplet configurations and processes  reach a steady state condition immediately following photoexcitation29. In other words, triplet diffusion or the triplet-triplet annihilation rate constant are assumed to be infinitely fast leading to only one triplet at a time. This limit effectively reduces singlet-triplet interactions to a simple two-state description where the system spends most time in either the lowest energy triplet (T1) or ground electronic state (S0)29 although crucial details of multi-exciton interactions are lost.Unfortunately, resolving multi-excitonic interactions at the materials level in polymers is complicated from a myriad of competing decay channels arising from intermolecular interactions and aggregation35. Although it is assumed that triplet formation follows a conventional perturbative mechanism, these systems are excellent models for understanding the full implications of heavy atom substitution at the SPC level in addition to resolving interactions between multiple triplets and triplet population dynamics. Quenching behavior is modeled by calculating the time-dependent probabilities of n = 0, 1, 2, … nth triplet population dynamics using a stochastic photodynamic model based on the Smith-Ewart differential difference equation originally developed to describe polymerization/emulsion kinetics40. We use the basic formalism of Barzykin and Tachiya41 and employ the approach of Birtwistle and co-workers39 to discretize and solve the Smith-Ewart model using the Gauss-Seidel iterative approach. Unlike effective two-state models mentioned earlier, this model incorporates finite triplet diffusion and triplet-triplet annihilation rate constants resulting in nonzero probabilities of multiple triplets at the SPC level. Simulations of triplet population and fluorescence quenching dynamics of P3HS and P3HT SPCs show good agreement with experiment when triplet-triplet annihilation rate constants are comparable to the natural first order triplet decay rate constant. Interestingly, despite larger reported triplet formation and decay rate constants in P3HS34, we observed larger steady-state probabilities of multiple triplets in P3HT chains. This unexpected result highlights the importance of accounting for all kinetic factors regulating population dynamics of multiple triplets that are difficult to obtain from ensemble level studies. Furthermore, singlet-triplet interactions were found to be much stronger in P3HS probably from substantial red-shifts in singlet exciton electronic transitions due to the heavier selenium heteroatom42 leading to improved spectral overlap between singlet donors and triplet acceptors. Overall, we demonstrate a robust and informative method for resolving the evolution of multiple triplet excitons to help bridge the gap in understanding of population dynamics on longer time scales.We use single molecule excitation intensity modulation spectroscopy to probe triplet induced fluorescence quenching dynamics in SPCs of single poly(3-hexylthiophene) (P3HT) and poly(3-hexylselenophene) (P3HS), which have reported triplet yields exceeding 30%−3) due to efficient triplet formation lead to low signal-to-noise ratios and individual spots often show ‘streaking’ due to singlet quenching by triplets. SPC fluorescence emission and quenching dynamics are resolved by exciting individual molecules using sequences of rectangular shaped laser pulses displayed in Fig. 43. Like other fluorescence-based single molecule probes, this technique relies on a loss of signal as an indicator of singlet-triplet interactions which, in the case of the target materials, can be very large leading to rapid quenching and low steady-state intensities. This effect can be seen in Fig. 0) no triplets are present but, over time, triplet populations increase causing fluorescence quenching via singlet-triplet energy transfer (annihilation). Average triplet populations are also affected by intrinsic decay  and triplet-triplet annihilation that depend on SPC structural and electronic factors44. Taken together, these processes cause the initial intensity, I(0), to decay to a steady-state level, Iss, and quenching depths can serve as a useful metric of both time-dependent triplet occupancies and the strength of singlet-triplet interactions. For ease of comparison with simulated quenching curves (vide infra), we report fluorescence quenching depths as the fraction of quenched fluorescence normalized to I(0), i.e., I(t)/I(0).Single molecule excitation intensity modulation spectroscopy was used to interrogate the presence of multiple interacting triplet excitons. Figure 45, we used a modified version of excitation intensity modulation technique described earlier45 where two rectangular pulses of the same duration and intensity are temporally delayed and I(0) values of each are recorded with pulse delay time and converted into I(t)/I(0) curves. Figure I(0), respectively, . The larger average quenching depths and faster quenching dynamics of P3HS SPCs are consistent with larger triplet occupancies and singlet-triplet quenching rates but, it is not possible to ascertain the relative magnitudes of associated rate constants by inspection alone. The former depend on triplet formation yields as well as the triplet and fluorescence lifetimes, however, it is not straightforward to directly infer relative contributions of triplet-triplet annihilation from I(t)/I(0) curves46.Because of the large variation in responses from SPCs of both polymers41 had previously overcame the limitations imposed by assumptions of infinitely fast triplet-triplet annihilation by introducing an expanded stochastic photodynamic model to simulate the time-dependent evolution of multiple triplet populations from fluorescence intensity modulation data. Importantly, these authors only considered quenching behaviors from conjugated polymers with relatively low triplet yields (<10%) and, consequently, smaller populations of multiple triplet exciton configurations. We now simulate population dynamics of multiple triplets and their interactions with emissive singlets by adopting a similar approach as Barzykin and Tachiya, which benefits from the fact that triplet decay kinetics are orders of magnitude slower than singlet decay time scales41. The model accounts for the existence of multiple chromophores where each can occupy either S0, S1 or T1 states at any given time where only the numbers of each state determine the overall configuration and not the specific location on the SPC41. The kinetic scheme for triplet formation and decay is given as follows for n triplets on an SPC that follows the same format as the original Smith-Ewart model,kf,n is the forward rate constant for generating a triplet on an SPC with n triplets. It is assumed that triplet formation and decay mechanisms are governed by intersystem crossing via spin-orbit singlet-triplet