E6/E7 mRNA testing for human papilloma virus-induced high-grade cervical intraepithelial disease (CIN2/CIN3): a promising perspective
Published: 29/04/2015 Received: 10/12/2014

Massimo Origoni email@example.com
Department of Gynaecology & Obstetrics
School of Medicine
Vita Salute San Raffaele University
20132MilanoItaly

Paolo Cristoforoni
Polo Oncologico Villa Montallegro
16145GenovaItaly

Guia Carminati
Department of Gynaecology & Obstetrics
School of Medicine
Vita Salute San Raffaele University
20132MilanoItaly

Chiara Stefani
Department of Gynaecology & Obstetrics
School of Medicine
Vita Salute San Raffaele University
20132MilanoItaly

Silvano Costa
M.F. Toniolo Hospital
40141BolognaItaly

Maria Teresa Sandri
Division of Laboratory Medicine
European Institute of Oncology
20141MilanoItaly

Luciano Mariani
HPV-UNIT
Regina Elena National Cancer Institute
00144RomaItaly

Mario Preti
Unit of Preventive Gynaecology
European Institute of Oncology
20141MilanoItaly

E6/E7 mRNA testing for human papilloma virus-induced high-grade cervical intraepithelial disease (CIN2/CIN3): a promising perspective
Published: 29/04/2015 Received: 10/12/201410.3332/ecancer.2015.5331 Review ecancer 2015, 9:533 Correspondence to: Massimo Origoni. Review 2 www.ecancer.org ecancer 2015, 9:533human papillomavirusHPVE6E7mRNAcervical cancerHPV-DNA
Since the introduction of biomolecular testing for the identification of high-risk human papillomavirus DNA (hrHPV-DNA) in cervical cancer preventive strategies, many interesting aspects have emerged in this field; firstly, HPV-DNA testing has been demonstrated to have better sensitivity than conventional cytology in several settings: screening, triage of ASC-US and in follow-up after treatment. Despite this, some limitations of these new technologies have also been underlined: the major issue is the low specificity of the tests, which cannot discriminate between regressive and progressive infections. Thus, recent research has moved the attention towards novel markers of progression that could more precisely detect cases at real risk of cancer development. In view of the fact that progression to cancer is dependable of the E6/E7 proteins integration and transforming action, the overexpression of E6/E7 transcripts has been seen as a valuable marker of this risk. This review aims to summarise the literature data on this topic and to provide a clear view of the emerging perspectives.

Background

To reduce morbidity and mortality caused by cervical cancer, in the past 40 years, most developed countries have introduced cervical screening programmes based on cytological examination of cervical smears (Pap test). However, cervical cytology demonstrated a less than optimal sensitivity, and advances in the knowledge of the natural history of the disease have more recently suggested alternative approaches. Persistent infection with high-risk genotypes of the human papillomavirus (hrHPV) has been recognised as the necessary cause of cervical cancer [1]. This led initially to the adoption of molecular tests evaluating hrHPV-DNA presence for the triage of minimally abnormal Pap smears. More recently, evidence has accumulated which implies that a significant improvement of the effectiveness of cervical cancer screening can be achieved by using hrHPV-DNA testing as a primary screening tool. As a matter of fact, hrHPV-DNA testing has a better sensitivity for clinically relevant cervical lesions (cervical intraepithelial neoplasia 2 or worse, CIN2+) than cytology. Moreover, molecular testing proved to offer better protection against high-grade CIN and cancer than cytology in subsequent screening rounds, allowing extension of the screening intervals with favourable logistic and economic implications. Nonetheless, hrHPV infections are rather common in the general population and most of them are transient, spontaneously regressing in a 18-24 months' period. In fact, the positive predictive value (PPV) of a positive hrHPV-DNA test is rather low (i.e., only a small proportion of the women who test positive for high-risk HPV-DNA will have CIN2+ lesions at the time of the test or will develop it in the next few years), and up to 10% of the screened population test positive and will consequently need some form of second-level triage. The research effort has therefore been focused on potentially alternative or supplementary testing methods capable to limit unnecessary follow-up procedures of women with clinically not relevant, transient hrHPV infections. So far, most of the national guidelines on cervical cancer prevention consider reflex cytology (i.e., the lecture of the cytological sample collected at the time of primary HPV-DNA testing but stored and not initially evaluated) a valuable triage tool for hrHPV-DNA-positive women. Nevertheless, up to 8% of hrHPV-positive women with normal cytology have or will develop in the subsequent years a CIN2+ lesion, and the previous knowledge of HPV-DNA status might influence the subjective reading of cytology. Therefore, there is a strong need for biomarkers that will allow risk-stratification of HPV-positive women with normal cytology or, alternatively, capable of replacing cytology as a triage tool. Considering that the progression to cervical malignancy requires the overexpression of the E6 and E7 genes of the integrated hrHPV genome, demonstration in cervical samples of hrHPV E6/E7 transcripts might be more specific than hrHPV-DNA testing alone for the detection of CIN2+ lesions. Nowadays, transcript analysis is feasible on cervical scrapings, because the introduction of liquid-based cytology has resulted in collection media that preserve RNA sufficiently to allow in vitro amplification and detection.

hrHPV E6/E7 mRNA detection

Currently, three commercial tests exist for detecting hrHPV E6/E7 messenger RNA (mRNA). The PreTect HPV-Proofer (NorChip AS, Klokkarstua, Norway) and the NucliSENS Easy Q HPV (bioMérieux) are based on the same technology, but are produced by different companies, with small differences in mRNA extraction protocol and data analysis [2]. These tests are nucleic acid sequence-based amplification (NASBA). The assay is an isothermal, RNA real-time amplification method that detects E6/E7 mRNA and perform genotyping of the five more oncogenic hrHPV types (16, 18, 31, 33, and 45) [3]. The NASBA amplification is based on primer extension and transcription by coordinated activities of three enzymes (RNase H, reverse transcriptase and RNA polymerase). The amplification reaction is isothermal and performs at 41°C; due to this relatively low temperature, contaminating DNA cannot be amplified. Real-time detection using molecular beacons is based on the measurement of the time-related detection of increase in a fluorescent signal. This signal is produced only after specific binding of the molecular beacon to an amplicon (specific HPV mRNA) ( Figure 1). The measurement is performed in combination with the amplification step (real time) [4]. Both tests received the CE-IVD mark. (Figure 1) The APTIMA HPV Assay (Hologic, San Diego, CA ) is a target amplification nucleic acid probe test for the qualitative detection of E6/E7 viral mRNA from 14 hrHPV types (16/18/31/33/35/39/45/51/52/56/58/59/66/68). The assay involves three main events in a single tube: target capture, target amplification by transcription-mediated amplification (TMA) and detection of the amplification products. TMA is a transcription-based nucleic acid amplification method that involves two enzymes, reverse transcriptase and RNA polymerase ( Figure 2). The reverse transcriptase is used to generate a DNA copy of the target mRNA sequence containing a promoter sequence for RNA polymerase. RNA polymerase produces multiple copies of RNA amplicon from the DNA copy template [5]. The detection of the amplicons is performed by the hybridisation protection assay (HPA) using single-stranded nucleic acid probes with chemiluminescent labels that are specifically complementary to the target amplicons. The selection reagent differentiates between hybridised and unhybridised probes by inactivating the label on the unhybridised probes. During the detection step, light emitted from the labelled RNA/DNA hybrids is measured as photon signals in a luminometre and are reported as relative light units (RLU) [6]. APTIMA assay could also perform genotyping for HPV 16 single and 18-45 in pool but requires a separate APTIMA 16, 18/45 genotype assay. The APTIMA HPV assay is a FDA and CE-IVD approved test. www.ecancer.org ecancer 2015, 9:533

