Eleocharis dulcis, which grows in a highly acidic swamp in Vietnam. Inoculation with CA42 allowed Oryza sativa to grow in the presence of 300 μM AlCl3 at pH 3.5, and biofilms were observed around the roots. Using 16S rRNA gene sequencing analysis, the strain was identified as Pullulanibacillus sp. CA42. This strain secreted large amounts of an extracellular polysaccharide (CA42 EPS). Results from structural analyses on CA42 EPS, namely methylation analysis and nuclear magnetic resonance (NMR), indicated that the chemical structure of CA42 EPS was a glycogen-like α-glucan. Purified CA42 EPS and the commercially available oyster glycogen adsorbed aluminum ions up to 15–30 μmol/g dry weight. Digestion treatments with α-amylase and pullulanase completely attenuated the aluminum ion-adsorbing activity of purified CA42 EPS and oyster glycogen, suggesting that the glycogen-like structure adsorbed aluminum ions and that its branching structure played an important role in its aluminum adsorbing activity. Furthermore, the aluminum tolerance of CA42 cells was attenuated by pullulanase treatment directly on the live CA42 cells. These results suggest that CA42 EPS adsorbs aluminum ions and is involved in the aluminum tolerance mechanism of Pullulanibacillus sp. CA42. Thus, this strain may be a potential plant growth-promoting bacterium in acidic soils. In addition, this study is the first to report a glycogen-like polysaccharide that adsorbs aluminum ions.A novel aluminum-tolerant bacterial strain CA42 was isolated from the aquatic plant  It is estimated that this kind of soil covers approximately 500,000 kmorldwide  and is eorldwide . Below porldwide  and ironorldwide , become orldwide , leadingHowever, few plants can survive in such extreme environments. Since it has become clear that acid- and aluminum–resistant microorganisms coexist in the rhizosphere of such plants, such microorganisms may be used for AASS bioremediation and crop cultivation. The use of these microorganisms in protecting the microenvironment of the plant surface and rhizosphere may yield a new and effective bioremediation method that can be used to recover AASS. Furthermore, protecting only the plant surfaces and rhizosphere is economical and has a minimal environmental impact.Acidocella aluminiidurans AL46 from Panicum repens grown in a highly acidic swamp in the AASS of Vietnam .In our previous study on developing bioremediation measures for AASS, we isolated several bacteria associated with plants grown in the highly acidic aquatic environments of AASS in Southeast Asia and reported new bacterial species , 2010a,b Vietnam . AL46 gr Vietnam . An alumIn the present study, we identified and characterized CA42 and determined the structure and aluminum-adsorbing ability of the EPS it produces. Furthermore, we validated the involvement of EPS in the aluminum tolerance of CA42.2.2.1.E. dulcis by using diluted tryptic soy broth 1:10 (1/10 TS) agar plates, which were prepared by diluting 2.75 g tryptic soy without glucose broth  per liter of water and solidified with 15.0 g of agar (BD) per liter (pH 4.0). Acidic plates were prepared by mixing double-strength TS medium component without agar and 3% agar solution that had been autoclaved separately to prevent agar hydrolysis. The growth of CA42 in the presence of AlCl3 was tested using modified TS (MTS) medium 2SO4; pH 4.0).CA42 was isolated from 3 tolerance of CA42 was examined through a growth promotion test (inoculation experiments) on rice plants (IR36) in Yoshida’s solution (pH 3.5) containing 300 μM AlCl3. Briefly, IR36 seeds were surface-sterilized as described by −2 s−1 illumination from cool-white fluorescent lamps at a 12-h photoperiod at 30°C.Furthermore, the AlCl2.2.The 16S rRNA gene of strain CA42 was amplified via PCR using universal primers , and theThe production of indole acetic acid was tested by using 1/10 TS containing 1% (w/v) glucose and 5 mM tryptophan (pH 3.5 and 6.0).2.3.CA42 EPS was extracted from cells cultivated on 1/10 TS agar plates and purified using enzymatic treatments, phenol-chloroform treatment, and ethanol precipitation, as previously described .2.4.CA42 EPS (50 μg) was completely hydrolyzed using 2 M trifluoroacetic acid (TFA) at 100°C for 3 h. The obtained monosaccharides were labeled with 4-aminobenzoic acid ethyl ester (ABEE) and analyzed using high-performance liquid chromatography , as described previously . To dete2.5.1H) and 125 MHz (13C) using an ECA 500 instrument  or at 600 MHz (1H) and 150 MHz (13C) with an ECZ 600 instrument (JEOL Ltd.). Chemical shifts were administered in parts per million (ppm), with acetone  used as an internal reference for samples measured in D2O solutions. Signals were assigned based on the results of the heteronuclear single-quantum coherence (HMQC) and heteronuclear multiple-bond coherence (HMBC) experiments. 1H NMR chemical shifts of the overlapping signals were obtained from the center of the cross peaks in the 2D spectra.NMR spectra were recorded at 500 MHz  or α-amylase  in 0.1 M acetate buffer (pH 5.0) at 37°C overnight. After denaturation of the enzyme via heat treatment at 100°C for 5 min, the products were recovered via freeze-drying. Oyster glycogen  and amylose (Sigma-Aldrich Co. LLC) were added using the same procedure.2SO4 method , with 0.2 M acetic acid as the eluent. Fractions containing saccharides were monitored using the phenol-H4 method .2.8.3 to a final concentration of 25 μM, and the concentration of free aluminum ions was measured by determining the adsorbance of PCV at 585 nm. The reduction in free Al3+ ions observed in the presence of glucans was considered as the amount of Al3+ adsorbed by the glucan.The aluminum ion-adsorbing ability of CA42 was measured according to methods reported by 2.9.g for 10 min. Cells were resuspended in 1 mL of 0.1 M acetate buffer (pH 5.0). Then, 30 μL of pullulanase was added, and the cells were incubated at 37°C for 4 h. Pullulanase-treated cells were recovered using centrifugation at 10,000 × g for 10 min, and the cells were rinsed with acetate buffer. Then, 5 μL of 1 M AlCl3 solution was added, and the cells were incubated at 28°C for 4 h. The cells exposed to aluminum ions were rinsed with 0.85% (w/v) saline, while the number of living cells was measured by calculating the number of colony-forming units on the 1/10 TS agar plates. Escherichia coli BW25113 was used as a control to evaluate aluminum toxicity. The survival rate of each strain was compared to that of cells that were not exposed to aluminum ions.CA42 was cultured in 1/10 TS broth at 28°C for 2 d, and cells were recovered using centrifugation at 10,000 × 2.10.p values <0.01 were considered significant.SPSS software  was used. Normality of distribution and equality of variance were assessed using the Shapiro–Wilk’ and Levene’s tests. 3.3.1.E. dulcis using 1/10 TS agar plates containing 5 mM AlCl3 (pH 3.5), strain CA42 restored the growth of rice plants in Yoshida’s solution (pH 3.5) containing 300 μM AlCl3 (Among the bacteria isolated from μM AlCl3 . In addiμM AlCl3 . The orgP. naganoensis (AB021193) and P. uraniitolerans (AM931441), respectively. The partial 16S rRNA gene sequence of CA42 was submitted to the GenBank/EMBL/DDBJ databases (Accession number: AB520692). Furthermore, phylogenetic analysis results revealed that strain CA42 belonged to the genus Pullulanibacillus -2-butyl derivatives. The results showed that the glucose had the 1H and 13C NMR spectra, respectively  and eight at δ 70–79 (C-2–C-5). The 1H NMR spectrum displayed several signals at δ 3.31–3.84 (H-2–H-6). The 1H chemical shifts of the CA42 EPS were assigned using two-dimensional total correlation spectroscopy (TOCSY) and two-dimensional double quantum filtered-correlation spectroscopy (DQF-COSY) experiments and showed two spin systems  can be found at: TA and MU contributed equally to the concept, design, and analysis of the data, as well as the writing of the manuscript. JS and SS contributed to the majority of experiments. TY and YM contributed to the structural polysaccharide analysis. All authors contributed to the article and approved the submitted version.This work was partially supported by the MEXT KAKENHI (grant numbers 20 K05735 and 21 K12298) and Nihon University College of Bioresource Sciences Research Grants for 2018, 2019, and 2020.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Human papillomavirus (HPV) E6 and E7 oncogene expression is essential for cervical carcinogenesis. Evidence exists that E6/E7 variants may have different transforming activities while the risk of HPV-16 variants (A/D) differs by race/ethnicity. We determined the type-specific diversity of HPV infection in women with high grade cervical disease or cervical cancer in Ghana and investigated naturally occurring E6/E7 DNA variants in this population. HPV genotyping was carried out on 207 cervical swab samples collected from women referred to a gynaecology clinic at two teaching hospitals in Ghana. HPV-16, HPV-18 and HPV-45 were detected in 41.9%, 23.3% and 16.3% of cases respectively. HPV-16 E6/E7 DNA sequencing was performed in 36 samples. Thirty samples contained E6/E7 variants of the HPV-16-B/C lineage. 