mixing although the specific triplet generation mechanism is inconsequential. Now, the full form of kf,n is,kexc is the excitation rate constant, kISC is the intersystem crossing rate constant, τfl is the fluorescence lifetime, and kQST is the singlet-triplet quenching rate constant. Triplets decay to the S0 state via first order reverse intersystem crossing which is given by kb , can now be obtained by solving the Smith-Ewart differential difference equation40,The time-dependent probability of n triplets, I(t), is expressed as41,I(0) is the initial  intensity (t = 0). Details for solving Eq. kf,0. As triplet occupancies increase, new rates of loss processes become important. In addition to population losses through reverse intersystem crossing, which varies linearly with the current number of triplets, triplet-triplet annihilation becomes operative for more than one triplet proportional to the square of the current occupancy. Importantly, this model implies that, if probabilities of any two states are equal and non-zero, the current state (Pn(t)) will always gain infinitely more triplets from annihilation of a higher state ) than losing triplets from annihilation to a lower state, ).And the time-dependent fluorescence intensity, kf,n is constant . This regime enables estimates of Pn(t) at long times, or, Pn(∞) without the need to solve the Smith-Ewart equation exactly41, which is helpful for estimating steady-state multi-triplet populations using only known photophysical constants (vide supra). In an earlier study of P3HS SPCs, we used this assumption and found non-zero probabilities of more than one triplet on an SPC when kTT was similar to 43. These preliminary estimates of steady-state triplet populations revealed that the assumption of infinitely large kTT is not valid in SPCs with large triplet yields (large kISC). This result can be checked easily by using the condition kTT ≫ kQST to achieve I(t)/I(0) comparable to experiment41. In order to accurately reproduce experimental fluorescence quenching behavior, the Smith-Ewart equation must be solved numerically to calculate Pn(t). We applied the general method of Birtwistle et al.39 to solve the Smith-Ewart model and estimate kTT and kQST with significant nonzero multi-triplet populations.It is first instructive to consider a useful approximation where kTT in either P3HS or P3HT at the ensemble level usually due to the disappearance of triplet signatures or ambiguous dynamics47 necessitates first exploring realistic ranges of possible values. This was accomplished by varying kTT and comparing calculated quenching behavior to experiment for fixed values of kQST. Figure I(t)/I(0) behaviors for varying kTT at fixed kQST values referenced to the reported fluorescence lifetimes, τfl. Estimates of kQST from other polymers at the SPC level were typically in the range of ~108–1010 s−1 which provides an additional benchmark for establishing realistic limits of this parameter29. We imposed a lower limit on kTT corresponding to kTT based on averaged experimental steady-state intensities (Iss) values of ensemble quenching curves in Fig. kTT in P3HS based on experimental quenching depths indicating stronger singlet-triplet interactions. It is also interesting to note that triplet lifetimes decrease drastically with increasing triplet densities due to faster triplet-triplet annihilation16. Although the presence of multiple triplets in P3HT and P3HS is apparent, it is doubtful that the relatively low excitation intensities used here (ca. 10–100 W/cm2) ever enter regimes where the bimolecular annihilation process vastly exceeds linear triplet decay mechanisms.Unfortunately, the difficulty in extracting reliable values of kQST accompanies increases in kTT values due to the fact that fewer triplets are present. Furthermore, we show in the following that experimentally measured quenching depths and dynamics place fundamental limits on kQST that support our assumption of kTT is comparable to kQST is small in both polymers (kQST * τfl < 10−2), I(t)/I(0) behaviors are practically invariant of kTT indicating no interactions between singlets and triplets regardless of their occupancies. This regime could represent the case when SPC conformations are extended and low excitation intensities , thus requiring excitons to diffuse over longer distances, and triplet lifetimes are short compared to τfl. Additionally, if τfl is relatively small , larger triplet occupancies or singlet-triplet interactions (viz. kQST) are needed to produce appreciable quenching.The results also reveal that an increase in kQST is next varied over several decades to assess the sensitivity of singlet-triplet interactions and their effect on I(t)/I(0). Figure kQST are required for P3HS consistent with stronger singlet-triplet interactions. This effect can be rationalized by the fact that the heavier selenium atom causes significant red-shifting of singlet electronic transitions42 which should result in better spectral overlap between singlet donor emission and triplet acceptor absorption and, consequently, larger kQST. Furthermore, based on experimental I(t)/I(0) curves, P3HS displays substantially larger quenching depths which implies that kQST must be sufficiently large compared to the excited state lifetime. Additional insights for validating the choice of kTT and kQST values can be obtained from triplet formation quantum yields (ΦISC). According to experimental estimates of kISC and τfl from previous ensemble measurements provided earlier and in Table ISC values for P3HT are ~0.5 compared to P3HS of ~0.8 leading to the expectation of larger triplet occupancies in the former. However, triplet decay by reverse intersystem crossing back to S0 and triplet-triplet annihilation is faster in P3HS, reflected in estimates of 5 s−1 compared to 1 × 104 s−1 of P3HT43. Rapid triplet population decay from larger triplet-triplet annihilation and reverse intersystem crossing rate constants in P3HS drastically reduce triplet occupancies despite their larger formation yields, requiring larger values of kQST to achieve fluorescence quenching depths comparable to experiment.Using the assumption, mentclass2pt{minimI(t)/I(0)) behavior and triplet populations for P3HS and P3HT SPCs using Eqs kTT and kQST from Figs kexc were generated assuming an absorption cross-section of ~10−15 cm2 for each polymer with an excitation power density of ~10 W/cm2 at 568 nm. To facilitate comparison between polymers we plot I(t)/I(0) curves by multiplying the simulation time step by kQST and multiple triplets. As predicted in Figs kQST) or faster triplet-triplet annihilation as well as shorter-lived singlet exciton (S1) states. Because measured τfl values are over an order of magnitude larger in P3HT compared to P3HS, the latter possibility can be easily ruled out. Earlier work on SPCs containing only light atoms reported kQST