Detergent-free solubilization of human Kv channels expressed in mammalian cells.

Styrene-maleic acid (SMA) copolymers are used to extract lipid-encased membrane proteins from lipid bilayers in a detergent-free manner, yielding SMA lipid particles (SMALPs). SMALPs can serve as stable water-soluble nanocontainers for structural and functional studies of membrane proteins. Here, we used SMA copolymers to study full-length pore-forming α-subunits hKCNH5 and hKCNQ1 of human neuronal and cardiac voltage-gated potassium (Kv) channels, as well as the fusion construct comprising of an α-subunit hKCNQ1 and its regulatory transmembrane KCNE1 ß-subunit (hKCNE1-hKCNQ1) with added affinity tags, expressed in mammalian COS-1 cells. All these recombinant proteins were shown to be functionally active. Treatment with the SMA copolymer, followed by purification on the affinity column, enabled extraction of all three channels. A DLS experiment demonstrated that negative stain electron microscopy and single particle image analysis revealed a four-fold symmetry within channel-containing SMALPs, which indicates that purified hKCNH5 and hKCNQ1 channels, as well as the hKCNE1-hKCNQ1 fusion construct, retained their structural integrity as tetramers.

The worldwide diversity of scalp seborrhoea, as daily experienced by seven human ethnic groups.

OBJECTIVE: The re-greasing process and kinetics of the human scalp, post-shampooing, have been previously documented, in vivo, on a few Caucasian subjects. The objective of the presented research was to extend such knowledge over seven different ethnic groups. METHODS: The post-shampooing re-greasing kinetics of the scalp was studied on 1325 subjects (women and men of two distinct age classes) from seven different ethnic groups in their residential and native country. Sebum amounts were determined onto small shaved scalp areas at various times post-shampooing, using the Sebumeter® technique. RESULTS: As previously published on Caucasian subjects, scalp re-greasing process follows a hyperbolic-like kinetics over days. However, amounts of collected sebum highly vary with ethnicity. As recorded through the casual level (CL) at the equilibrium phase, 2-3 days post-shampooing, the highest amount of sebum was found in African American subjects, followed in descending order by Caucasian American, Japanese, Chinese, Thai, Caucasian European and Indian subjects, the latter showing very low values. Lower amounts of sebum were recorded in the older age class in all ethnics, as compared to the younger one, and male subjects were found higher sebum producers than women, irrespective of ethnicity. CONCLUSION: The kinetics and slopes of the re-greasing process of the human scalp appear similar in all ethnic groups studied. However, striking quantitative differences are found between the seven ethnic groups, resulting from different sebaceous production levels and scalp hygiene routines.

The diversity of the human hair colour assessed by visual scales and instrumental measurements. A worldwide survey.

OBJECTIVES: To study (i) the diversity of the natural colour of the human hair through both visual assessment of hair tone levels and colorimetric measurements of hair strands collected from 2057 human male and female volunteers, from 23 regions of the world and (ii) the correlation between visual assessments and colorimetric measurements. METHODS: Hair strands were analysed by a spectrocolorimeter under the L*, a*, b* referential system and scored in vivo by experts before sampling, through standardized visual reference scales based on a 1-10 range. RESULTS: Results show that from a typological aspect, black or dark brown hairs largely predominate among studied ethnic groups, whereas Caucasian or derived populations exhibit the widest palette of medium to fair shades, partly explaining some past interbreeding among populations. Instrumental measurements clearly confirm that a given colour of a pigmented hair, at the exclusion of red hairs, is mostly governed by two components, L* and b*, from the L*, a*, b* reference system. CONCLUSION: The comparisons between visual assessments and instrumental data show that these appear closely linked. Darker hairs show close or subtle variations in L*, a*, b* parameters, making their individual colour differentiation calling for technical improvements in colorimetric measurements. The latter are likely governed by other physical factors such as shape, diameter and shine.

What is a Caucasian 'fine' hair? Comparing instrumental measurements, self-perceptions and assessments from hair experts.

OBJECTIVE: To determine the various factors contributing to what Caucasian women describe as 'fine hair'. METHODS: Three complementary approaches were used, namely self-evaluation by the volunteer, assessment by a sensorial expert and instrumental measurements, in order to determine some of the possible parameters taken into account by Caucasian women when they describe the notion of fine hair. One hundred fifty one women of Caucasian origin participated in the study. They varied in age, and varied in that some considered themselves as having fine hair, and others not. The instrumental measurements carried out included hair diameter measurements, hair density measurements, hair breakage force, hair flexibility and scalp sebum levels. RESULTS: From six parameters defined initially, four parameters were found to be in common with the three approaches: hair abundance (density), hair thickness, hair resistance and the volume of the hair on the head. The commonly used term 'body' was only common to self and expert evaluation, whereas the influence of curliness was only common to expert evaluation and instrumental measurements. CONCLUSIONS: This study has shown close agreement between sensorial and instrumental findings, and also illustrates how the women participating can subtly and adequately describe their own hair. It is important to note that the words 'fine hair' describes a lot more than just physically thin hair fibres. Ageing is an additional factor that clearly impacts certain parameters associated with 'fine hair' among the volunteers.