21/36 samples were of the HPV-16C1 sublineage variant and all contained the E7 A647G(N29S) single nucleotide polymorphism (SNP). This study reveals the diversity of E6/E7 DNA and the dominance of HPV16 B/C variants in cervicovaginal HPV infection in Ghana. Type-specific HPV diversity analysis indicates that most Ghanaian cervical disease cases are vaccine preventable. The study provides an important baseline from which for the impact of vaccine and antivirals on clinically relevant HPV infection and associated disease can be measured. Next to cancer of the breast, cancer of the cervix is the second most prevalent female cancer with 3151 new cases and 2119 associated deaths recorded in 2018 ,5. As a HPV is the etiological agent of almost all 99.3%) cervical cancer cases globally % cervica,7 and thE6 and E7 are the two main HPV oncoproteins and HPV-16 E6 and E7 gene sequence variants are associated with persistent HPV infection and progression to cancer . Among cAround 89% of cervical cancers are attributable to HPV-16/18/31/33/45/52/58 . Although globally, HPV-16 is the predominant cancer-causing genotype, type-specific prevalence of medically important high risk HPVs (HR-HPVs) varies according to geographic area ,18. In SThe current study sought to determine the type-specific prevalence of cervicovaginal HPV genotypes in a contemporary cohort of cervical lesions and cervical cancers in Ghana. Moreover, we aimed to determine the common, clinically significant, naturally occurring cervicovaginal HPV-16 E6/E7 gene variants in this population. These data will set a baseline to help determine the nature of HPV-associated disease in Ghana and the potential impact of current and future vaccination strategies.22.1Ethical review and approval for the study was obtained from the Committee on Human Research Publication and Ethics (CHRPE), School of Medical Sciences, Kwame Nkrumah University of Science and Technology (KNUST-SMS)/Komfo Anokye Teaching Hospital, Kumasi  and Scientific Technical Committee/Institutional Review Board, Korle-Bu Teaching Hospital, Accra .2.2This cross-sectional study was conducted at the colposcopy service points and Combined Obstetrics and Gynaecology OPD/Family Planning Clinic, Obstetrics and Gynaecology Directorate, of Komfo Anokye Teaching Hospital, Kumasi and the cervical cancer treatment and/or management clinic, Department of Radiotherapy/Nuclear Medicine and the Family Planning Unit, Obstetrics and Gynaecology Department, Korle-Bu Teaching Hospital, Accra.2.3The study population included women with clinical suspicion of cancer of the cervix or confirmed diagnosis of CIN or CxCa who were referred for further assessment and/or treatment/management. Individuals without histology results at the time of sampling were primarily being referred for further assessment via colposcopy and/or biopsy to rule out CxCa. Those individuals already diagnosed with cervical precancer or cancer were recruited before the commencement of treatment. Histology or cytology analysis for diagnosis was made at laboratory centres and reported as part of routine clinical practice. All cervical cancer cases in this study were diagnosed via histology. All study participants presented with at least one gynaecological symptom associated with CxCa. Data collected included histology, age group (in years) 20–39, 40–59, 60–79, ≥80, HPV genotype profile .2.4th October 2018 to 10th November 2020 from consented individuals during routine medical examination. The cervical swab samples were preserved in a 20 ml solution  and stored at 4 °C. 209 out of 280 samples were available for HPV genotyping within the timeframe of the project. 10 ml aliquots of samples were transported at ambient temperature for centralized testing at the Scottish HPV Reference Laboratory (SHPVRL), in Edinburgh, for DNA extraction and HPV genotyping. Nucleic acid was extracted from 1 ml of the samples using the QIAamp Media MDx DNA extraction Kit (Qiagen) via the MDX platform and HPV infection in the samples was detected using the Optiplex HPV genotyping kit (a multiplex HPV genotyping assay by L1 DNA PCR/Luminex-detection) and following the manufacturer's instruction . Optiplex HPV genotyping is a Fluorescent Bead Assay (FBA) for diagnostic determination of 24 HPV types including the 12 established oncogenic high-risk (HR) HPV types (HPV-16/18/31/33/35/39/45/51/52/56/58/59), HPV-68 (probably carcinogenic HPV type), HPV-26/53/66/70/73/82  and HPV-6/11/42/43/44 (low-risk (LR), HPV types). A random selection of HPV-16 L1 DNA positive samples were selected for E6/E7 DNA analysis .Cervical swabs  were collected from from 92.52+, ultra-pure dNTPs, Accuzyme polymerase (high yield-high fidelity polymerase)) (Bioline-UK). Briefly, a total 25 μl PCR volume was used; DNA template; 1x ACCUZYME™ Master Mix (Bioline-UK), 0.2 μM HPV-16 E6/E7 forward (FW) primer; 0.2 μM reverse (RV) primer (Eurogentec-UK)   and nucl2.6https://blast.ncbi.nlm.nih.gov/Blast.cgi). Using the alignment report and printed text file, nucleotide variations observed were summarized in an excel spreadsheet. The classification of HPV-16 variants (lineages/sublineages) in the study was carried out according to the HPV-16 E6 lineage-specific diagnostic and non-lineage SNPs [PCR product purification and Sanger sequencing was carried out by Eurofins (UK). Two replicate sequencing reactions were carried out for each of the PCR-positive samples using forward and reverse sequencing primers as detailed in age SNPs ,20. The age SNPs .2.7The classification of HPV-16 variants in the study samples was carried out as previously reported for lineage-specific diagnostic, likely sublineage diagnostic and non-lineage specific SNP patterns observed within the E6 gene region . Briefly33.1th October 2018 to 10th November 2020 at two teaching hospitals in Ghana  from 280 consented individuals recruited from 9in Ghana . 209 out3.2In total, 22 out of the 24 different HPV genotypes included in the genotyping assay were detected amongst the samples that were HPV-positive . 172/207The three most common HPV types (HPV-16/18/45) were detected in 70 (76.9%) of the 91 CxCa cases positive for a single HPV genotype. HPV-16 alone was found in 39/91 cases (42.7%) and with other HPV genotypes in 55/135 cases (40.7%). HPV-18 was present alone in 19/91 (20.9%) of cases or with other genotypes in 36/135 (26.7%) of cases while HPV-45 was found alone in 12/91 (13.2%), or with other genotypes in 21/135 (15.6%) of cases. One SCC samples was positive for a single HPV-73 (Possibly Carcinogenic HR-HPV Type) and another for HPV-73/82 (possibly carcinogenic HR-HPV Types).For this study population, the majority were in the 40–59 year age range, four were aged between 60 and 79 years and one was in the age range 20–39 years. The average age was 51 years. 77.8% (14/18) of CIN/AIS cases were HPV-positive. Nine were positive for one HPV type and five for more than one HPV type. 55% (11/20 samples) of cases of “other” histology , which r3.3C143G/G145T/T286A/A289G/C335T) specific for HPV-16 B/C variants were observed in 32/36 (88.89%) samples. E6 SNPs specific for HPV-16 B1/C1 sublineages (including the C1 non-lineage specific SNP T109C) were present in 26 out of these 32 samples. Specifically, 20/26 samples had C1 SNPs (T109C/G132T/403G) and 6/26 samples had B1 SNPs (A83C/G132C). Additionally, two samples were classified as HPV-16C2. The remaining four samples mapped to the HPV-16 B/C lineage variant but could not be classified into sublineages. Two of these contained E6 SNP A131 but nucleotides were unchanged at T295 or T350 with respect to HPV-16 reference genome A1. They are likely diagnostic for the B2 lineage [HPV-16 genome variants, including SNPs within HPV oncogenes E6/E7, and the inherent risk associated with some variants (lineages-A/D) differ by race/ethnicity, but African studies are limited. Thus, E6/E7 PCR was performed on a random selection of 48 of the 72 samples  that wer lineage . In a fu3.4Only 4/36 of the studied samples had an E7 DNA sequence that was similar to the reference HPV-16 genome (A1: K02718.1) . The HPV3.5G; A312T; C315T; C620T). Furthermore, another novel SNP candidate, A619T, was also detected in three samples . The other nucleotide changes appear to be novel SNP candidate  general population. In the absence of such a control population we cannot make statements on viral fitness. Thus, this cross-sectional study was carried out to determine the diversity of cervicovaginal HPV genotypes and characterise the naturally occurring HPV-16 E6/E7 DNA SNPs (and lineage/sublineage variants) in individuals with clinical suspicion of cancer of the cervix or diagnosed with CxCa or precancer of the cervix. HPV genotyping indicated 83% of samples were HPV-positive. Of the cervical cancers, all were positive for at least one high-risk type, with 67.4% being positive for type 16 and/or 18.Most of the cervical cancer cases were squamous cell carcinoma (SCC) with a lower percentage of adenocarcinoma (ADC) giving an ADC to SCC ratio of about 1:11, which is typically observed in LMIC that are yet to implement nationwide, organised cervical cancer screening programmes. This ADC to SCC ratio is consistent with previous Ghanaian studies by Denny et al. (2014) who reported a ratio of 1:10  and Awua et al. (2016) who reported a ratio of 1:12 . A worldwide study reported an ADC to SCC ratio of 1:18 (5.3%)  while st87.1% of CxCa cases were HPV-positive. This is similar to some worldwide studies which reported 85%–89.9% positivity ,25. HPV As a single genotyping test was employed in this study and given that some cervical cancers are associated with an HPV-independent pathway to transformation, some HPV-negative CxCa cases were expected. We acknowledge that some of the HPV-negative CxCa cases in this study