values within the same range as generated here although these studies also assumed infinitely fast kTT. Since this latter regime allows only one triplet quencher at a time, larger kQST are needed to achieve substantial quenching depths and faster quenching dynamics. For example, comparison of quenching behavior between earlier studies and ours indicate that exceptionally large kQST values  would be necessary to produce observed quenching depths found in P3HS and P3HT chains. This observation confirms that triplet-triplet annihilation must be held finite in systems with larger triplet yields and occupancies.We now simulate time-dependent fluorescence quenching (I(t)/I(0) curves –allowing for up to 15 triplets–reveals that P3HS triplet dynamics are complete by ~3 ± 1 µs compared to ~25 ± 6 µs in P3HT resulting in steady-state triplet populations (Pn(∞)) of ~2 ± 2 triplets for P3HS compared to ~4 ± 3 triplets on P3HS chains. The lower values of Pn(∞) from P3HS chains are a consequence of both faster triplet relaxation and triplet-triplet annihilation for multiple triplet configurations coupled with a very short effective excited state lifetime. In contrast, weaker singlet-triplet interactions, slower triplet-triplet annihilation and longer-lived excited states in P3HT result in larger triplet occupancies despite having lower triplet formation yields.Time-dependent triplet occupancies for ves Fig.  are nextPn(t) dynamics in Fig. I(t)/I(0) behavior of both systems. This was particularly apparent in the case of large quenching depths and faster quenching dynamics in P3HS despite having smaller triplet occupancies at longer times. This result appears counterintuitive when only considering triplet formation yields which have been shown earlier to depend on the heteroatom. It is also informative to point out how the choice of kTT and kQST used in the simulations of I(t)/I(0) curves and Pn(t) dynamics in Figs kTT > kTT < kQST to tune quenching responses that can be compared directly to experiment. We set an upper limit of ~1011 s−1 which is comparable to the fastest observed singlet-triplet quenching rate constant reported in conjugated polymers29. When kTT > Pn(∞) shifts to lower values) requiring larger kQST to achieve the same quenching depth seen experimentally . However, upon comparison with experimental I(t)/I(0) curves, poor agreement arises from quenching dynamics behaviors, i.e., simulated quenching dynamics are much faster. On the other hand, by allowing kTT < kQST decreases drastically as shown in Fig. Pn(∞) shifts to larger values). Although previous pulse radiolysis work on larger polymer chains found evidence that an SPC can support up to 30 triplets44, it is doubtful that such large steady-state occupancies are possible here since both polymers are relatively small (ca. 30 KDa).Plots of kTT and kQST to fluctuate with sample preparation conditions (viz. solvent) and from molecule-to-molecule. In fact, previous single molecule spectroscopic investigations of singlet-triplet interactions found drastic variations in fluorescence responses with molecular size32 and order45. We expect that SPC conformational qualities should have a large impact on kTT. Here, we do not expect either P3HS or P3HT to be able to self-aggregate which should attenuate triplet diffusion and triplet-triplet annihilation. Further examination of this effect is beyond the scope of the present paper due to large variations in solubility of both polymers although large variations in fluorescence quenching often appear with very subtle changes in chain conformation and order50. In addition to triplet-triplet annihilation, self-aggregation in larger polymer chains has been proposed to increase singlet-triplet interactions52. However, reliably sorting out the dominant energy transfer mechanism has proven difficult. Nonetheless, long-range, Forster-type energy transfer mechanisms should not be affected by conformation as much as exchange mediated, Dexter-type energy transfer.Lastly, photophysics of polymers are highly dependent on the SPC conformational qualities and we expect that values of We have demonstrated new perspectives of kinetic factors governing the occupancies of triplets in conjugated polymers with large triplet yields on time scales relevant to optoelectronic devices. This feature, along with the multi-chromophoric nature of polymers, increases the likelihood of multiple excitonic species existing on an SPC at any given time. Our simulations have revealed several critical points that should be taken into consideration when designing excitonic materials for optoelectronic applications. Specifically, multi-exciton generation and heavy atom substitution approaches to generate and harvest many triplets. Perhaps the most noteworthy of these involves the kinetic competition between triplet formation and decay mechanisms that determine triplet occupancies on longer time scales. This effect was most pronounced in P3HS where, despite larger triplet yields, faster relaxation through first and second order processes lowered triplet occupancies compared to P3HT. We expect these processes in addition to singlet-triplet interactions to be strongly dependent on the SPC conformation, which can be simulated by adjusting the associated rate constants. Overall, our approach can now provide a clearer link between triplet formation  on sub-picosecond time scales and triplet interactions on much longer time scales.42. Dilute solid dispersions of each polymer were prepared by dissolving in chlorobenzene (~10−9 M) which was added to a polystyrene solution. Thin films were deposited by spin coating on rigorously cleaned glass coverslips and sealed with an aluminum layer (~100 nm) from vacuum deposition. Single molecule excitation intensity modulation experiments were performed on a scanning confocal based microscope spectrometer described earlier45. Briefly, SPCs were excited by a series of rectangular laser pulses  and fluorescence intensity trajectories were averaged many thousands of cycles with an multi-channel analyzer (Fast Com Tec). Triplet quenching dynamics were also measured by using a delayed two-pulse excitation arrangement where one rectangular pulse excites the SPC to produce a steady-state triplet population distribution followed by a second identical pulse. By delaying the second pulse in time, initial intensities (I(0)) can be used as a metric of triplet relaxation in the absence of light. Details of this approach were provided in ref.45. Time-dependent triplet populations and triplet-induced fluorescence quenching behaviors were simulated by solving the Smith-Ewart equation using the approach of Birtwistle and co-workers39. Details of the computational approach are provided in the Supplemental Information Section.The synthesis and characterization of P3HS and P3HT  were described in detail previouslySupp. Info.