State of the art

In 2009, a group of researchers from Catholic University, Rome, published their findings on 180 colposcopically and histologically evaluated women, studied for the presence of HPV-DNA (HC II) and mRNA (NucliSENSE) [7]. As expected, fewer women tested positive for mRNA transcripts than hrHPV-DNA, and there was a fairly good correlation between the degree of positivity and the severity of the lesion. DNA from hrHPVs was found in 57.8% of the specimens; E6 and E7 transcripts were found in 45%. The rates of detection of HPV-DNA and of E6 and E7 transcripts were 33.3% and 25%, respectively, for specimens with normal findings; 51.4% and 31.9%, respectively, for specimens with cervical intraepithelial neoplasia grade 1 (CIN1); and 61.1% and 44.2% for specimens with CIN2, respectively. All specimens with CIN3 and 95.5% of specimens from patients with squamous cell carcinoma were positive by both assays. Overall, the mRNA tests showed a better specificity than the DNA tests for high-grade lesions (72.7% and 56.2%, respectively) and a higher positive predictive value (59.3% and 49.0%, respectively). The authors suggested that mRNA assays could be more powerful than DNA testing for predicting the risk of progression and offer a strong potential as a tool for triage and patient follow-up. In 2011, Burger from Norway performed a systematic review to determine the test performance of HPV mRNA testing compared to DNA testing using CIN2+ as the target condition [2]. The reference standard used to diagnose precancerous lesions was histologically confirmed cervical intraepithelial neoplasia 2+ (CIN2+). Sensitivity, specificity, positive and negative likelihood ratios and diagnostic odds ratios were evaluated for each study. The included studies (11 of 3271 publications) were of varying methodological quality and predominately performed in a secondary screening setting. Eight studies investigated the performance of the PreTect HPV-Proofer/NucliSENS EasyQ, two studies investigated the performance of the APTIMA assay, and one study investigated both mRNA tests on the same patient samples (Table 1). Due to few studies and considerable clinical heterogeneity, pooling of data was not possible; the authors, however, compiled a 'best evidence synthesis' for E6/E7 mRNA HPV testing. Sensitivities ranged from 0.41 to 0.86 and from 0.90 to 0.95 for the PreTect HPV-Proofer/Easy Q and APTIMA assay, respectively. Specificities ranged from 0.63 to 0.97 and from 0.42 to 0.61 for the PreTect HPV-Proofer/Easy Q and APTIMA assay, respectively. The SROC curves for both mRNA tests were to the left of the diagonal, and the APTIMA assay performed closest to the DNA tests. The authors concluded that mRNA tests have diagnostic relevance, but additional studies and economic evaluations have to be performed in order to make a solid conclusion regarding the clinical applicability of HPV mRNA testing. In 2012, Monsonego published the results from the FASE Study (French APTIMA screening evaluation), a multicentre trial involving 5,000 French women designed to assess the performance of APTIMA HPV assay (AHPV), hybrid capture 2 (HC2), in-house PCR genotyping, and ThinPrep LBC in population-based screening [8]. AHPV had the highest absolute risk of both histological endpoints, detecting 5% to 15% more CIN3+ and CIN2+ lesions, respectively, than LBC. Compared to the HC2 assay, the relative risk of AHPV positivity was 24% to 29% higher, with a significant difference in CIN2+ detection. With LBC as reference, AHPV had the best sensitivity/specificity balance measured by AUC (area under ROC curve) comparison test (significant for CIN2+), and the colposcopy referral rate (9.2%) comparable to that of LBC (8.7%). Data from the FASE study corroborate the suitability of AHPV in a primary cervical cancer screening. Several studies have investigated the PreTect HPV-Proofer assay on biopsy and cervical scraping samples and showed that the ratio of hrHPV E6/E7 mRNA positivity to hrHPV-DNA positivity increased along with the histological severity of dysplasia. This suggests a higher specificity of this mRNA assay for high-grade cervical lesions compared to HPV-DNA assays [9]. In a recent study by Rijkaart [10], 375 women among 13.401 women participating in a population-based cervical cancer screening programme in the Netherlands were stratified for CIN2+ risk according their hrHPV E6/E7 mRNA status. Study populations included 202 women with normal cytology, 88 with borderline or mild dyskaryosis (BMD) and 85 with moderate dyskaryosis or worse (>BMD); all of the women tested positive for hrHPV-DNA by the GP5+/6+ PCR assay used in the screening programme. A positive mRNA test result conferred an increased CIN2+ risk in hrHPV-DNA-positive women with normal cytology (0.55, 95% CI: 0.34-0.76) versus 0.20 (95% CI: 0.07-0.33) in mRNA-negative women. In hrHPV-DNA-positive women with BMD or >BMD, the result of the mRNA test did not influence the CIN2+ risk. Therefore, the authors concluded that mRNA testing by PreTect HPV-Proofer might be of value to select hrHPV-DNA-positive women with normal cytology at an increased risk and therefore requiring immediate referral to colposcopy. A very recent study from Korea evaluated in a series of 337 ThinPrep samples a commercial diagnostic kit targeting a HPV E6/E7 mRNA based on RT-qPCR assay and reported 91% sensitivity and 98.6% specificity for CIN2+ cervical lesions [11]. The overall positive rates of the HPV E6/ E7 mRNA RT-qPCR assay were 100% (24/24), 86% (38/44), 100% (7/7), 37% (10/27), 15% (5/32), and 1% (3/203) in SCC, HSIL, ASC-H, LSIL, ASC-US, and normal samples, respectively. The tested kit showed a remarkably higher sensitivity (91 vs. 41%) compared to the realtime NASBA assay, considered in the study as the reference standard. The tested assay showed also a better correlation between rate of positivity and severity of the cytological lesion and stressed the possible relevance of mRNA detection assays in the clinical management of women screened for cervical cancer precursors. In the discussion section, however, the authors underlined that the poor performance of the NASBA assay could be related to a series of technical limitations due to the adopted method of RNA extraction and amplification www.ecancer.org ecancer 2015, 9:533 and stressed the importance of larger studies evaluating a more representative patient population as well as the importance of taking into account geographical differences, due to variable HPV genotypes prevalence in cervical cancer specimens. In summary, the performance in terms of sensitivity/specificity of the 5 hrHPV mRNA tests (PreTect HPV-Proofer and NucliSENS EasyQ HPV) cannot be pooled with the 14-h HPV mRNA test APTIMA. The APTIMA test is more similar to HPV DNA test like Hybrid Capture 2 regarding sensitivity and specificity, while the five genotype HPV E6/E7 mRNA tests have much higher specificity than the APTIMA assay [12,13].