may represent false negatives due to HPV genome integration leading to a lack of detection by L1 PCR. A reassessment of the HPV L1-negative CxCa samples by targeting other HPV genome region(s) including E6/E7, and through performing deep whole genome sequencing, is likely to increase the percentage occurrence of HPV-positivity in the CxCa cases reported in this study.In the current study, the three most common HPV types detected in CxCa cases positive for a single HPV genotype were HPV-16 (42.9%), −18 (20.9%), and −45 13.2%). These genotypes are the most predominant HPV types in CxCa cases globally (∼74.5%), including in Africa. In West Africa, the percentage occurrence of HPV-16/18/45 in CxCa cases was previously reported to be about 70.1%; HPV-16 (35.5%), HPV-18 (20.1%), HPV-45 (16.5%) .2%. Thes. In a prThis study reported a high prevalence of HPV-16 in high grade lesions and cancer in Ghana, with comparable prevalence to the work of Denny et al., 2014 , howeverOur data is observational but adds to current understanding of HPV E6/E7 variant prevalence and distribution in Africa. Viral oncogene variants have been linked with progression to cancer. This study provides preliminary data in Ghana on E6/E7 DNA sequence polymorphism of HPV-16 within a highly-disease enriched population. The data can serve as the basis for epidemiological studies to ascertain the spread and impact of these HPV type-specific variants in the Ghanaian population, which may be important for screening, prevention, and treatment/management decisions. In future, we would like to complement the work to ascertain the pattern of variants in the healthy population. HPV-16 variants are population specific, and the B/C lineages are dominant in Sub-Saharan Africa . Most GhOur study also confirms B/C lineages to have the E7 SNPs (T789C/T795G) previously reported as non-lineage specific as they are also observed in the A and D lineages ,34. The G; A312 A/T; C315 C/T; C620 C/T with dual nucleotide changes observed this study, only one of the nucleotide changes (un-bolded text) was unchanged as in the HPV-16 reference genome (K02718.1) and/or previously reported [T, was also detected in 3 samples. Thus, five SNPs (C245G/312T/315T/A619T/C620T) detected in two or more samples are being proposed as naturally occurring, candidate novel SNPs [A minority of the study samples (6/36 samples) positive for a single HPV type (HPV-16) showed dual nucleotide changes in a single position possibly due to the presence of multiple HPV-16 variants in the same sample ,37. Therreported ,17,20,38vel SNPs ,38,39.In this study histology was carried out at centres providing routine pathology services in Ghana and a consensus review of histology was not performed as a consequence of this research. We accept that a consensus opinion would have made histology data more robust technically (particularly for the CIN cases). However, we were also keen to deliver a study that reflected the HPV prevalence in routinely diagnosed high grade lesions and cancer in Ghana. Our study is also limited by sample size, especially considering the limited number of samples positive for the HPV-16-B lineage variant. However, the cases were sampled from the two major teaching hospitals in Ghana. Therefore, the distribution of HPV-16 variants we observed among cervical tumours may reflect the wider distribution in the Ghanaian population. Although eleven out of the 48 samples gave no E6/E7 PCR product, all were positive by β-globin and GAPDH control PCR analysis. Factors such as the presence of other HPV genotypes, loss of HPV E6/E7 DNA and/or low viral copy number, might have impacted the PCR outcome. Nonetheless, it would be interesting for future work in Ghana to look at case-control analysis of the spread and impact of HPV variants in Ghana, especially HPV-16-sublineage-C1, with E6 SNPs (109C/132T/403G) detected together with E7 SNP 647G (N29S).4.1This study confirms the dominance of the HPV types-16/18/45 with a high percentage occurrence of single HPV-16 infection in cervical cancer cases in Ghana using L1 DNA genotyping. The study provides the first dataset describing the naturally occurring HPV-16 E6/E7 DNA sequence variance in cervix cancer/precancer cases in Ghana and confirms the dominance of the B/C lineage variant and the rarity of the A lineage in Africa. The study documents the dominance of the HPV-16 C1 lineage variant and the presence of the E7 647G (N29S) SNP in all our C1 lineage cases. Characterisation of E6/E7 DNA sequence variants can be very important when newer treatment/management of cervical cancer cases, that involves HPV-16 E6 and/or E7 is to be considered in the future, in particular if therapeutic E6/E7 vaccines become available. Our data will also serve as a contemporary baseline for future trials where the impact of prophylactic and therapeutic vaccine and HPV antivirals are measured.GK was a recipient of a Commonwealth Split-Site Scholarship grant number GHCN201823 and also received a Ghana Public Universities Senior Members Book and Research allowance for research activities carried out in Ghana.GK: Data curation. Visualisation. Formal analysis. Funding acquisition. Writing – original draft. AS: Data curation, methodology, resources. SAS: Supervision. Writing – original draft review and editing. TOK: Supervision (KATH), Project administration. RB: Data curation. NAT: Conceptualization, Supervision. SAO: Supervision (KBTH), Project administration. FA-Y: Conceptualization, Supervision. KC: Conceptualization. Funding acquisition. Supervision. Writing – review and editing. SVG: Conceptualization. Funding acquisition. Supervision. Project administration. Writing –original draft, review and editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Differences in healthcare delivery systems and pathways to mental healthcare for Canadian Armed Forces (CAF) members and Veterans may contribute to variations in mental health services use (MHSU) and the factors associated with it. We: (1) estimated the prevalence of past 12-month MHSU ; and (2) identified sociodemographic, military-, trauma-, and health-related variables associated with MHSU among CAF members and Veterans.The current study used data from the 2018 CAF Members and Veterans Mental Health Follow-Up Survey (CAFVMHS). Model variables were selected a priori, and their respective associations with MHSU were estimated among (1) CAF members and (2) Veterans using separate multivariable logistic regression models.Similar proportions of CAF members and Veterans reported past 12-month MHSU . For both CAF members and Veterans, meeting criteria for at least one past 12-month MH disorder was associated with past 12-month MHSU [adjusted odds ratio (AOR) = 7.80, 95% confidence interval (CI) = 7.18–8.46; and AOR = 11.82, 95% CI: 11.07–12.61, respectively). Past-year suicide ideation, a history of sexual trauma, and endorsement of adverse childhood experiences were also significantly associated with MHSU among CAF members and Veterans.Similar to previous research, meeting screening criteria for a past 12-month MH disorder was strongly associated with MHSU among both samples. This study extends our existing knowledge about factors associated with MHSU among CAF members and Veterans, and offers direction for future research to increase MHSU. Military careers carry unique occupational risks, including the possibility of exposure to psychologically distressing events. Previous research has documented elevated rates of mental health (MH) disorders, such as major depressive disorder and posttraumatic stress disorder (PTSD), among military members and Veterans compared to the general population –4. A recWithin Canada, pathways to healthcare, including mental healthcare, differ for Regular Force (RegF) members of the Canadian Armed Forces (CAF) vs. Reserve Force (ResF) members and Veterans of the CAF. While serving in the CAF, RegF members, who are full-time employees of the CAF, receive their healthcare through the Canadian Forces Health Services Group, a CAF-specific, federally-overseen healthcare system . Within Upon exiting the CAF and transitioning to civilian life, Veterans of the CAF transition to provincial healthcare coverage . One of The first nationally representative MH survey of CAF members, the Canadian Community Health Survey – Canadian Forces Supplement (CCHS-CFS), was conducted in 2002 and revealed a number of important findings, including a higher past 12-month prevalence of depression and panic disorder among CAF RegF members compared to the general Canadian population  and a reIn the years following the 2002 CCHS-CFS administration, the CAF made significant changes to its MH system, bolstering its capacity to address MH concerns via strategies such as pre- and post-deployment MH screening and improved access to specialized mental healthcare . The 201Among Veterans, existing research related to MHSU has primarily been conducted using smaller national surveys or population-level data from the province of Ontario. The Life After Service Survey (LASS) is a national survey of Canadian Veterans that was administered in 2013, 2016, and 2019 . FindingDespite existing research in Canadian military populations, the impact of differential access to mental healthcare between CAF members and Veterans on MHSU remains understudied. The overarching aim of this study was to describe MHSU among Veterans and currently-serving CAF members, using a follow up sample of those who served as CAF RegF