Correlations between lung function measures of BEC donors, and COL1A1, HAS2, and α-SMA gene expression, as well as supernatant concentrations of HA, pro-collagen Iα1, hyaluronan (HA), and PGE2 were assessed. We observed that expression of α-SMA and COL1A1 by HLFs co-cultured with asthmatic BECs was negatively correlated with BEC donor lung function. BEC-HLF supernatant concentrations of pro-collagen Iα1 were negatively correlated, and PGE2 concentrations positively correlated, with asthmatic BEC donor lung function. Expression of HAS2, but not α-SMA or COL1A1, was greater by HLFs co-cultured with asthmatic BECs from donors with a history of severe exacerbations than by HLFs co-cultured with BECs from donors who lacked a history of severe exacerbations. In conclusion, α-SMA and COL1A1 expression by HLFs co-cultured with BECs from asthmatic children were negatively correlated with lung function measures, supporting our hypothesis that epithelial regulation of HLFs and airway deposition of ECM constituents by HLFs contributes to lung function deficits among asthmatic children. Furthermore, epithelial regulation of airway HAS2 may influence the susceptibility of children with asthma to experience severe exacerbations. Finally, epithelial-derived PGE2 is a potential regulator of airway FMT and HLF production of collagen I that should be investigated further in future studies.Airway remodeling may contribute to decreased lung function in asthmatic children. Bronchial epithelial cells (BECs) may regulate fibroblast expression of extracellular matrix (ECM) constituents and fibroblast-to-myofibroblast transition (FMT). Our objective was to determine if human lung fibroblast (HLF) expression of collagen I (COL1A1), hyaluronan synthase 2 (HAS2), and the FMT marker alpha-smooth muscle actin (α-SMA) by HLFs conditioned by BECs from asthmatic and healthy children correlate with lung function measures and exacerbation history among BEC donors. BECs from asthmatic (n = 23) and healthy children (n = 15) were differentiated at an air-liquid interface (ALI) and then co-cultured with HLFs for 96 hours. Expression of COL1A1, HAS2, and α-SMA by HLFs was determined by quantitative polymerase chain reaction (qPCR). FMT was quantified by measuring HLF cytoskeletal α-SMA by flow cytometry. Pro-collagen Iα1, hyaluronan (HA), and PGE Longitudinal studies in asthmatic children have demonstrated lung function deficits that persist into adulthood3. One possible mechanism explaining differences in lung function observed between asthmatic and healthy individuals is airway remodeling. Airway remodeling encompasses multiple pathologic changes that have been observed in asthmatic airways4. In adult asthma, basement membrane thickening has been well studied and is considered pathognomonic of the disease. Fewer studies of biopsy specimens exist in children; however, both qualitative6 and quantitative8 data have demonstrated increased airway basement membrane thickness in children with asthma. Additional studies have shown that basement membrane thickness at infancy does not predict subsequent asthma9. Taken together, data from both epidemiologic and pathologic studies support the premise that airway remodeling in asthmatic individuals is not present early in life, but evolves during childhood and persists into adulthood.Asthma is the most prevalent chronic lung disease of childhood affecting an estimated 14% of the world’s pediatric population10. Prior work from our laboratory has demonstrated increased expression of pro-remodeling signaling mediators by primary BECs obtained from asthmatic children in well-differentiated air-liquid interface (ALI) cultures11. Further studies have demonstrated that healthy human lung fibroblasts (HLFs) co-cultured with differentiated BECs display greater production of extracellular matrix (ECM) components including type I and III collagen, hyaluronan (HA), and fibronectin when co-cultured with primary BECs obtained from asthmatic donors12. Separate studies have also confirmed increased expression of alpha smooth muscle actin (α-SMA) and tropomyosin-I from HLFs co-cultured with asthmatic BECs compared to healthy BECs indicative of a greater fibroblast to myofibroblast transition (FMT)13.There has been increasing focus on the role of the airway epithelium as a driver of asthma pathogenesis given that bronchial epithelial cells (BECs) are the initial point of contact between the environment and the hostex vivo BEC regulation of HLFs, and the lung function and exacerbation history of asthmatic BEC donors, we utilized our primary differentiated BEC/HLF co-culture model and medical history and spirometry data from healthy and asthmatic children who donated BECs. We tested the hypothesis that lung function and/or exacerbation history of BEC donors would be associated with the expression of genes related to airway remodeling by HLFs conditioned by BECs from children with asthma. Specifically, we hypothesized that expression of genes related to FMT (α-SMA) and ECM production [collagen I (COL1A1) and hyaluronan synthase 2 (HAS2)] by HLFs co-cultured with BECs would be correlated with spirometry data obtained from BEC donors, and/or associated with a history of severe exacerbations among asthmatic BEC donors. Furthermore, we assessed correlations between prostaglandin E2 (PGE2) in BEC-HLF supernatant and lung function measures among asthmatic BEC donors given the potential role that this epithelial derived soluble mediator may play in regulating airway stromal cells. Some of the results of these studies have been previously reported in abstract form14.To investigate potential associations between NO) compared to healthy subjects . Only 5 asthmatic subjects had a bronchodilator response during pulmonary function testing (defined as ≥12% increase in forced expiratory volume in 1 second (FEV1)). Asthmatic donors had elevated IgE levels compared to the healthy subjects . Forced expiratory volumes and flows were significantly lower in the asthmatic group and consistent with airway obstruction. Expression of α-SMA, COL1A1, and HAS2 by HLFs co-cultured with BECs from healthy (n = 15) and