HPV mRNA testing in the triage of LSIL

HPV-DNA tests have low specificity and a high positivity rate in LSILs, making HPV-DNA test not particularly useful in triage of LSIL. The 14 genotype APTIMA test and especially the five genotype HPV E6/E7 mRNA tests have higher specificity and a low positivity rate in LSIL. A low positivity rate translates into a low referral rate for colposcopy, which is very appealing for triage situations [13][14][15][16]. Women with minor cervical lesions have a small but significantly increased risk of developing cervical cancer compared to women with normal smears. The purpose of triage is to identify women needing further follow-up by colposcopy and biopsy to detect high grade cervical dysplasia or cancer (CIN2+). In cases of atypical squamous cell of undetermined significance (ASC-US), it is now widely recognised that triage with an HPV-DNA test is more sensitive, but less specific than repeat cytology [17,18]. For low-grade squamous intraepithelial lesions (LSIL), however, recommendations regarding the best triage method are conflicting and vary from repeat cytology, HPV testing or direct referral to colposcopy [19,20]. Meta-analyses indicate that triage of LSIL with a high-risk HPV DNA test is not more sensitive and substantially less specific than repeat cytology [15,16]. In some studies, HPV-based triage of LSIL in women older than 30-35 years has been suggested [21,22], but other studies have not confirmed this [23,24]. Defining the best strategy to triage LSIL lesions is, therefore, identified as a priority for research [25]. In a Norwegian study of 522 women with LSIL, 207 had biopsies and 125 of them had CIN2+. The sensitivity and specificity of repeat cytology (ASC-US or worse) were 85.7% (95% (CI): 72.1, 92.2) and 54.4 % (95% CI: 46.9, 61.9), respectively. The sensitivity and specificity of the HPV mRNA test were 94.2% (95% CI: 88.7, 99.7) and 86.0% (95% CI: 81.5, 90.5), respectively. The PPV of repeated cytology was 38.4% (95% CI: 29.9, 46.9) compared to 67.0% (95% CI: 57.7, 76.4) of the HPV mRNA test. In conclusion, five genotype HPV E6/E7 mRNA testings were more sensitive and specific than repeated cytology in triage of women with LSIL cytology. In addition, the HPV mRNA test showed higher PPV. These data indicate that the HPV mRNA test is a better triage test for women with LSIL than repeated cytology [15,16]. In a meta-analysis by Arbyn, the pooled sensitivity and specificity of APTIMA to triage LSIL were 96.7% (95% CI: 91.4-98.9%) and 38.7% (95% CI: 30.5-47.6%) for CIN3+. APTIMA was as sensitive as HC2 but more specific (ratio: 1.35; 95% CI: 1.11-1.66). In both triage of ASC-US and LSIL, APTIMA is as sensitive but more specific than HC2 for detecting cervical pre-cancer [14]. In follow-up of patients with negative colposcopy/negative cervical histology, the five genotype HPV E6/E7 mRNA tests have a higher specificity and a higher PPV for CIN2+ than repeated cytology, suggesting a 'test and treat' approach for HPV mRNA-testing in women above 40 years [26]. In clinical studies, histology is used as a gold standard for detecting CIN2+. Unfortunately, colposcopy does not have optimal sensitivity for CIN2+. The National Health Service Cervical Screening Programme (NHSCSP) Guidelines for Colposcopy and Programme Management, which guides British practice, asks for evidence of a colposcopic accuracy of 65% [27]. Zuchna, reported 66.2% sensitivity of CIN2+ when up to three guided cervical biopsies were taken regarded as a diagnostic test with the cone specimen as reference standard [28]. Using digitised cervical images from 919 women referred for equivocal or minor cytologic abnormalities into the ASCUS-LSIL Triage Study, Massad, reported 39% sensitivity for CIN2+ [29]. Hence, all women with negative colposcopy and biopsies after abnormal cytology and/or HPV testing have to be followed.

HPV mRNA testing in the follow-up after conservative treatment for CIN2/CIN3 lesions

Women conservatively treated for CIN2-3 lesions are considered at high risk of developing invasive carcinoma for many years, and therefore, they require continued surveillance. In particular, between 5% and 20% of women treated for CIN2+ will develop recurrent disease within 3 years [30]. For this reason, an adequate follow-up is mandatory. Currently, despite the now perceived low sensitivity, Pap test is widely used in the follow-up of patients treated for intraepithelial neoplasia and European guidelines for cervical screening policy recommend 6-, 12-and 24-month cytology after CIN treatment [31,32]. Observational studies demonstrated that HPV-DNA testing has a significant higher efficacy compared to cytology in predicting persistent disease or relapse, and hence, the detection of a persistent infection with hrHPV genotypes has the potential to improve patient management. In point of fact hrHPV-DNA testing is significantly more sensitive (90%) compared with follow-up cytology (70%) [33,34] and currently ASCCP recommends the use of HPV-DNA testing with cytology (co-testing) at 12 and 24 months following treatment for CIN2/3 [35]. Actually, eradication of the clinical lesion does not necessarily mean eradication of all the infected tissue and up 40% of treated women still remains hr-HPV positive due to the fact that hrHPV-DNA testing does not distinguish between a persistent infection from a new, transient one [36,37]. This is the reason why hrHPV-DNA testing owns a lower specificity than cytology in identifying relapse or persistent disease. Recent prospective studies have documented that rates of residual or recurrent disease in women with persistent hrHPV 16 and/or 18 are higher than in women with other hrHPV types, stressing the importance of type-specific genotyping determination after treatment for CIN2+ [38][39][40]. As hrHPV-genotyping assays have become increasingly utilised in clinical practice, further research will be needed to determine whether type-specific HPV results can improve the identification of women most likely www.ecancer.org ecancer 2015, 9:533 at higher risk of developing cervical disease after treatment. A recent systematic review highlights that new HPV infections are a potential source for future disease risk after treatment for cervical pre-cancer and cancer [41]. More recently, it has been suggested that the oncogene activity by hr-mRNA transcripts [42] may be a better indicator of women at risk of persistence or relapse of the disease, showing an higher specificity than DNA-based assays, having a better specificity and negative predicting value (NPV) than hrHPV-DNA testing [43], while others underline that HPV E6/E7 mRNA is not useful to detect relapse [2]. In order to assess the performance of different biomolecular tests, alone or in cotesting with cytology, an Italian prospective study is on-going to evaluate the prediction of persistence/recurrence rate in women treated for CIN2+. Preliminary results seem to indicate that cotesting Pap + HPV E6/E7 mRNA shows the best specificity, while hrHPV genotyping retains the higher prediction for CIN2+ persistent or recurrent disease.