members in 2002. The specific objectives of this manuscript are to: (1) assess the proportion of CAF members and Veterans engaging in past 12-month MHSU; and (2) identify sociodemographic, military-, trauma-, and health-related variables associated with MHSU among CAF members and Veterans. Despite a seemingly more accessible pathway to mental healthcare, we hypothesize that CAF members may be more reticent to report MHSU and therefore have a lower reported prevalence of past 12-month MHSU than Veterans; and that for both samples, health-related variables will be most strongly associated with past 12-month MHSU.The Canadian Armed Forces Members and Veterans Mental Health Follow-Up Survey (CAFVMHS) is a follow-up survey of current and former members of the CAF who participated in the 2002 CCHS-CFS. Interview data from the in-person, computer-assisted interviews were collected between January 8 and May 31, 2018 . Detailsn = 256) were also randomly selected for exclusion from the CAFVMHS due to budget restrictions; and an additional 1,316 opted out of participating in the CAFVMHS, leaving a total sample size of 2,941 before longitudinal weights provided by Statistics Canada were applied and rounded to a base of 20, resulting in a weighted sample of 18,120 CAF members and 34, 380 Veterans. A previous analysis found the attrition rate among eligible participants between the original 2002 CCHS-CFS survey and the CAFVMHS to be 31.2%, which the authors indicated is somewhat lower than studies in the general population with a similar period of follow up time. Being younger, a member of the Army, and a lifetime history of suicide attempt at baseline were associated with increased odds of attrition, while being female, separated/divorced/widowed, or an officer were associated with exclusion from the follow up survey  (CAF members and Veterans were eligible to complete the 2018 CAFVMHS if they: (1) were members of the RegF at the time of the 2002 CCHS-CFS; (2) completed the 2002 CCHS-CFS interview; (3) were still alive at the time of data collection in 2018; (4) could be located for the follow-up survey; (5)  agreed to have the 2018 CAFVMHS data linked to their 2002 CCHS-CFS data. A small number  and a three-level categorical variable for Veterans . Age categories for CAF members and Veterans varied slightly due to differences in age distributions. We did not include age as a continuous variable in the regression model, primarily due to the large number of parameters to be estimated relative to the expected number of events. Sex was assessed as a dichotomous variable . Marital status was dichotomized as married/common-law vs. widowed/separated/divorced/single. Finally, highest level of education was dichotomized as completed secondary school or less vs. any post-secondary education.Veteran status was dichotomized based on whether a respondent was currently serving in the CAF as either a RegF or Reserve Force (ResF) member (CAF member) or had released from the CAF (Veteran) at the time of survey administration. Current military component (for actively serving members) and military component at time of release (for Veterans) was dichotomized as RegF vs. ResF. A history of ever being deployed outside of Canada, regardless of mission type, was assessed using a dichotomous yes/no variable for both CAF members and Veterans. Current rank (for CAF members) and rank at time of release (for Veterans) was operationalized as a categorical variable ; and length of service was assessed as a continuous variable, in years.A history of ever experiencing sexual trauma was assessed with a categorical variable . Adverse childhood experiences were evaluated using nine questions from the Childhood Experiences of Violence Questionnaire . ParticiPresence of selected past 12-month MH disorders were assessed using the World Health Organization Composite International Diagnostic Interview (WHO-CIDI), and were derived based on the Diagnostic and Statistical Manual, Fourth Edition (DSM-IV-TR) . MH disoC statistics were used to explore the crude associations between each variable of interest and the MHSU outcome.Descriptive statistics  were used to describe the sample characteristics by Veteran status. Bivariate analyses in the form of simple logistic regression models and C statistics. All analyses were conducted with longitudinal weights provided by Statistics Canada to provide estimates that were representative of the original 2002 CCHS-CFS sample. 95% CIs were calculated using the 500 bootstrapped weights provided by Statistics Canada.Multivariable logistic regression models were employed to identify variables independently associated with MHSU among (1) CAF members and (2) Veterans. Model fit was assessed with All statistical analyses were conducted using SAS statistical software, version 9.4 .n = 4,880; 26.9%, 95% CI = 23.7–30.2; Veterans: weighted n = 9,460; 27.6%, 95% CI = 25.4–29.8). A slightly higher proportion of Veterans reported engagement in past 12-month MHSU with a psychiatrist or psychologist, whereas almost twice as many active CAF members endorsed MHSU with a nurse/physician assistant or social worker compared to Veterans  = 1.01, 95% CI = 0.91–1.13, .80] see . Among VC-statistic for the final CAF member model was 0.771, whereas the C-statistic for the final Veteran model was 0.847. Models are typically considered a reasonable fit when the C-statistic is higher than 0.7 and a good fit when the C-statistic is higher than 0.8 , any adverse childhood experience, past 12-month SI, and any past 12-month MH disorder. Rank and length of military service were removed due to multicollinearity issues with age. The than 0.8 . The resDespite differences in pathways to mental healthcare, similar proportions of active CAF members and Veterans – slightly more than one-quarter – reported engaging in past 12-month MHSU. A higher proportion of CAF members reported past 12-month MHSU with a nurse/physician assistant or social worker than Veterans, which is likely a reflection of care providers and pathways within the CAF healthcare system. Among both the CAF member and Veteran models, meeting criteria for any past 12-month MH disorder was strongly associated with MHSU. This finding is similar to previous research exploring predictors of MHSU among active CAF members . Past-yeDeployment out of Canada was significantly associated with MHSU among Veterans but not CAF members in both the crude and adjusted regression models. This finding may be at least partially attributable to the healthy soldier effect . SpecifiAfter including all variables in the regression model, age, sex, and highest level of education were statistically significantly associated with MHSU among Veterans, but not CAF members. The non-significant finding for sex among CAF members in the multivariable model was likely due to collinearity between sex, age, education, and other variables included in the model, as these variables were all significant in the simple logistic regression models. In the absence of significant collinearity, a theoretical explanation could be that sex-specific differences in health-seeking behaviors are minimized in still-serving populations, where contact with health services is common ; and thaIn the adjusted model, RegF vs. ResF membership was not significantly associated with MHSU among either the active CAF or Veteran subsample. This is somewhat different than previous research, which has demonstrated lower likelihood of MHSU among ResF members compared to RegF members . Within Our study had a number of notable strengths. First, it included a reasonably large follow-up sample of CAF members and Veterans. The use of survey weights also helped to ensure the generalizability of our findings to the original CAF RegF military population in 2002. The survey design also allowed us to investigate MHSU within current CAF members and Veterans who served during a similar period of time . Additionally, the computer-assisted structured interview used in this study was designed to aid in recall and ensure interview questions were asked in a standardized manner, which reduced the likelihood of interviewer bias.In terms of limitations, it is possible that individuals who declined participation in the CAFVMHS differed from those who did agree to participate, although a previous study exploring attrition between the CCHS-CFS and CAFMVHS surveys found no associations between military status, MH conditions, traumatic experiences, adverse childhood experiences, and attrition . This paThe current study explored MHSU across CAF members and Veterans, and assessed variables associated with MHSU among these subsamples. We found that similar proportions of CAF members and Veterans reported engaging in past 12-month MHSU; and that meeting screening criteria for a past 12-month MH disorder was more strongly associated with MHSU among both samples than any of the other sociodemographic, military-, trauma-, and health-related variables considered in the analysis. Demographic variables, such as sex and highest level of education attained, were associated with MHSU only among the Veteran subsample. This finding may help identify potential avenues for future research related to understanding facilitators and barriers to mental healthcare when accessing it outside of the internal CAF healthcare system, such as access and attitudes toward mental healthcare. This study also extends our existing knowledge about variables associated with MHSU, which