asthmatic (n = 23) children was measured by quantitative polymerase chain reaction (qPCR) and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). In BEC-HLF co-cultures, α-SMA, COL1A1, and HAS2 expression were all significantly greater by HLFs co-cultured with asthmatic as compared to healthy BECs  and asthmatic donors (n = 23) to generate ALI cultures used in BEC/HLF co-cultures. Clinical characteristics for each group are shown in Table 5 and astECs Fig.  and HAS2ECs Fig.  expressi1   ratio , however, concentrations of hyaluronan (HA) in BEC-HLF co-culture supernatant were not significantly different between asthmatic BEC donors with and without a history of severe exacerbations .We assessed associations between the expression of α-SMA, COL1A1, and HAS2 by HLFs cultured with asthmatic BECs and history of severe asthma exacerbations among BEC donors, defined as an acute asthma exacerbation requiring treatment with systemic corticosteroids in the emergency department or hospital. Expression of α-SMA and COL1A1 by HLFs cultured with BECs from children with asthma and a history of severe exacerbations was not significantly different than expression by HLFs cultured with BECs from asthmatic children without a history of severe exacerbations Fig. . HAS2 exons Fig. , P = 0.12 in supernatant of asthmatic BEC-HLF supernatant (n = 21) following HLF co-culture with BECs. PGE2 concentrations were positively correlated with FEV1  demonstrated an absolute decline in post-bronchodilator FEV1 (% predicted) in longitudinal studies of individuals with severe asthma, which correlated with airway thickness observed on CT scans of the lung17. No differences were observed in pre-bronchodilator FEV1 (% predicted) in the SARP study.While multiple cross-sectional and longitudinal studies of lung function in adults exist, fewer studies have examined the longitudinal changes in lung function seen in asthmatic children. Data from the Tucson Children’s Respiratory Study suggest that pulmonary function in children who develop persistent symptoms of asthma is normal at birth, with subsequent evidence of airflow limitation manifesting as early as 6 years18. Additional studies have provided insight into mechanisms of disordered wound repair also contributing to remodeling, which may be regulated by the epithelium10. From a lung mechanics standpoint, airway remodeling is a biologically plausible explanation for the reduced lung function seen in asthma19. While the concept of airway remodeling refers in general to a complex process that includes several pathological features, changes in the basement membrane have been the most extensively described findings in asthmatic airways20. Biopsy studies have provided insight into the role of basement membrane thickening and decreased lung function. In adults, evidence of airflow limitation correlated with increased airway wall thickness related to thickening of both the epithelial and basement membrane layers21. These findings are consistent with several prior reports of increased deposition of ECM constituents in the subepithelial layer in adults with asthma24. Payne and colleagues compared basement membrane thickness in airway biopsies obtained from children with asthma with samples obtained from adults with asthma. In that study, the authors demonstrated a similar degree of thickening in samples obtained from asthmatic children compared to adults regardless of asthma severity7. These data suggest that establishment of the thickened basement membrane seen in asthma occurs during childhood and persists into adulthood.Accumulating data have provided further insight into the factors contributing to basement membrane thickening in asthmatic airway remodeling. Once thought to be the product of chronic airway inflammation, more recent studies suggest that remodeling may occur in parallel with inflammatory changes or may be independent of airway inflammation26. Myofibroblasts are a primary source of types I and III collagen in fibrotic lesions and represent a contractile phenotype that may directly participate in scar formation and contraction in the asthmatic airway30. Other important ECM constituents such as HA are also primarily secreted by fibroblasts and may play important roles in airway remodeling. HA is a major component of the ECM that has been shown in animal models to be involved in inflammatory cell recruitment and retention during wound repair, is directly influenced by transforming growth factor beta (TGFβ) signaling31, and its clearance is essential for resolution of local inflammation during acute injury33. Our observations of greater HLF expression of the HA synthetic enzyme HAS2 when cultured with asthmatic BECs from donors with a history of severe exacerbations is particularly interesting within the context of these prior findings relating HA to inflammatory cell recruitment and retention33. Furthermore, HA in bronchoalveolar lavage samples is higher in adults with asthma and correlates with asthma severity34. Interestingly, HLFs from patients with airway hyper-responsiveness demonstrate greater overall production of ECM constituents than those from healthy individuals35. Using a similar BEC/HLF co-culture model system, our group has previously reported that that HLFs co-cultured with BECs obtained from asthmatic donors drive co-cultured HLFs to produce greater amounts of important ECM constituents including types I and III collagen and HA12, and that HLFs co-cultured with BECs obtained from asthmatic donors displayed a greater degree of FMT compared to HLFs co-cultured with BECs obtained from healthy donors13. In recent studies we have provided both indirect and direct mechanistic evidence that airway epithelial TGFβ2, PGE2, and follistatin-like-3 (FSTL3)/activin A signaling influence FMT and HLF expression of ECM constituents36. In the present study, the positive association between PGE2 concentrations in co-culture supernatant and measures on lung function among asthmatic BEC donors provides additional evidence in support of the hypothesis that epithelial PGE2 may play a role in regulating FMT and HLF expression of ECM constituents, although future