Conclusions

In general, HPV E6/E7 mRNA testing has been demonstrated in several published experiences to have the characteristic of an accurate diagnostic tool in the field of cervical cancer prevention [44]. For this reason, current evidence suggests that mRNA testing may be regarded with promising possibilities in terms of implementation of the diagnostic accuracy of pre-neoplastic cervical lesions in different settings [45][46][47][48][49][50], including glandular lesions and adenocarcinoma [51]. Primary screening has obviously been investigated more widely, representing the most interesting application option in view of the recent evidence-based shift from cytology to molecular-based screening programmes worldwide. Compared to hrHPV-DNA testing, which actually represents the most validated alternative to cytology in screening settings, mRNA tests present the valuable improvement of a better specificity, and consequently, higher positive predictive value (PPV) towards highgrade cervical lesions (CIN2+) [52]. In terms of test of cure after treatment, though with the significant limitation of a lack of large controlled trials, interesting preliminary data indicate a possible integration of mRNA testing with cytology. HPV E6/E7 mRNA testing may serve as a more specific discriminator between transient cervical dysplasia and potentially progressive lesions. Accordingly, testing for high-risk HPV E6/E7 mRNA might reduce the psychological burden associated with HPV-DNA testing [53].

In conclusion, since the beginning of the molecular era in the prevention of cervical cancer, many steps forward have already been made to overcome the limitations of cervical cytology alone; mRNA testing actually represents a new challenge, with the promising possibility of being integrated in the pool of valuable molecular tools that will hopefully lead to the elimination of invasive cervical cancer in women in the near future. www.ecancer.org ecancer 2015, 9:533

www.ecancer.org ecancer 2015, 9:533

Figure 1 .
1The NASBA RNA amplification technology.

Figure 2 .
2The TMA RNA amplification technology. www.ecancer.org ecancer 2015, 9:533

Table 1 .
1Performance of E6/E7 HPV mRNA and DNA tests. www.ecancer.orgecancer 2015, 9:533

AcknowledgmentsThe authors wish to acknowledge the guidance and mentorship of Mario Sideri in this work and the entire activity of the Italian HPV Study Group (IHSG). He has been among the most intelligent and visionary colleagues we ever encountered and we were blessed with his friendship. The 'Mario Sideri Italian HPV Study Group -IHSG' endeavours to continue its research and teaching initiatives in his memory.
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Distributed under Creative Commons CC-BY 4.0 Effect of shampoo, conditioner and permanent waving on the molecular structure of human hair
Published 1 October 2015

Yuchen Zhang
Department of Physics & Astronomy
McMaster University
HamiltonONCanada

Richard J Alsop
Department of Physics & Astronomy
McMaster University
HamiltonONCanada

Asfia Soomro
Department of Physics & Astronomy
McMaster University
HamiltonONCanada

Fei-Chi Yang
Department of Physics & Astronomy
McMaster University
HamiltonONCanada

Maikel C Rheinstädter
Department of Physics & Astronomy
McMaster University
HamiltonONCanada

Distributed under Creative Commons CC-BY 4.0 Effect of shampoo, conditioner and permanent waving on the molecular structure of human hair
Published 1 October 201510.7717/peerj.1296Submitted 2 June 2015 Accepted 15 September 2015Corresponding author Maikel C. Rheinstädter, firstname.lastname@example.com Academic editor Li Zuo Additional Information and Declarations can be found on page 13 OPEN ACCESSHair care productsHuman hairMolecular structureX-ray diffractionShampooConditionerPermanent waving
The hair is a filamentous biomaterial consisting of the cuticle, the cortex and the medulla, all held together by the cell membrane complex. The cortex mostly consists of helical keratin proteins that spiral together to form coiled-coil dimers, intermediate filaments, micro-fibrils and macro-fibrils. We used X-ray diffraction to study hair structure on the molecular level, at length scales between ∼3-90Å, in hopes of developing a diagnostic method for diseases affecting hair structure allowing for fast and noninvasive screening. However, such an approach can only be successful if common hair treatments do not affect molecular hair structure. We found that a single use of shampoo and conditioner has no effect on packing of keratin molecules, structure of the intermediate filaments or internal lipid composition of the membrane complex. Permanent waving treatments are known to break and reform disulfide linkages in the hair. Single application of a perming product was found to deeply penetrate the hair and reduce the number of keratin coiled-coils and change the structure of the intermediate filaments. Signals related to the coiled-coil structure of the α-keratin molecules at 5 and 9.5Å were found to be decreased while a signal associated with the organization of the intermediate filaments at 47Å was significantly elevated in permed hair. Both these observations are related to breaking of the bonds between two coiled-coil keratin dimers.SubjectsBiophysics How to cite this article Zhang et al. (2015), Effect of shampoo, conditioner and permanent waving on the molecular structure of human hair. Astbury WT, Sisson WA. 1935. X-ray studies of the structure of hair, wool, and related fibres. iii. The configuration of the keratin molecule and its orientation in the biological cell. Doucet J. 2000. Exploring a biological tissue from atomic to macroscopic scale using synchrotron radiation: example of hair. Cellular and Molecular Biology 46:1005-1016. Busson B, Briki F, Doucet J. 1999. Side-chains configurations in coiled coils revealed by the 5.15-meridional reflection on hard-keratin X-ray diffraction patterns. Journal of Structural Biology 125:1-10 DOI 10.1006/jsbi.1999.4082. Busson B, Engstrom P, Doucet J. 1999. Existence of various structural zones in keratinous tissues revealed by x-ray microdiffraction.

INTRODUCTION

The human hair has a complex internal structure consisting of many layers including, from outside in, the cuticle, the cortex and the medulla, all bound by the cell membrane complex. These structures and corresponding length scales are shown in Fig. 1. The medulla is a loosely packed, disordered region at the centre of the hair shaft. It is surrounded by the cortex, the structure that makes up majority of the hair fibre and contains keratin proteins and structural lipids. The individual keratin fibres coil together in an organized fashion to form higher level structures, including dimers, protofilaments and intermediate filaments.

The cuticle is a layer of overlapping dead cells that form a protective barrier against the outside environment (Robbins, 2012).  Figure 1 Schematics of the X-ray setup and example X-ray data. The hair strands were oriented in the X-ray diffractometer with their long axis along z. Two-dimensional X-ray data were measured for each specimen covering distances from about 3-90Å including signals from the coiled-coil α-keratin phase, the intermediate fibrils in the cortex and from lipids in the membrane complex. The 2-dimensional data were integrated and converted into line scans and fit for a quantitative analysis.

Details of hair structure organization have been studied in the past with X-ray diffraction. In particular, the α-helical keratin structure within the cortex has been extensively studied since the 1930s (Astbury & Street, 1932;Astbury & Woods, 1934;Astbury et al., 1935;Pauling & Corey, 1951;Fraser & Macrae, 1958;Fraser, MacRae & Rogers, 1962;Fraser, MacRae & Rogers, 1964;Busson, Briki & Doucet, 1999;Er Rafik, Doucet & Briki, 2004). In addition to X-ray diffraction, other techniques have been employed. For instance, electron microscopy has significantly contributed to studying the organization of intermediate filaments . X-ray microdiffraction contributed to exploring the structure of the cuticle, cell membrane complex, lipids and keratin (Busson, Engstrom & Doucet, 1999;Kreplak et al., 1999b;Ohta et al., 2005;Kajiura et al., 2006). Infrared microspectroscopy was used to further elucidate the complex hair structure (Briki et al., 2000).