offers direction for future research to identify opportunities to increase MHSU. Future research may also benefit from oversampling ResF members so that variables associated with MHSU can be assessed within this subpopulation, and from asking CAF members and Veterans specifically about their health-seeking behaviors and the factors that increase or reduce their access to mental healthcare.STATCAN.infostats-infostats.STATCAN@canada.ca.The data analyzed in this study are subject to the following licenses/restrictions: Data are not publicly available, but access may be obtained from a Statistics Canada Research Data Centre. Requests to access the datasets should be directed to Statistics Canada's Statistical Information Service at The studies involving human participants were reviewed and approved by Statistics Canada. The patients/participants provided their written informed consent to participate in this study.KS and AN conceptualized the study, while KS, AN, AL, RP, MN, SW, and BB conceptualized the analysis plan. AL had access to the data and conducted the analysis. KS drafted the initial manuscript, which was critically reviewed by all authors. All authors reviewed the analytic output and contributed to the interpretations and read and approved the final manuscript as submitted and agree to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Its localization in the outer layer of the Blue–DTAB co-assembly explains the uniaxial growth of spheroidal DTAB micelles to wormlike micelles with increasing [Blue] : [DTAB] ratio from 0 : 1 to 1 : 3. This is in line with the concept of the packing parameter for amphiphilic substances.Contrast variation in small-angle neutron scattering (SANS) was successfully applied to localize the anionic azo dye Blue in co-assemblies with the cationic surfactant dodecyltrimethylammoniumbromide (DTAB). For this purpose, the scattering contrast between DTAB and the aqueous solvent was eliminated by SANS contrast matching, leaving only the scattering signal from Blue to be detected. Results obtained by contrast matching were confirmed by NOESY NMR-spectroscopy, showing that Blue interacts with the positively charged DTAB head groups and with up to the 4 The complementary use of SANS contrast matching and NMR-spectroscopy permits the unambiguous localization of an anionic dye in dodecyl trimethyl-ammonium bromide micelles. Such a response results in a viscosity increase of the solution as directly observable parameter.2–4 Depending on the system, responsivity to composition, pH, ionic strength, temperature, redox reactions, light and even CO2 may be achieved.2,5–11 WLMs are frequently formed by small amphiphilic surfactant molecules upon concentration increase or addition of inorganic salt, another surfactant or hydrotropic solutes.4,9,10,12–14 This provides a versatile, simple and huge toolbox for stimuli-responsive systems.2 Given the variety of possible components of WLM-forming systems, it is important to fundamentally understand intermolecular interactions leading to observable responses. Responsivity to pH, redox conditions or irradiation is frequently achieved by the interaction of surfactant micelles with a compound sensitive to corresponding changes.2,7,15,16 In order to understand the mechanism of responsivity to permit systematic improvement or rational design of similar systems, the localization of such a compound within the surfactant micelle needs to be known.Systems capable of interacting with and responding to their environment are interesting for the design of sensor applications or molecular switches.17,18 In some cases, however, an unambiguous localization of a solute in a co-assembly is not possible with NOESY due to overlap of resonances or ambiguity in peak assignment.5,19 Furthermore, NOESY does not provide information on the morphology of the co-assemblies.It is possible to obtain information on the localization of a compound in a surfactant micelle using nuclear magnetic resonance (NMR)-spectroscopy. Use of the 2-dimensional Nuclear Overhauser Effect spectroscopy (NOESY) is particularly helpful in this regard, as it provides information on spatial proximity.Small-angle neutron scattering (SANS) permits the elucidation of assembly morphology while also providing the possibility to locate an added compound within that assembly using contrast variation. This technique can either be used in combination with NOESY to confirm and extend findings, but may also be applied as stand-alone-method.20,21 It was furthermore successfully used to locate the protein hydrophobin in the outer shell of its co-assemblies with either ionic or non-ionic surfactant and to identify its folding state.22 Penfold et al. located small organic fragrance molecules in micelles formed by the non-ionic surfactant dodecaethylene glycol monododecyl ether (C12E12) by recording SANS curves under different contrast conditions.23 However, Penfold et al. did not perform contrast matching, resulting in ambiguity concerning the solubilisation locus of phenyl ethanol.SANS contrast variation was previously employed to explain morphological transitions of mixed surfactant micelles upon variation of the ratio between both surfactants by identifying their mutual arrangement in the mixed micellar assembly.Herein, the value and feasibility of SANS contrast matching for the localization of an organic azo dye in micelles of a low molecular weight surfactant is demonstrated. The SANS experiments are complemented by NMR experiments and the results jointly discussed.14,24,25 Apart from hydrophobic and electrostatic interactions, π–π stacking of the dye was suggested to have a template-effect on assembly morphology.26 The co-assembly of Blue and DTAB was studied in an aqueous NaHCO3/Na2CO3 buffer with pH = 10.5 and ionic strength I ≈ 0.25 M.WLM formation of the cationic surfactant dodecyltrimethylammoniumbromide (DTAB) and the anionic azo dye Blue  was stud34-DTAB, 99%) was obtained from abcr GmbH, Germany. Completely deuterated dodecyltrimethylammoniumbromide  was obtained from INNOVACHEM SAS, France. Tail-deuterated dodecyltrimethylammoniumbromide  was obtained from C/D/N Isotopes Inc., Canada. The buffer salts sodium carbonate Na2CO3 (≥99.8%) and sodium bicarbonate NaHCO3 (≥99.7%) were obtained from Sigma Aldrich Chemie GmbH, Germany. MilliQ water was used to prepare the NaHCO3/Na2CO3 buffer solutions  for samples for viscosity measurements, Cryo-TEM measurements and determination of the phase diagram by visual inspection. D2O was used to prepare the NaHCO3/Na2CO3 buffer solutions  for SANS and NMR samples. A mixture of 50 vol% H2O and 50 vol% D2O was used to prepare the NaHCO3/Na2CO3 buffer solutions  for the SANS sample containing Blue at a concentration of [Blue] = 5 mM and h34-DTAB at a concentration of [h34-DTAB] = 30 mM. In all cases, the buffer contained NaHCO3 at a concentration of 0.021 M and Na2CO3 at a concentration of 0.079 M, which resulted in the given ionic strength (I ≈ 0.25 M) and pH = 10.5 (pD = 10.7). D2O (99.90% D) was obtained from Eurisotop, France or Sigma Aldrich Chemie GmbH, Germany. Chemicals were used without further purification. Samples were prepared from stock solutions, followed by a minimum equilibration time of 20 h at room temperature prior to analysis.Blue  was provided by KAO GmbH, Germany. Hydrogenated dodecyltrimethylammoniumbromide  was prepared with MilliQ water and used as a solvent in all cases. After each addition of dye stock solution, the sample was vortexed for approximately 30 s and its visual appearance recorded immediately. Some samples were kept and visually inspected after 24 h to investigate long-term stability. Phase diagrams were established at room temperature (≈22 °C).The phase diagram was established by stepwise addition of a 15 mM stock solution of Blue to solutions containing h3/Na2CO3 buffer  were deposited on the surface of a lacey carbon film coated grid  and vitrified by a Leica blotting and plunging device operating at room temperature . The samples were plunged into liquid ethane, which was cooled with liquid nitrogen to achieve sufficiently fast cooling and freezing without formation of ice crystals. Afterwards the sample was transferred into a cryo transfer and tomography holder . The images captured were afterwards processed with a digital imaging processing program .The cryo-transmission electron microscopy (Cryo-TEM) measurements were performed on a JEOL JEM-2200FS electron microscope  operating at 200 kV acceleration voltage and equipped with an “OneView” CMOS camera . 