studies are needed to more directly test this potential mechanism by blocking epithelial PGE2 signaling. The novel observations of the current study are that we have for the first time demonstrated associations between ex vivo BEC-HLF crosstalk and clinical features among asthmatic BEC donors, namely lung function measures and history of severe asthma exacerbations.Previous work has demonstrated that fibroblasts are the predominant source of many ECM constituents. This is particularly true for HLFs that have undergone FMTex vivo will be an important future direction of investigation. For this study, we used a common HLF cell line from a healthy individual in all experiments to limit biological variability and isolate the differences to the primary BECs. We acknowledge that there are likely important differences in behavior in HLFs obtained from asthmatic and healthy children; however, this is beyond the scope of our present study. Finally, in this study, we provide indirect evidence to support a role of epithelial PGE2 as a regulator of airway FMT and HLF production of collagen I, however, we did not directly test this mechanism through blockade of epithelial-derived PGE2, which we hope will be an important aspect of future investigations using our model system.Despite the unique strengths of our study design, it does have some important limitations. The subjects recruited into our cohort displayed mild airflow obstruction, and only 5 subjects exhibited bronchodilator responsiveness, indicative of a milder asthma phenotype. It is important to note that baseline spirometry obtained in our asthmatic subjects was significantly lower for all measured parameters compared to healthy subjects. Measurements obtained in our cohort may be indicative of an asthmatic population that has been appropriately treated with maintenance therapies as the majority of our subjects were taking daily inhaled corticosteroids. Given that only a small minority of asthmatic BEC donors exhibited bronchodilator responsiveness, we had insufficient power to assess potential associations between outcome measures in our BEC-HLF model system and bronchodilator responsiveness among BEC donors. Given that BECs were used in co-culture studies after two passages and several media changes, the likelihood that any residual corticosteroids would remain present in the culture media is extremely low; however, we are unable to fully exclude the possibility that lasting effects of these medications via epigenetic or other mechanisms exist in this model system. If these effects were present, they would likely bias data toward the null hypothesis. Studies designed to test the effects of steroids in BECs ex vivo BEC-HLF cross-talk to clinical features among BEC donors, together with our previous reports of asthmatic BECs driving an activated, pro-remodeling phenotype in healthy HLFs13 in ex vivo studies, are consistent with airway biopsy data obtained from children with asthma, and provide additional translational data implicating the airway epithelium and airway remodeling as factors leading to lower lung function among children with asthma8. Additional studies are needed to further elucidate the signaling mediators involved in the intercellular crosstalk between BECs and HLFs  and may provide valuable insight into novel therapeutic targets to prevent the establishment of airway remodeling in asthma.In this study, we have demonstrated an inverse correlation between expression of the myofibroblast marker α-SMA and COL1A1 by HLFs co-cultured with differentiated BECs obtained from asthmatic children and measures of lung function from BEC donors, and that HAS2 expression by HLFs co-cultured with BECs was greater when HLFs were co-cultured with BECs from asthmatic donors with a history of severe exacerbation. These novel findings relating Children ages 6–18 years who were undergoing an elective surgical procedure requiring endotracheal intubation and general anesthesia were recruited for this study as either atopic asthmatic or healthy non-atopic non-asthmatic subjects. A detailed medical history was obtained to ensure that participants met the following inclusion/exclusion criteria. Children with asthma had at least a 1-year history of physician-diagnosed asthma, used a short-acting beta-agonist ≥twice a month or were taking a daily maintenance medication, and were not premature. Children with asthma had one or more of the following atopic features: history of a positive skin prick test or positive RAST for a common aeroallergen, elevated serum IgE (>100 IU/mL), history of physician-treated allergic rhinitis, history of physician-treated atopic dermatitis. Healthy subjects lacked a history of any of the above features and were excluded if they had any other history of atopy.D. farina and D. pteronyssinus), cat epithelium, dog epithelium, Alternaria tenuis, Aspergillus fumigatus, and timothy grass. At a subsequent follow-up visit, FENO was measured according to American Thoracic Society (ATS) guidelines using a NIOX MINO nitric oxide analyzer 37. Spirometry was performed using a VMAX® series 2130 spirometer  to quantify FVC, FEV1, and FEF25–75 according to ATS guidelines. In children with asthma, spirometry was repeated 15 minutes following administration of 2 puffs of albuterol.A blood sample was drawn from each subject and used to measure total serum IgE and RAST allergen-specific IgE to dust mites  and were maintained for 96 hours at which point samples were isolated.Methods for BEC/HLF co-cultures have been described previously12. qPCR was performed using validated TaqMan® probes  for COL1A1, HAS2, and α-SMA using standard methodologies.Total RNA was isolated from HLF cells co-cultured with BECs grown at an ALI as previously described2 were measured using an ELISA from Cayman® Chemicals  per manufacturer instructions, and concentrations of hyaluronan (HA) were measured using an ELISA from R&D Systems® .Supernatant from BEC-HLF co-cultures was collected at the conclusion of experiments and frozen at −80 °C until constituents were measured. BEC-HLF supernatant concentrations of pro-collagen Iα1 were measured using an