The development of a breast cancer screening protocol using X-ray diffraction of human hair has been extensively studied by James and Corino (James et al., 2000;Corino & French, 2000;Corino et al., 2009). This screening protocol is already in the clinical trial stage, with an overall accuracy of greater than 77% and a negative predictive value of 99% (Corino et al., 2009). Aside from breast cancer, there exist other diseases that affect hair molecular structure. For instance, Giant Axonal Neuropathy (GAN) is a rare, fatal genetic disease with the presentation of curly hair that significantly differs from that of the parents (Kuhlenbäumer, Timmerman & Bomont, 2014). Similarly, Menkes disease affects hair. Infants having Menkes disease will exhibit short, coarse and twisted hair due to disorders of copper transport (Kaler, 2010). Both these diseases exhibit pili torti, the twisting of the hair shaft by 180 • , which is characteristic of defective internal hair structure (Kaler, 2010;Kuhlenbäumer, Timmerman & Bomont, 2014).

There is potential in studying the X-ray diffraction patterns of the hair from individuals afflicted with these diseases in the search of specific disease markers. However, such a diagnostic tool is only valuable, if common hair care products and chemical treatments, such as shampoo, conditioner and permanent waving, do not affect the internal hair structure. Studies in the past have determined that hair care products may affect hair structure, as observed via X-ray diffraction, electron microscopy, and atomic force microscopy (AFM) (Sano, 2006;Nishikawa et al., 1998;Gould & Sneath, 1984;Tate et al., 1993;Bhushan, 2008).

In a previous study, we used X-ray diffraction to analyze the structure of human scalp hair of various individuals with differing characteristics (Yang, Zhang & Rheinstädter, 2014). However, the subjects in this study used differential amounts of shampoo and conditioner on a regular basis. While we found no relationship between this differential use and their respective hair patterns, the comparison was done across various individuals. In this work, we conducted a study on a single subject to solely examine the effect of hair care products and treatments uninfluenced by other factors.

The purpose of this project was to observe the effect that hair products and treatments have on the X-ray profiles using hair from a single subject to maintain baseline consistency in other characteristics. Shampooing, conditioning, perming and various combinations of these treatments were performed on the subject's hair, the strands were scanned using X-ray diffraction, and the resulting data were analyzed for differences in signals.

Effect of shampoo and conditioner on hair

The main purpose of shampoo is to remove dirt and oil from the surface of the hair fibres and the scalp, while the main purpose of conditioner is to ensure that the hair is smooth for combing. Some commercial shampoos may also have additional components to control dandruff and condition hair. Similarly, conditioners may also prevent static electricity, improve the cosmetic shine and increase protection. Due to the multitude of purposes of these hair care products, they contain a long list of ingredients with various effects on the hair. Shampoos typically contain a primary and a secondary surfactant for thorough cleaning, a viscosity builder, a solvent, conditioning agents, pH adjuster and other non-essential components such as fragrance and colour for commercial appeal. Conditioners usually contain silicone polymers to increase shine and soften hair, cationic polymers such as quaternized nitrogen compounds to reduce static electricity, bridging agents to increase absorption, viscosity builder, pH adjuster and components for commercial appeal (Robbins, 2012;Preedy, 2012).

Since shampoo removes soil and lipids at the surface level, and the active conditioner ingredients only adsorb to the surface, most of the shampoo and conditioning interactions can be expected to occur at the first few layers of the cuticle. The cortex will only be impacted if damage to the cuticle is extensive and the cortex has been exposed. There is evidence that shampooing can result in hair damage in this case; however, the effect is believed to be mostly restricted to the cuticle level. For instance, abrasive action of the shampooing process can damage the keratin and non-keratinous structures at the surface of the hair (Gould & Sneath, 1984). Yet, other studies suggest that the application of conditioner after shampoo increases the protection at the surface of hair fibres, preventing damage from destructive processes such as grooming and bleaching (Tate et al., 1993).

Studies have shown that application of shampoo once can extract approximately 50% of total extractable lipids in hair (Shaw, 1979), and that 70-90% of total lipid can be extracted with repeated shampooing (Koch, 1982). However, it is believed that largely surface level lipids are removed. The cell membrane complex at the cuticle level contains covalently bonded 18-methyl eicosanoic acid (18-MEA) lipids attached to a proteinous cell membrane. At the hair's outer surface, free lipids within the 18-MEA lipid layers are removed during shampooing (Robbins, 2012). Internal lipids found deeper within the hair shaft are not affected to the same extent as the surface lipids. Internal lipids can travel to the surface layers via the process of diffusion after repeated shampooing (Robbins, 2012).

In summary, shampooing has the effect of extracting surface lipid matter and repeated shampooing could result in damaging structures at the hair surface. These changes could be reflected in the X-ray profiles, particularly the lipid signals. Conditioner ingredients adsorb to hair surface and act as a protective layer preventing this damage. Thus conditioner application, either alone or after the use of shampoo could also affect the X-ray signals.

Effect of permanent waving on hair

Permanent waving is the process of converting straight hair into curled hair using a chemical process. This process takes many steps to complete and involves the breaking and restructuring of the disulfide bonds within hair. In the first step, disulfide bonds are cleaved using reducing agents such as thioglycolates and bisulfites. The reduction permeates throughout the cuticle and into the outer layers of the cortex. Alkaline reductive solutions are used to lift the scales of the cuticle such that the reducing agent can effectively diffuse into the cortex. In a second step, the hair fibres are shaped into the preferred curl and held using curlers. During this time, molecular reorganization occurs in the cuticle layers, as well as in the outer levels of the cortex. Lastly, the fibres are neutralized with an oxidizing agent such as hydrogen peroxide (Robbins, 2012;Bolduc & Shapiro, 2001).

The damaging effects of permanent waving are widely known. Particularly, the use of hydrogen peroxide in the oxidation step increases surface damage and decreases the stiffness of the fibre (Tate et al., 1993). Studies have shown structural changes in both protein composition and lipids after perming (Johnson, 1997). A recent study suggests that prolonged exposure of over 30 min to reducing/oxidizing agents during the permanent waving process reduces the contents of α-helical proteins. This effect is observable in the wide angle X-ray diffraction (WAXD) pattern as a reduction in the intensity of signals related to packing at the level of individual keratin fibres (Nishikawa et al., 1998). The decrease in keratinous protein has also been observed by another group of researchers using a novel method for quantitative analysis of hair components (Kon et al., 1998). Additionally, the permanent waving process was found to extract internal lipids from the cell membrane complex of the fibre, as one study discovered a significant decrease in polar internal lipids after perming treatment (Hilterhaus-Bong, 1989). Moreover, the permanent waving process converts the hydrophobic hair surface to a more hydrophilic one. Thus, in interactions with shampoos, this would result in greater lipid removal from the cuticle (Robbins, 2012).

In summary, permanent waving produces more significant changes to hair structure than shampooing and conditioning, and its effects permeate into the cortical layers of hair fibres. Observable changes to the X-ray profile are, therefore, expected to incur as a result of perming.

MATERIALS & METHODS

Preparation of hair samples

This research was approved by the Hamilton Integrated Research Ethics Board (HIREB) under approval number 14-474-T. Written consent was obtained from the participating individual. Samples were obtained from a single subject to eliminate the effect of other potential factors on the observed signals, since the X-ray profiles of various individuals were observed to be different in the previous study (Yang, Zhang & Rheinstädter, 2014). Using samples from a single subject ensures that any changes observed in the signals are the result of the hair treatments. The subject is female, of Asian descent and naturally has slightly wavy and black hair.