3 μL of a sample containing Blue at a concentration of 10 mM and DTAB at a concentration of 30 mM in an aqueous NaHCOν of solutions containing Blue and h34-DTAB in an aqueous NaHCO3/Na2CO3 buffer  were determined using an Ubbelohde viscometer . Viscosity measurements were performed at 25 °C. The kinematic viscosity was obtained by multiplying the viscometer constant K by the time t the solution needed to drop from the first to the second mark of the Ubbelohde viscometer: ν = K × t.The kinematic viscosities After their preparation from stock solutions, SANS samples were filtered  into dust-free sample vials and equilibrated for a minimum of 20 h at room temperature.25-DTAB/d34-DTAB mixture to the NaHCO3/Na2CO3 buffer prepared in 100% D2O was performed. For this purpose, four solutions containing a total DTAB concentration of 30 mM but varying proportions of d25-DTAB and d34-DTAB were prepared and their SANS-curves recorded. Square roots of resulting forward scattering intensities  were plotted against the volume fraction of d25-DTAB contained in the sample and the match composition of d25-DTAB : d34-DTAB determined to 46 : 54 (v/v) by linear extrapolation towards . This match composition was experimentally confirmed by recording the SANS curve of a corresponding solution, which is shown in the ESI  differences and was estimated according to theoretical SLD differences to:34-DTABh is the theoretical SLD of h34-DTAB and SLDmatched DTAB the SLD of the mixture of d25-DTAB and d34-DTAB (46 : 54). SLD2O solvent100% D is the SLD of the solvent containing 100 vol% D2O. SLD2O solvent50% D is the SLD of the solvent consisting of 50 vol% H2O and 50 vol% D2O. This scaling was solely done for plotting the full contrast SANS curve of the sample with [Blue] = 5 mM and [DTAB] = 30 mM for comparison to full contrast SANS curves of other samples obtained under different SLD conditions. Fitting was performed with the unscaled data.In a second step, “full contrast” SANS curves were recorded, where Blue and DTAB show non-zero contrast relative to the solvent. In the first set of experiments, full contrast SANS curves were obtained by dissolving the above-mentioned 46 : 54 mixture of d25-DTAB : d34-DTAB in a NaHCO3/Na2CO3 buffer in 100% D2O was used.In a third step, SANS curves of “DTAB-matched” samples were recorded, where the scattering contrast of DTAB relative to the solvent is zero but Blue shows a scattering contrast with the respective solvent. For this purpose, the above-described mixture of 46 : 54 (v/v) mixture of dq-range of 0.002 Å−1 to 0.5 Å−1. A circular neutron beam with a diameter of 15 mm was used. (2) Full contrast and DTAB-matched samples containing [Blue] : [DTAB] at a ratio of 1 : 3 were measured at three sample-to-detector distances , ,  at a neutron wavelength of 6 Å to cover a q-range of 0.0014 Å−1 to 0.5 Å−1. A circular neutron beam with a diameter of 14 mm was used. (3) Full contrast and DTAB-matched samples containing [Blue] : [DTAB] at a ratio of 1 : 4 or 1 : 4.5 were measured at three sample-to-detector distances , ,  at a neutron wavelength of 6 Å to cover a q-range of 0.0014 Å−1 to 0.5 Å−1. A circular neutron beam with a diameter of 14 mm was used. Neutrons were detected with a 3He-detector (Reuter-Stokes multi-tube detector consisting of 256 tubes with a tube diameter of 8 mm and a pixel size of 8 mm × 4 mm), detector images azimuthally averaged, corrected to the transmission of the direct beam and scaled to absolute intensity using the Mantid software.27,28 Empty cell and solvent scattering were subtracted from the scattering curves.29 SANS data were collected at a sample temperature of 25 °C.SANS was performed at the small-angle neutron scattering instrument D11 at the Institut Laue-Langevin . Different setups were used: (1) identification of the match point and the measurement of the full contrast and the DTAB-matched sample containing [Blue] = 5 mM and [DTAB] = 30 mM were carried out at three sample-to-detector distances , ,  at a neutron wavelength of 6 Å to cover a 3/Na2CO3 buffer  prepared in D2O was used as a solvent in all cases. Sample solutions were filtered  into the NMR-tube. 1H-NMR and Nuclear Overhauser Effect spectroscopy (NOESY) spectra were recorded with a NMR Ascent 700 spectrometer (700 MHz) equipped with a cryogenic probe with z-gradient at 298 K. 1H-NMR chemical shifts were referenced to residual HDO.30Samples for NMR-spectroscopy were prepared from stock solutions. A NaHCOIq arising from particles with a volume Vpart and a volume fraction of ϕ = N × Vpart/Vtot with N being the number of particles and Vtot the total sample volume is generally described by the following equation:29K is the contrast factor and P(q) is the particle form factor, which describes q-dependent modulations in scattered intensity caused by interferences between neutron waves scattered from the same particle. It is therefore determined by particle geometry. For small q it approaches P(q → 0) = 1.29S(q) denotes the structure factor which arises due to spatial ordering of different particles. In case of negligible inter-particular interactions and ordering, e.g. in sufficiently dilute solutions, S(q) tends to one.29Bincoh is a background signal arising due to incoherent scattering and is therefore termed “incoherent background”. Scattering contrast is caused by differences in SLD between the particle and the solvent. In case of a simple system, where the particles possess a constant SLD1 which is homogeneously distributed throughout the whole particle, and the solvent possesses a homogeneous SLDsolv, K depends on their difference according to:29The absolute scattering intensity 31 For generating and fitting the form factor model of a triaxial ellipsoidal shell, the SASfit software package was used.32 For form factor fits, the volume fraction of scattering particles was derived from the known molar concentration of scatterers and their partial molar volumes. For this purpose, it was assumed that all Blue- and DTAB-molecules, which are present in solution, participate in micelle formation. Partial molar volumes are displayed in the ESI  preliminary information on the system, (2) characteristic features of the SANS-curves, (3) fit quality and (4) morphological transitions expected from existing models and similar systems. The SasView small-angle scattering analysis software with implemented form- and structure factor models was used for analysis of experimental SANS curves in most cases.Table SI1. SLD1, B33 The solid phase was observed to be a crystalline precipitate corresponding to highly ordered Blue–DTAB complexes, which are likely caused by lamellar assembly of both components.24,33ν of Blue–DTAB solutions at a constant concentration of Blue ([Blue]) as a function of DTAB concentration ([DTAB]) and at a constant concentration of DTAB as a function of [Blue] respectively. Both figures show that the kinematic viscosity of samples increases with increasing Blue : DTAB ratio, i.e. upon approaching the phase transition threshold. This increase is not linear but rises with proximity to the 2-phase region. Considering existing literature, similar trends were observed for the change in solution zero shear viscosity η0 upon addition of hydrotrope to surfactant solutions and are often related to morphological changes in the sample.9,14 The observation of a viscosity increase when approaching the phase transition threshold points towards the formation of WLMs, their entanglement and network formation.3,9,34,35To understand phenomena leading to phase separation, the viscosity of samples in the 1-phase region was investigated. 4 These include the addition of salt or hydrotropes such as phenol and phenolate derivatives or the addition of negatively charged azo dyes.9,10,12,14 For this reason, the presence of WLMs in solutions of the negatively charged azo dye Blue and the positively charged surfactant DTAB close to the phase transition threshold was strongly expected.WLM formation from cationic surfactants is a frequently observed phenomenon and can be induced by various triggers.A confirmation of WLMs was obtained from cryo-transmission electron microscopy (cryo-TEM) images of a solution containing [Blue] = 10 mM and [DTAB] = 30 mM. Two images of the same solution at different locations are shown in The formation of WLMs is likely caused by successive growth upon addition of Blue to DTAB. To investigate this mechanism further and to obtain ensemble-average information on the morphology of Blue–DTAB assemblies in solution, SANS was performed on samples with compositions indicated by white dots in q with increasing [Blue] : [DTAB] is evident. This indicates an increase in the radius of gyration of the scatterers and therefore an increase in assembly size.42 Furthermore, the appearance of a power law of Iq ∼ q−1 in the mid-q region with increasing [Blue] : [DTAB] suggests a morphological transition from spherical to cylindrical structures.7,43,44 The SANS curves of samples containing Blue and DTAB at a molar ratio of 1 : 3 furthermore show a crossover region between Iq ∼ q−1 and a power law Iq ∼ qx− with an exponent x > 1. This likely results from the appearance of flexibility upon elongation of the cylindrical structures. A power law behaviour of Iq ∼ q−2 is characteristic for the random walk configuration (θ solvents) and Iq ∼ q−5/3 is characteristic for SANS curves from polymer chains in the self-avoiding random walk configuration (good solvents).36,44 Once length scales probed by q approach the persistence length of those chains, a transition to Iq ∼ q−1 is observed.36 At higher q, a Iq ∼ q−4 decay is observed for all [Blue] : [DTAB] ratios due to the finite cross section of all micelles.4,361 of Blue–DTAB co-assemblies was fitted to account for SLD changes, which are introduced by a variation in the composition of such assemblies through variation of sample composition. The SLD of the solvent was kept constant and the volume fraction of scatterers was calculated as indicated in the section on Data analysis. Fitted SLDs  and of the polar radius of oblate spheroidal DTAB micelles (rminor = 14.0 Å) as well as the radius of the medium semi axis of the Blue–DTAB triaxial ellipsoidal micelles (rmajor = 22.0 Å) and the equatorial radius of DTAB micelles (rmajor = 22.4 Å). This could point towards an uniaxial growth of these micelles.4The SANS curve of the sample containing [Blue] = 5 mM and [DTAB] = 30 mM (ratio 