Enzyme-Linked Immunosorbent Assay (ELISA) from Abcam, Inc.  per manufacturer instructions, concentrations of PGE13.In a subset of BEC-HLF co-cultures , HLFs were mechanically detached from the culture wells and suspended in PBS. Samples were snap frozen in liquid nitrogen until use. Flow cytometry was used to quantify the percentage of myofibroblasts among HLFs as determined by cells staining positive for cytoskeletal α-SMA as we have previously described13.Sterilized 12 mm round glass coverslips were coated with type I collagen and placed in the bottom of one of the replicate chambers of the 12-well plates prior to seeding the HLFs. Following 96 hrs. of co-culture, the coverslips were carefully removed and placed in a separate 12-well plate for IHC. Coverslips containing cells were then washed 3 times in room temperature PBS to remove residual media and were then fixed with 50:50 methanol and acetone at 20 °C for 10 minutes. Coverslips were then washed with PBS and blocked with 10% FBS for 30 minutes. Following this, coverslips were washed again with PBS and then incubated with an α-SMA-FITC primary antibody  as we have previously described38 and Prism® 6.0 software . For comparisons between groups, unpaired t-test was used for normally distributed data, and the Mann-Whitney test was used for non-normally distributed data. To assess correlations between gene expression and lung function parameters, the Pearson correlation coefficient was used for normally distributed data, and the Spearman correlation coefficient was used for non-normally distributed data.For RT-PCR studies, the relative expression of genes was normalized using GAPDH as a non-regulated reference gene. Analyses of RT-PCR results were performed using GenEx version 5.0.1  based on methods described by PfafflApproval: The work presented in this study was approved by the Seattle Children’s Hospital Institutional Review Board. Accordance: The methods were carried out in accordance with relevant guidelines and regulations of the Seattle Children’s Research Institute and the Seattle Children’s Hospital Institutional Review Board. Informed consent: written informed consent was obtained from a parent or legal guardian for all subjects below the age of 18 years, or from the subject if age 18 years. In addition, written assent was obtained for children ≥age 10 years.Dataset 1


To the editor,GFAP) gene. Onset of symptoms can range from infancy to adulthood, with the infantile form being most common and the adult onset form accounting for one third of cases [GFAP gene, as 98% of the individuals have detectable single nucleotide or small indels pathogenic variants. Here we highlight the emerging importance of testing an exon only expressed in an alternative GFAP transcript, which has recently been shown to harbor pathogenic variation found in later-onset patient presentations. A brief survey of clinical testing laboratories reveals that not all include this exon in the reportable range of GFAP testing and consequently fail to reveal the correct genetic diagnosis. This point is highlighted in a 43-year-old male patient was referred to Clinical Genomics for evaluation of hereditary hemorrhagic telangiectasia (HTT) based on positive family history and a variant of uncertain significance (VUS) in ACVRL1 . In addition, he presented with a family history of an unidentified leukodystrophy in mother and maternal uncle, and a potential AD diagnosis based on the characteristic distribution of Rosenthal fibers in uncle’s autopsy and mother’s neurological symptoms. His mother was 67 years old and had history for about four years of progressive worsening gait, spasticity, and slurred speech. MRI revealed leukodystrophy. The patient was asymptomatic with a normal neurological examination  but a brain MRI demonstrated findings previously described in adult-onset AD  done at a CLIA laboratory revealed no pathogenic variants. Additional genetic evaluation for adult-onset leukoencephalopathies was also negative. Subsequent research whole exome sequencing (WES) detected a pathogenic variant in a deep intronic region of the canonical transcript (c.1171 + 472G > A) as well as in exon 7 of an alternative transcript (NM_001131019.2(GFAP):c.1289G > A; p.(Arg430His)). This variant has been previously reported in two affected individuals with clinical and MRI findings of AD and a variant in HDAC6, a possible modifier gene [HDAC6 or other known modifiers in our patient. The GFAP variant is present in 2 of 245,904 alleles in gnomAD [GFAP exon, making this variant a recurrent cause of adult-onset AD. We believe that the available clinical genetic testing for AD should be revisited and include this alternate exon.Alexander disease (AD) is an autosomal dominant progressive leukoencephalopathy caused by heterozygous mutations in the glial fibrillary acidic protein (of cases . A clini


The enclosed work focuses on the construction variables associated with a dual-antigen liposomal carrier, delivering encapsulated polysaccharides and surface-localized proteins, which served as a vaccine delivery device effective against pneumococcal disease. Here, the goal was to better characterize and compare the carrier across a range of formulation steps and assessment metrics. Specifically, the vaccine carrier was subjected to new methods of liposomal formation, including alterations to the base components used for subsequent macromolecule encapsulation and surface attachment, with characterization spanning polysaccharide encapsulation, liposomal size and charge, and surface protein localization. Results demonstrate variations across the liposomal constructs comprised two means of surface-localizing proteins . In general, final liposomal constructs demonstrated a size and zeta potential range of approximately 50 to 600 nm and −4 to −41 mV, respectively, while demonstrating at least 60% polysaccharide encapsulation efficiency and 60% protein surface localization for top-performing liposomal carrier constructs. The results, thus, indicate that multiple formulations could serve in support of vaccination studies, and that the selection of a suitable final delivery system