The shampoo and conditioner used in this experiment were a common, top-selling brand found in drugstores in US and Canada. When studying the list of ingredients we found that many shampoos contain conditioner ingredients and vice versa, such that a clear distinction is often difficult (Robbins, 2012). The particular brand was chosen for relatively little overlap between shampoo and conditioner ingredients. The active ingredient in the shampoo was sodium laureth sulfate and the active ingredients in the conditioner included cetearyl alcohol, stearamidopropyl dimethylamine and dimethicone. The permanent waving product used was an acid wave product with the main ingredient being glycerolmonothioglycolate (GMT). Since GMT is not stable in water, the reducing formula consisted of two parts that were mixed right before application to produce GMT (Robbins, 2012). The reducing formula was applied to virgin hair, which was subsequently held in position with curling rods. Though the particular product did not require heat application, the hair dresser chose to use an electric heater for thirty minutes to improve treatment result. After heat treatment, the reducing formula was washed off with warm tap water. Then neutralizing lotion was applied to entirety of the head and allowed to settle in for five minutes. Afterwards, the curling rods were removed and hair was rinsed with warm tap water. Lastly, the hair was blow dried.

At the beginning of the study, around 200 hair strands were obtained from the subject's frontal scalp region, less than 10 cm from the roots. These 200 strands were divided into four approximately equal portions with around 50 strands each. In preliminary experiments where we optimized the X-ray setup and hair preparation we found that a sample that contained ∼50 hair strands gave the best signal. Fewer hairs resulted in faint signals, thicker hair bundles led to absorption and a reduction of signal and increase of background. Shampoo and conditioner were applied differentially to each of the samples according to Table 1. Then the subject underwent permanent waving treatment in all regions of her scalp hair at a local barber shop using the procedure described above. Another 200 strands were obtained from the same area of the head. These 200 strands were again divided into four 50 strand portions and differential amounts of shampoo and conditioning was applied to each. The treatments applied to each sample can be found in Table 1.

Prior to shampoo and conditioning treatments for both non-permed and permed series, the subject did not wash her hair for the duration of one week to facilitate the build-up of lipids on the hair surface. A previous study noted that one application of shampoo can extract 50% of total extractable lipids in hair after not shampooing hair for 2.5-3 days (Shaw, 1979). Thus, the period of a week was determined to be enough time for lipid build-up. Shampoo and conditioning treatments were performed after the hair strands had been collected from the subject's scalp. Solutions of shampoo and conditioner were prepared in a 1:9 ratio with tap water to give a 10% solution. To shampoo or condition the sample, the hair was held using a tweezers at one end and rinsed in the corresponding 10% solution for 2 min continuously. Then the solution was replaced by tap water and the sample was rinsed for another 2 min. Each sample was allowed to dry for at least 1 hour to eliminate the effect of water on the keratin signals, since water is known to cause swelling of hair (Robbins, 2012). This process was designed to mimic the normal application of shampoo and conditioner during a single shower.

The treated samples were then cut into strands ∼3 cm long. Care was taken to prevent stretching or deforming the hair strands during this process. Each sample was mounted onto a flexible cardboard apparatus as shown in Fig. 2A. The cut-out at the middle of the apparatus allows for the scattering of X-ray signals on the hair sample. The cardboard apparatus is then mounted vertically onto the loading plate of the Biological Large Angle Diffraction Experiment (BLADE) using sticky putty as shown in Fig. 2B. All hair samples were scanned at ambient conditions, i.e., a room temperature and humidity of 28 • C and 50% RH.

X-ray diffraction experiment

X-ray diffraction data was obtained using the Biological Large Angle Diffraction Experiment (BLADE) in the Laboratory for Membrane and Protein Dynamics at McMaster University. BLADE uses a 9 kW (45 kV, 200 mA) CuKα Rigaku Smartlab rotating anode at a wavelength of 1.5418Å. Focusing multi-layer optics provides a high intensity parallel beam with monochromatic X-ray intensities up to 10 10 counts/(s × mm 2 ). By aligning the hair strands in the X-ray diffractometer, the molecular structure along the fibre direction and perpendicular to the fibres could be determined. We refer to these components of the total scattering vector, ⃗ Q, as q z and q ∥ , respectively, in the following. An illustration of q z and q ∥ orientations is shown in Fig. 1.

The result of such an X-ray experiment is a 2-dimensional intensity map of a large area of the reciprocal space of −2.5Å −1 < q z < 2.5Å −1 and −2.5Å −1 < q ∥ < 2.5Å −1 . The corresponding real-space length scales are determined by d = 2π/|Q| and cover length scales from about 2.5 to ∼200Å, incorporating typical molecular dimensions and distances for primary and secondary protein structures and lipid structures.

Integration of the 2-dimensional data was performed by a script developed in our lab using MATLAB (MathWorks, Natick, Massachusetts, USA). By integrating of the peak intensities along the q z and the q ∥ directions, one-dimensional data along each of the two directions were produced. The q z intensity was integrated azimuthally for an angle of 25 • over the meridian. The q ∥ intensity was integrated azimuthally for an angle of 25 • over the equator.

The instrumental background was determined from an empty scan, where no hair was mounted on the apparatus and resulted in a sharp ring at ∼3Å. This area was, therefore, excluded from the hair data analysis in the following. The corresponding area is covered by a red circle in the 2-dimensional data.

RESULTS

Two-dimensional X-ray intensity maps were collected for all hair samples. These graphs exhibit the characteristic hair signals observed in the previous study (Yang, Zhang & Rheinstädter, 2014). Similar to the previous study, the hair strands were oriented with In (A), the untreated sample is compared with the shampooed sample. In (B), the untreated sample is compared with the conditioned sample. In (C), the untreated sample is compared with the sample with both shampoo and conditioners applied. There were no major changes observed in signals of interest. The red outer ring is the result of a known background from the apparatus and has no relation to hair internal structure.

the long axis of the hair parallel to the vertical z-axis, and the displayed (q z ,q ∥ )-range was designed to cover the length scales of the features of interest. The assignment of biological structures to scattering signals is depicted in Fig. 1. The 2-dimensional data for all hair samples show very good agreement in signal position and shape, suggesting that the hair products used to not have a significant impact on the structure of hair on the observed length scales. However, the perming treatment lead to observable changes in intensity for some signals. Figure 3 show split comparisons between the original untreated hair and hair with various combinations of shampoo and conditioner treatments. The 2-dimensional intensity maps of the respective samples were cut in half and combined such that the left half shows one sample and the right half a different one. This presentation makes a qualitative comparison between the scans easy. The red rings in each of the 2-dimensional plots mark the position of a known background from the apparatus used, as discussed under Materials and Methods. There are no observable differences in the scattering from untreated hair and hair treated with shampoo or conditioner from untreated and shampooed or conditioned hair samples within the resolution of this experiment, suggesting that likely no internal lipid or keratin structures were significantly altered during the application of shampoo and conditioner. A single application of shampoo, conditioner or the combination of the two does not seem to have an effect on the observed signals within the resolution of this experiment.