1 : 6) is well described using the form factor of a triaxial ellipsoid. This resembles observations of Bergström and Pedersen who showed that with increasing ionic strength of the solution by NaBr addition, oblate spheroidal micelles turn into triaxial ellipsoids.tor fits  indicaterminor and rmajor) of Blue–DTAB triaxial ellipsoidal micelles .13 Furthermore, Hassan et al. suggested that the polydispersity obtained for the circular cross-section radius of cylindrical hydrotrope–SDS micelles likely results from an effective elliptical cross section of those micelles.47 Whereas cross section radii of Blue–DTAB assemblies remain approximately similar for all sample compositions  and [Blue] = 7.5 mM with [DTAB] = 30 mM (ratio 1 : 4) are well described using either the form factor of a cylinder with elliptical cross section or the form factor of a cylinder with circular, polydisperse cross section. The application of the model of a cylinder with elliptical cross section resulted in a better fit of experimental data. Furthermore, resulting cross section radii agree well with the minor and the medium semi-axes .48 This sequence of assembly morphologies goes along with a change in their spontaneous curvature from high for spherical aggregates to low for aggregates with locally flat interfaces such as bilayers.3The mechanism of the above-described morphological transition of DTAB micelles beginning with oblate spheroidal micelles 3,4Based on the packing parameter it can be understood that intermolecular interactions between surfactant molecules and additives may change the morphology of surfactant micelles by changing the geometry of the amphiphile. In other words, the effective size of the hydrophobic and hydrophilic sections may be influenced by their rigidity and interactions such as hydrogen bonding or electrostatic interactions. Moreover, salt or hydrotrope addition usually reduces the effective head group size of ionic surfactant molecules due to charge screening and a concomitant reduction in electrostatic repulsion between head groups.a0 due to shielding of electrostatic repulsion between positively charged DTAB head groups by negatively charged molecules of Blue. The reduction in a0 due to shielding of electrostatic repulsions must overcompensate an eventual increase of DTAB head group size caused by the geometrical inclusion of Blue into the head group layer to result in a net decrease of a0. This decrease of a0 leads to an increase of the packing parameter and explains the morphological transition from ellipsoidal to wormlike micelles upon successive addition of Blue. In addition to that, an increase of P can also be achieved if parts of the Blue molecules would contribute to the volume v of the hydrophobic surfactant chain. This would require a penetration of Blue into the palisade layer or core of the DTAB micelle. As Blue molecules carry a charge opposite to that of the DTAB head group and are well water soluble, they are expected to remain on the surface or outer layer of the micelle, rendering the reduction of effective head group size by charge screening the most likely reason for micellar growth. To confirm this hypothesis, it is necessary to locate Blue within the Blue–DTAB micelle. For this purpose, a combination of SANS contrast matching and NMR spectroscopy was used.Accordingly, the observed morphological transitions in solutions of Blue and DTAB can be reconciled with a reduction in DTAB head group size i.e. NaHCO3/Na2CO3 buffer prepared in D2O, was eliminated by using deuterated d25-DTAB and d34-DTAB at a volume ratio of 46 : 54, which corresponds to the experimental match composition.SANS contrast matching gives access to one component of the mixed Blue–DTAB micelle by eliminating the contrast of the other component relative to the surrounding medium upon isotopic substitution. Within the investigations presented here, the contrast of DTAB relative to the aqueous solvent, −6 Å−2) during fits. Two fitting strategies were employed: in a first attempt, the SLD of the shell was fixed to SLDshell = 3.028 × 10−6 Å−2. This is the SLD of the one-fold deprotonated Blue based on its elemental composition and on the experimentally determined partial molar volume. In a second attempt the SLD of the shell was fitted. This permits a less dense shell of Blue due to hydration and proximity of the hydrophilic head groups of DTAB. For this reason, the latter strategy is more realistic and resulted into a similar or slightly better fit quality in most cases.50 However, results have to be evaluated with care as size parameters, such as shell thickness, depend on the SLD.52The form factor models used to describe experimental data are visually indicated next to the respective SANS curve in q region, SANS curves containing [Blue] : [DTAB] = 1 : 3 for contrast matched DTAB were described using the form factor model of a rigid core–shell cylinder rather than a flexible core–shell cylinder. This does not depreciate the analysis, as the scattering signal in the evaluated high-q range does not carry information on flexibility of the overall assembly. Furthermore, information on overall assembly size were obtained from full contrast measurements. In addition to that, the evaluation of cross section dimensions should not be hampered by not considering low-q scattering. Therefore, lengths of core–shell cylinders used to describe SANS curves from DTAB contrast matched samples were fixed to values obtained from fitting corresponding full contrast SANS curves. Precise values can be found in the ESI  to the ratio between rmajor and rminor of the respective full contrast model  and, dependent on the employed strategy, SLDshell needed to be fitted. To this end, two strategies were applied. In one strategy, SLDshell was fixed and in the other strategy, SLDshell was fitted. rminor obtained for the elliptical cross section from fitting full contrast SANS curves.As many size parameters were adapted from fits to full contrast SANS curves, only the minor core radius th are larger when SLDshell was fitted and (2) fitted SLDshell are higher than the SLD of negatively charged Blue, leading to lower contrast relative to the solvent. Both observations are easily explained by considering that the shell does not solely consist of a dense layer of Blue, but also solvent molecules and head groups from the SLD-matched DTAB. This leads to an increase in SLD and, as shell thickness and SLD correlate, a thicker shell.52 As fitting the SLDshell resulted in more realistic thicknesses (>1 Å) compared to fitting with the SLDshell being kept constant, further discussion will be based on the fits where SLDshell was fitted.Comparing the results of both fitting strategies based on core–shell structures leads to two main observations: (1) shell thicknesses rcore,minor is smaller than rminor from analysis of full contrast curves in most cases except for the sample with [Blue] = 7.5 mM and [DTAB] = 30 mM. However, rcore,minor and rminor agree within uncertainty of the values for this sample. The relation rcore,minor < rminor implies that the “shell” containing Blue molecules extends into the DTAB micelle. Furthermore, the sum of rcore,minor and th, corresponding to the total minor radius of the elliptical cross section for the core–shell structure, is similar and systematically bigger than rminor for all samples. The relation rminor < rcore,minor + th implies that the shell likely ranges into the surrounding medium. Core radii and shell thicknesses vary only insignificantly with varying sample composition and therefore do not allow to discern any trends concerning the penetration depth of Blue into the DTAB micelle as a function of [Blue] : [DTAB] ratio.From As a major result, SANS based on contrast matching successfully demonstrates that Blue molecules are located close to the DTAB head groups in the Blue–DTAB co-assembly. From the presented results it is reasonable to further conclude that at least a part of the Blue molecules are localized next to the positively charged DTAB head groups.1H-NMR spectrum of pure DTAB is shown in green, along with the peak assignment.53 Peaks are narrow even though [DTAB] = 30 mM lays far above the cmc of 9 mM in the present solvent, where monomeric DTAB molecules and micellar aggregates exist in equilibrium. Narrow peaks for this system suggest rather loosely coupled DTAB molecules with a high degree of relative thermal mobility.54 From 55,56 The methyl group f experiences a small downfield shift. The upfield shift of most proton resonances is mostly caused by an increase in electron density upon addition of the negatively charged Blue. This results into shielding of protons close to the negative charge towards the external magnetic field.17,57,58 Furthermore, the ring current of aromatic moieties of the Blue molecule could affect the chemical shift of neighbouring protons. Depending on the localisation of these protons relative to the aromatic moiety, shielding or de-shielding can occur.59 However, the contribution of such ring current effects to changes in the chemical shift of DTAB proton resonances is assumed to be exceeded by the shielding due to the negative charge of Blue. Depending on the localisation of DTAB protons relative to the negatively charged phenolate group of Blue, some DTAB protons may experience stronger shielding and therefore a more pronounced upfield shift compared to others. This phenomenon may permit deductions on the localisation of Blue in DTAB micelles. To give an example, protons of type a or b experience a stronger change in their chemical shift upon Blue insertion than protons of type f, indicating that the negative charge of Blue is likely to be in direct proximity of type a or type b protons.17,58 Furthermore, the insertion of Blue causes a variation in electron density experienced by different protons of type e, resulting into splitting of the corresponding proton resonance.NMR experiments were performed to further investigate intermolecular interactions between Blue and DTAB in solution. 