would be dictated by preferences or requirements linked to target antigens and/or regulatory demands. Vaccines have a significant impact on overall global health and are expected to continue to serve as key prophylactic options against current and emerging infectious diseases and cancer ,2,3,4. TStreptococcus pneumonia (S. pneumonia) is responsible for pneumococcal disease, which initiates upon S. pneumoniae establishing an asymptomatic biofilm in a human host  (DGS)–NTA liposomal component containing the cobalt and zinc metals, lipid was firstly dissolved in chloroform. Either cobalt (II) chloride or zinc (II) chloride solutions (20 mg/mL (in deionized (DI) water)) were added, followed by brief sonication (to mix the solutions) and then dialyzed in 1× phosphate-buffered saline (PBS). The resulting DGS–NTA(Co) or DGS–NTA(Zn) lipid components were then used in the final liposomal formulation process. To the lipid mixtures indicated in Relative lipid molar amounts for liposomal formulations are presented in In order to separate liposomes encapsulating polysaccharide 19F from free polysaccharides, 500 µL of the post-extrusion sample was transferred to a 300-kDa centrifugal tube  and centrifuged for 5 min at 4 °C and 1200 relative centrifugal force (rcf). The resulting filtered sample volume was adjusted to the initial volume using PBS and then subjected to protein binding by incubation with GFP (280 µg) for 30 min at room temperature. To separate unbound protein from GFP-bound liposomes encapsulating polysaccharide 19F, samples were subjected to centrifugation purification, and resulting liposomes were further purified with an additional centrifugation step .w/v) phenol and 1.5 mL of concentrated sulfuric acid and vortexed for 5 s. The resulting solution (250 µL) was transferred to a Falcon® 96-well microplate, covered, and incubated for 15 min in a 92 °C water bath, followed by incubation at room temperature for the same duration, allowing the well plates to cool to the point of colorimetric analysis at 480 nm using a Synergy Multi-Mode Microplate Reader . Encapsulated polysaccharide values were calculated by comparison to a standard calibration curve and then ratioed to the initial amount of polysaccharide introduced to the liposomal production process to calculate percentage encapsulation values.Liposome samples (0.6 mL) were mixed with 3 mL of 5%  to determine particle diameter and zeta potential of liposomes at 25 °C with a 4-mW, 633-nm He–Ne laser as the light source at a fixed measuring angle of 90° to the incident laser beam.Liposomal samples were assessed for GFP surface binding efficiency via fluorescence analysis at an excitation wavelength of 359 nm and an emission wavelength of 508 nm using a Synergy 4 Multi-Mode Microplate Reader (BioTek Instruments Inc.). Resulting values were compared to a standard GFP calibration curve and ratioed to the initial amount of protein introduced to the liposomal surface binding step to calculate percentage encapsulation values.Error bars associated with data represent results from three different experimental efforts.A refined process for liposomal formulation based upon a concept called liposomal encapsulation of polysaccharide (LEPS)  was compIn this study, we sought to better characterize and compare the variety of LEPS particles across parameters of liposomal integrity, polysaccharide encapsulation, and protein surface binding. In particular, we analyzed each step of the liposomal formulation process and evaluated new steps in particle purification in an attempt to refine and better characterize the process of LEPS generation. Initially, however, empty liposomal products were tested for size and zeta potential, as presented in S. pneumoniae serotype capsular polysaccharide. Resulting encapsulation results are presented in The LEPS formulation process was next tested for the encapsulation of polysaccharide 19F as a representative Prior to efforts of protein surface binding, a purification centrifugation method was applied to the extruded liposomal products . In so dThe final series of characterization experiments assessed key metrics associated with macromolecule co-localization, namely, polysaccharide encapsulation and protein surface attachment. Protein binding to the surface of the liposomal variants is presented in Complete liposomal products resulting from the protein surface attachment process were finally assessed for particle size distribution as indicated in The enclosed study provided a comparative analysis across the LEPS delivery system for co-localized polysaccharide and protein antigens. In particular, liposomal formulations were generated for different means of protein surface attachment. In the past, we used both metal chelation and biotinylation to secure protein molecules to the surface of liposomal products . As suchThis result suggests that the zinc liposomes are generally less stable than the other variants tested in this study. Comparisons of zeta potential values across Furthermore, the loss of polysaccharide encapsulation for the Co and Zn samples when comparing Finally, we note that, while the Ni sample lost the clean uniform size distribution in A liposomal dual-antigen delivery vehicle was extensively characterized across base construction components involved with surface localization of protein agents. Those components studied in this work that were not designed for protein attachment  demonstrated consistent and uniform polysaccharide encapsulation (≥30% efficiency), size (~100 nm), and zeta potential . Of those liposomes containing metals capable of chelating histidine-tagged proteins (GFP in this study), the nickel sample demonstrated the most consistency across the metrics of size (~200 nm), zeta potential (approximately −30 mV), polysaccharide encapsulation (≥60% efficiency), and protein surface binding (~60% efficiency) and, thus, shows the best potential for utilization as a co-antigen delivery carrier when compared to samples utilizing cobalt or zinc, which resulted in less uniform characterization metrics  and decreased polysaccharide retention following protein exposure (≤30% efficiency).