Effect of shampoo and conditioners

Effect of perming

The effect of permanent waving on hair structure is illustrated by the split comparison 2-dimensional intensity map in Fig. 4A. Similar to the shampoo and conditioner trials, position of peaks associated with individual keratin and lipids agree between samples. Differences in peak intensities are observed suggesting that perming effects the molecular organization within the hair. There are two observable differences between the untreated and the permed samples at the small angle q ∥ region, as shown in Fig. 5. The signals corresponding to this region are best visualized using the small angle X-ray diffraction (SAXD) technique to quantitatively analyze the position and amplitude of signals. The signals at 90Å, 47Å and 27Å were observed in the q ∥ direction of the SAXD data, in agreement with the results of the previous study (Yang, Zhang & Rheinstädter, 2014). As seen in Fig. 5, the difference between untreated and permed hair observed on the 2-dimensional maps and the line graphs is represented by the disproportional increase in the 47Å signal and slight reduction of the 27Å signal after perming. This is expected to be the region were keratin intermediate filaments demonstrate packing along the cross-sectional plane of the hair into macro-fibrils (Yang, Zhang & Rheinstädter, 2014). This suggests that structural changes do occur at the intermediate filament level after permanent waving.

Another change is observed after integrating the WAXD data along both the q ∥ and q z directions. In the q ∥ -direction, the peak at 9.5Å is noticeably decreased in comparison to the 4.3Å peak after perming. In the q z -direction, the peak at 5.0Å is also decreased in comparison to the peak at 4.3Å. This change is visualized in Fig. 6. Both of these peaks The wide angle X-ray diffraction data for the q z direction. There is a noticeable decrease of the 5.0Å signal corresponding to α-keratin helix distances in comparison with the lipid signal at 4.3Å. (B) The wide angle X-ray diffraction data for the q ∥ direction. There is a noticeable decrease of the 9.8Å signal corresponding to α-keratin helix distances in comparison with the lipid signal at 4.3Å.

refer to the coiled-coil protein structure, i.e., the α-helical keratin packing distances, while the peak at 4.3Å refers to lipid packing. The peak at 2.08Å −1 or ∼3Å is the result of instrumental background and has no relation to hair structure.

Shampoo and conditioner usage is often advertised to repair damage to hair ("liquid hair repair," "advanced care and repair" or "keratin repair shampoo"). In this study, we applied combinations of shampoo and conditioner to permanently waved hair to examine whether these hair care products had an impact on the changes incurred during perament waving. As shown in Figs. 4B-4D, the signal in the SAXD region at 47Å remained after a single application of shampoo or conditioner. Thus, a single application of shampoo and conditioner was found to have no restorative effect to the changes sustained by hair fibres during permanent waving. Moreover, the wide angle region of the 2-dimensional data remains unchanged. As with untreated hair, a single application of shampoo or conditioner to permed hair did not result in changes to internal hair structure on the length scales of the intermediate filaments or keratin proteins studied in this experiment.

DISCUSSION

Shampoo and conditioner appear to have no observable effect on the internal keratin and lipid signals visualized using X-ray diffraction. This is in congruence with the fact that these hair care products should solely act at the outer cuticle layer of the fibre. The potential extraction of free lipids by a single use of shampoo is not significant enough to be observed. It is also possible that the lipid signals observed in the experiment scatter from covalently bonded internal lipids rather than free lipids. Moreover, the single use of shampoo is not expected to damage cortical structures. Damage to hair reported from shampoo likely arises from repeated applications, leading to erosion of surface structures. The data also suggests that the adsorption of conditioning ingredients to the outer surface of hair fibres will not impact the X-ray signals.

Since the visualized X-ray profiles are not affected by application of shampoo and conditioner, it is possible to use this method to study the disorganization of keratin fibres without concern over subjects' differential use of these hair care products.

However, for permanently waved hair samples, differences were observed in the intensities of signals. At the wide angle region, there is a reduction of α-helical signals seen in the treated samples along both equatorial and meridional directions in comparison to the lipid signals. The keratin proteins in the cortex are known to organize in bundles, whose structure is dominated by α-helical coiled-coils (Pauling & Corey, 1950;Pinto et al., 2014;Yang et al., 2014;Yang, Zhang & Rheinstädter, 2014). Coiled coils consist of α-helices wound together to form a ropelike structure stabilized by hydrophobic interactions. The coiled-coil motif is found in about 10% of the proteins in the human genome (Neukirch, Goriely & Hausrath, 2008).

The main features of this motif is a ∼9.5Å (corresponding to q ∥ ∼ 0.6Å −1 ) equatorial reflection corresponding to the spacing between adjacent coiled-coils and a ∼5.0Å meridional reflection (corresponding to q z ∼ 1.25Å −1 ) corresponding to the superhelical structure of α-helices twisting around each other within coiled-coils (Crick, 1952;Cohen & Parry, 1994;Lupas & Gruber, 2005). These signals were observed in the X-ray data. A decrease of the corresponding peak intensities in permed hair is not related to a decrease in the number of keratin proteins in the hair, however, indicative of a reduced number of coiled-coil proteins, i.e., indicative of a breaking of the bonds between two coiled dimers. We note that any X-ray diffraction experiment is not sensitive to protein monomers but the scattered signals stem from protein dimers. Perming products, therefore, seem to deeply penetrate the hair and break up hydrophobic bonds between coiled-coil keratin proteins. The basic building block of each intermediate filament is such a dimer of a coiled-coil pair of proteins. The SAXD signals observed in this experiment agree with those of previous experiments. Signals in the SAXD region of 90Å, 47Å and 27Å along the cross-sectional direction have long been recognized as representative of distances between microfibrils immersed in a sulfer rich matrix (Er Rafik, Doucet & Briki, 2004). Many models have been developed in the past 50 years with an attempt to characterize the arrangement of microfibrils in the formation of intermediate filaments, with some assuming a ring-core arrangement while others stating a uniform profile (Er Rafik, Doucet & Briki, 2004). Yet the actual structure of intermediate filaments remain elusive and different model are used to describe SAXD profiles in the literature.

The signal at 47Å was found to be significantly increased in the permed samples in Fig. 5. This effect was observed in all four permed samples in comparison to the non-permed samples. This is in agreement with the observation that the permanent waving process produces changes at the intermediate filament level (Robbins, 2012).

The small angle peak pattern in Fig. 5 with peaks at 90Å, 47Å and 27Å is compatible with a 7 dimers arranged on a hexagonal lattice, as shown in Fig. 7A  of this structure was calculated and shows excellent agreement with the experimental data.

Perming was found to have two effects on the hair structure: (1) a decrease of the coiledcoil signal indicative of a reduced number of protein dimers and (2) an increase of the 47Å signal. The fact that the small angle peak positions do not change after perming is indicative that the overall structure of the intermediate filaments is not affected by perming. However, decomposition of the keratin coiled-coils may lead to the internal fibre structure shown in Fig. 7B, where individual keratin molecules arrange in a hexagonal pattern. We have calculated the diffraction patterns of different filament arrangements. The small angle signals in our experiments are consistent with a hexagonally packed organization of dimers, as shown in Fig. 7A, with a spacing between the dimers of 90Å. Decomposition of the keratin coiled-coils into single helices may change the internal fibre structure as shown in Fig. 7B, where individual keratin molecules arrange in a hexagonal packing. The corresponding calculated peak pattern results in an increase in the 47Å signal, in agreement with the experimental findings. While the calculations assumed that all dimers split into monomers, the slight decrease in the coiled-coil peaks indicate that most proteins still form coiled-coils.