53,55–57 Increasing the [Blue] : [DTAB] ratio increases the fraction of DTAB molecules interacting with Blue and therefore the weighting of corresponding resonances for the time-averaged NMR spectrum, resulting into an upfield shift of time-averaged signals.The continuous upfield shift of NMR resonances with increasing [Blue] : [DTAB] ratio can be understood considering an equilibrium of multiple DTAB states with protons experiencing different local magnetic environments and therefore exhibiting different chemical shifts. Given a sufficiently fast exchange between these states, a single time-averaged spectrum is obtained.53,58,60 The observations are therefore consistent with the previously made observations of aggregate growth with increasing dye-to-surfactant ratio.Apart from an upfield shift of most DTAB resonances, the addition of Blue results in peak broadening. This may be caused by two effects: (1) a penetration of Blue into the DTAB micelle likely decreases the mobility of DTAB molecules, favouring spin–spin relaxation and leading to peak broadening. (2) The formation of cylindrical micelles and micellar growth with increasing Blue : DTAB ratio results in a slowing of the end-over-end tumbling motion of rod-like micelles in the isotropic micellar phase. This causes a broadening of their proton resonances.1H-NMR spectrum of Blue in the absence and in presence of DTAB. First and foremost, an obvious downfield shift of Blue proton resonances upon addition of DTAB is observed. This is likely caused by electrostatic interactions between Blue and DTAB and corresponding proximity of the positively charged DTAB head group to Blue, which results in a removal of electron density and subsequent de-shielding of Blue proton resonances upon DTAB addition.1758 The observed downfield shift is highest for resonances from protons b′ and c′ in meta-position to the phenolate group of the phenolic sub-unit with Δσ of 0.97 ppm and 0.88 ppm for the sample containing [Blue] : [DTAB] = 1 : 6 compared to pure Blue. The signal position of proton a′ is affected less with Δσ = 0.46 ppm, which is likely due to the delocalization of the negative phenolate charge into its ortho- rather than meta-position based on structural resonance formula.61 Therefore, a′ is likely less affected by the de-shielding effect caused by the proximity of the positively charged DTAB head group than b′ and c′. Proton resonances of the benzothiazole aromatic sub-unit were affected less leading to Δσ = 0.49, 0.33 and 0.55 for d′, e′ and f′ respectively when comparing chemical shifts from the 1H-NMR spectrum of the sample with [Blue] : [DTAB] = 1 : 6 to pure Blue. Considering the stronger de-shielding for resonances of phenolic protons b′ and c′, the phenolate subunit is likely located closer to the positively charged DTAB head group than the benzoisothiazole subunit  can be calculated from 55σfree is the chemical shift of a proton resonance of Blue in the free state, not interacting with DTAB  × 106 L3 mol−3 for a stoichiometry of [Blue] : [DTAB] = 1 : 3.33 Accordingly, with decreasing DTAB excess, the fraction of Blue molecules interacting with DTAB micelles decreases.Previous UV/vis spectroscopic investigations on the presented system indeed showed that the mole fraction of Blue molecules interacting with DTAB changes significantly from 0.95 for [Blue] : [DTAB] = 1 : 6 over 0.85 for [Blue] : [DTAB] = 1 : 4 to 0.73 for [Blue] : [DTAB] = 1 : 3. These values were calculated from the Blue–DTAB association constant of 171H resonances are shifted relative to each other due to variation in the fraction of Blue interacting with DTAB, which makes it possible to display all three NOESY spectra in the same graph. Intensities of cross peaks are negative in all cases, which means that the zero-quantum transition W0 dominates over the double-quantum transition W2 causing the NOE.17,18 This points towards the presence of large aggregates.Spatial proximity of protons up to a distance of about 5 Å can be studied by observing the Nuclear Overhauser Effect (NOE) with 2-dimensional NMR-spectroscopy by recording NOESY spectra.26 Cross peaks between resonances of DTAB were not analysed due to strong overlap. Cross peaks between resonances of Blue and resonances of DTAB carry valuable information on the penetration of Blue into the DTAB micelle and are therefore discussed in the following.Several cross peaks are visible and can be distinguished into (1) cross peaks between resonances of Blue, (2) cross peaks between resonances of DTAB and (3) cross peaks between resonances of Blue and resonances of DTAB. Cross peaks between resonances of Blue suggest spatial proximity among Blue molecules within Blue–DTAB co-assemblies. This proximity could be promoted by the formation of intermolecular π–π-stacking interactions between aromatic moieties of Blue. Such a mechanism was previously suggested for the interaction between oppositely charged dye and surfactant and would promote the formation of elongated, cylindrical assembly structures.et al., where the structure of DTAB micelles was studied using SANS contrast variation and who found, that the first four methylene groups of the DTAB alkyl chain are part of the hydrated shell of the DTAB micelle.50 For this reason, cross peaks should also be observed between Blue proton resonances and resonances of DTAB protons of type d. Unfortunately, an unambiguous assignment of DTAB alkyl chain resonances to corresponding protons is not possible due to peak broadening and shifts induced by the addition of Blue to DTAB solution. However, all Blue proton resonances show one or two additional cross peaks with resonances from protons in the DTAB alkyl chain. Due to the strongly upfield shifted spectrum of DTAB in Blue–DTAB solutions compared to pure DTAB solution, these cross peaks are attributed to the spatial proximity of Blue-protons and the four DTAB protons of type d. As discussed before, the strong upfield shift of these resonances results from an increase in electron density and subsequent shielding of proton resonances due to the interaction of DTAB with negatively charged Blue.17 Finally, the observation of cross peaks between DTAB proton resonances and all Blue proton resonances confirms the penetration of Blue molecules into DTAB micelles rather than a part of the Blue molecules sticking out of the micelles  the existence of various states of Blue and DTAB, i.e. Blue molecules interacting with DTAB micelles and Blue molecules being dissolved as single molecules in solution, generates a time-average resonance due to fast exchange between these two states. This resonance shifts with an increasing or decreasing fraction of Blue molecules interacting with DTAB. (2) A change in Blue- and DTAB-resonances due to continuous shielding or de-shielding effects upon variation of the composition of Blue–DTAB micelles.To summarize results from the NMR-spectroscopic study, Blue was found to penetrate into the hydrated part of the DTAB-micelle, which corresponds to the Stern layer or outer region .49,50 Th3/Na2CO3 buffer with ionic strength of I = 0.25 M was investigated in detail. Small-angle neutron scattering clearly showed that addition of Blue in successive steps induces a uniaxial growth of pure, oblate spheroidal DTAB micelles to prolate ellipsoidal micelles and further on to cylinders and WLMs.The co-assembly of the azo dye Blue and the cationic surfactant DTAB in an alkaline NaHCOth methylene group, thereby showing that the penetration boundary of Blue molecules reaches the inner boundary of the outer layer  a reduction in the effective surface area per DTAB molecule or (2) an increase of the DTAB hydrophobic chain volume. Following the localisation of Blue close to the surfactant head group, an increase in the DTAB hydrophobic chain volume upon addition of Blue may be ruled out as the principal cause for the ellipsoid-to-cylinder transition. Therefore, Blue addition must cause a reduction of the effective surface area per DTAB molecule. This is likely caused by partial neutralisation of the positive DTAB head group charge upon interaction with negatively charged Blue, which overcompensates an increase in the DTAB head group size due to inclusion of the Blue molecule.The complementary use of SANS contrast matching and NMR-spectroscopy is powerful for the localization of solutes in surfactant micelles. Not only does each method reveal a set of highly relevant information, but also does their complementarity reduce doubts in data interpretation, which could be caused by ambiguities of results obtained by just one method. As the feasibility of performing SANS contrast matching experiments for the localization of solutes in small surfactant micelles was demonstrated, such use may be particularly helpful in systems, where NMR peak assignment is hampered by peak overlap or unexpected changes in chemical shifts.There are no conflicts to declare.NA-005-D3NA00556A-s001