Technically, packing of the filaments in non-permed hair was described by a primitive monoclinic unit cell, space group P2, with lattice constants a = c = 90Å and α = 90 • , β = 60 • and γ = 90 • . After perming, a base centered monoclinic lattice, space group C2, with lattice constants a = c = 90Å and α = 90 • , β = 60 • and γ = 90 • well describes the experimental findings. The corresponding diffraction patterns were calculated and convoluted with the instrumental resolution, which leads to a finite peak width. The results are shown in Fig. 7 and the corresponding [hkl] reflections are marked in the figure. The pronounced change between the two peak patterns is the increase in the [200] reflection, indicative of the occurrence of monomers at positions between two dimers.

In future studies involving subjects with recent permanent waves, care must be taken to rule out the smaller intensity of the coiled-coiled keratin signals and the increased intensity of the 47Å signal as a possible structural defect. However, other abnormalities observed in the profile, such as a split, extra or shifted signal could be assigned to structural abnormalities independent of the perming treatment. CONCLUSIONS X-ray diffraction is able to visualize signals corresponding to keratin coiled-coils, intermediate filaments and lipids in the cell membrane complex. The hair structure of a single individual under shampoo, conditioning and permanent waving treatments was studied in the natural state using wide angle and small angle X-ray diffraction. Signals corresponding to the coiled-coil structure of keratin fibres and lipids from the cell membrane complex identified previously study were observed. For shampooing and conditioning treatments, no differences were observed before and after treatment within the resolution of this experiment, suggesting that their effects are limited to the surface of the hair fibre, leaving the inner cortex unchanged. However, permanent waving is a more invasive chemical treatment that results in both a decrease in α-keratin signals and the changes of a signals at the intermediate filament level related to breaking of the bonds between two coiled dimers. Otherwise, the general shape of both equatorial and meridional profiles remain unchanged. Thus, use of hair care products, such as shampoo and conditioners, would not impact the molecular structure of hair, while permanent waving treatments lead to slight alterations. These are aspects that one should keep in mind when interpreting the results of X-ray diffraction profiles in relation to structural abnormalities.

ADDITIONAL INFORMATION AND DECLARATIONS Funding

Figure 2
2Experimental setup. (A) Apparatus used to mount hair fibres in the experiment; (B) shows how the hair samples were mounted on the X-ray diffractometer.

Figure 3
3Zhang et al. (2015), PeerJ, DOI 10.7717/peerj.Split graphs comparing the effects of shampoo and conditioner on internal hair structures.

Figure 4 Figure 5
45et al. (2015), PeerJ, DOI 10.7717/peerj.Split graphs comparing the effects of shampoo and conditioner on permanently waved hair. In (A), the untreated sample is compared with the permed hair. In (B), the permed hair is compared with the permed and shampooed sample. In (C), the permed hair is compared with the permed hair treated with conditioner and in (D) the permed sample is compared with the permed sample with both shampoo and conditioners applied. Similar to Fig. 3, the red outer ring is the result of the instrumental background. Small angle X-ray scattering (SAXS). (A) Two-dimensional intensity map of untreated hair vs permed hair. While the majority of signals corresponding to keratin fibres and lipids remain unchanged, the area highlighted by the close-up view of (A) showed slight differences at the region corresponding to intermediate filaments. (B) and (C) the SAXD data of untreated hair vs. permed hair. Peaks are shown at 90Å, 47Å and 27Å. Increased 47Å peak and decreased 27Å peak intensities are observed.

Figure 6
6Wide angle X-ray scattering (WAXS). (A)

Figure 7
7Calculated diffraction patterns. (A) A hexagonal packing of the coiled-coils well describes the experimental small angle data in Fig. 5B in non-treated hair. (B) The second peak was found to be increased in intensity after perming, consistent with a structure where some coiled-coils are dissolved into monomers and occupy positions in between the dimer locations. [hkl] indices of the primitive and base centred monoclinic lattices used for the calculation are marked. Cartoons show the arrangements of dimers and monomers within the filaments.

Table 1
1Treatment sequences for each of the eight hair samples. Treatments include cleaning with tap water, shampooing, conditioning and permanent waving. Permanent waving was done directly on the subject's head in a hair salon while shampooing and conditioning were performed in petri dishes using 10% shampoo and conditioner solutions.Perm treatment
Hair care product treatments

Sample 1
Tap water rinse
-
-
-
Sample 2
Shampoo
Tap water rinse
-
-
Sample 3
Conditioner
Tap water rinse
-
-
Sample 4

No

Shampoo
Tap water rinse
Conditioner
Tap water rinse
Sample 5
Tap water rinse
-
-
-
Sample 6
Shampoo
Tap water rinse
-
-
Sample 7
Conditioner
Tap water rinse
-
-
Sample 8

Yes

Shampoo
Tap water rinse
Conditioner
Tap water rinse

. The structure factor0

0.05
0.1
0.15
0.2
0.25
0.3
0.35

0.1

0.2

0.3

0.4

0.5

0.6

S(q

||

)
S(q

||

)

0.1

0.2

0.3

0.4

0.5

0.6

q (Å

−1

)

a)
b)

0
0.05
0.1
0.15
0.2
0.25
0.3
0.35

q (Å

−1

)

||
||

[100]

[201]
[200]

[302]
[300]

[100]
[201]
[200]

[302]
[300]

Zhang et al. (2015), PeerJ, DOI 10.7717/peerj.1296 4/16
Zhang et al. (2015), PeerJ, DOI 10.7717/peerj.1296 9/16
Zhang et al. (2015), PeerJ, DOI 10.7717/peerj.1296 13/16
Zhang et al. (2015), PeerJ, DOI 10.7717/peerj.1296 16/16
This research was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC), the National Research Council Canada (NRC), the Canada Foundation for Innovation (CFI) and the Ontario Ministry of Economic Development and Innovation. RJA is the recipient of an NSERC PGS-D. MCR is the recipient of an Early Researcher Award of the Province of Ontario. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Grant DisclosuresThe following grant information was disclosed by the authors: Natural Sciences and Engineering Research Council of Canada. National Research Council Canada. Canada Foundation for Innovation. Ontario Ministry of Economic Development and Innovation. NSERC PGS-D. Early Researcher Award.Competing InterestsThe authors declare there are no competing interests.Author Contributions• Yuchen Zhang and Richard J. Alsop conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.• Asfia Soomro performed the experiments, contributed reagents/materials/analysis tools.• Fei-Chi Yang contributed reagents/materials/analysis tools, prepared figures and/or tables.• Maikel C. Rheinstädter conceived and designed the experiments, performed the experiments, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.Human EthicsThe following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers): Hamilton Integrated Research Ethics Board (HIREB) under approval number 14-474-T.Supplemental InformationSupplemental information for this article can be found online at http://dx.doi.org/ 10.7717/peerj.1296#supplemental-information.
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