[How I treat dandruff using a shampoo with a sulfate-free tensio-active]. / Comment je traite un état pelliculaire par un shampoing à base lavante «sulfate-free¼.

A new formulation of an anti-dandruff shampoo containing a surfactant devoid of a sulfate-type anionic part has been assessed in vivo in 41 subjects. Introducing a welltried agent, the 0.5 % piroctone olamine confers to this shampoo a high anti-dandruff efficacy, a reduction in the density in Malassezia spp, as well as a good remanence after shampoo. In addition, piroctone olamine appears to influence the sebum rheology by reducing the hair greasing.

Une nouvelle formulation d'un shampoing antipelliculaire, basée sur l'utilisation de tensio-actifs sans groupe anionique de type sulfate («sulfate free¼) a été évaluée in vivo sur 41 sujets. L'introduction d'un agent éprouvé, la piroctone olamine à 0,5 %, confère à ce shampoing une efficacité antipelliculaire élevée, une réduction de la densité de Malassezia spp, ainsi qu'une bonne rémanence de l'effet après shampoing. La piroctone olamine semble, de plus, modifier la rhéologie du sébum en ralentissant le phénomène de regraissage des cheveux.

Changes in Chinese hair growth along a full year.

OBJECTIVE: To confirm the existence of seasonal hair growth cycle among Chinese subjects and objectivize the seasonal effect of hair loss; the hair growth parameters of Chinese volunteers were followed monthly for an entire year on the same area of vertex. METHODS: The hair growth parameters of 41 Chinese volunteers (women and men), free from alopecia, were recorded monthly along an entire year using the phototrichogram technique. RESULTS: Results show an increased rate of telogen hairs (growing arrest) around August-September in the study group, as previously reported in European subjects albeit of a lower extent and remaining within the normal range of healthy head hair parameters. The possible effects of latitude and daylight duration are discussed. CONCLUSION: Data confirm that Chinese hairs present characteristics of the most developed and fast growing terminal fibres, as compared to other non-Asian ethnics.

Greying of the human hair: a worldwide survey, revisiting the '50' rule of thumb.

BACKGROUND: While numerous papers have reported on the biological mechanisms of human hair pigmentation and greying, epidemiological descriptions of both natural hair colour and the greying process, worldwide, remain scarce. OBJECTIVES: To assess hair colour and greying in a large world sample of human subjects, and to revisit the validity of the 50/50/50 rule of thumb, which states that 'at age 50 years, 50% of the population has at least 50% grey hair'. METHODS: The natural hair colour of 4192 healthy male and female volunteers was assessed using a sensorial expert evaluation through the comparison of each volunteer's hair with standard swatches. Hair colour was studied according to age, gender and ethnic or geographical origin. RESULTS: Overall we observed that between 45 and 65 years of age, 74% of people were affected by grey hair with a mean intensity of 27%. Men harboured significantly more grey hair than women. Both age at onset and rate of greying with age appeared to be clearly linked to ethnic/geographical origin. Subjects of Asian and African descent showed less grey hair than those of caucasian origin, at comparable ages, confirming previously reported data. CONCLUSIONS: Calculating the percentage of people showing at least 50% grey hair coverage at age 50 years leads to a global range of 6-23%, according to ethnic/geographical origin and natural hair colour: well below that expressed by the '50' rule of thumb.

Age-dependent changes in eumelanin composition in hairs of various ethnic origins.

Hair pigmentation is one of the most conspicuous phenotypes of humans. From a chemical point of view, however, data remain scarce regarding human hair pigmentation characteristics. To determine melanin content and composition in human eumelanic hair from individuals of different ethnic origins and at different ages, we collected hair from 56 subjects with eumelanic hair from each group of African-American, East Asian, and Caucasian origin. The 56 subjects consist of 14, seven each of males and females, each from four age classes of younger than 11, between 12 and 19, between 20 and 45, and older than 46. We analysed hair colour scale, total melanin value, and contents of pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3-dicarboxylic acid (PDCA). We measured age-dependent increases in the relative quantity of eumelanin in pigmented human hairs in the three ethnic groups. Regarding melanin composition, we observed an increase in the PDCA/PTCA ratio with age in African-American and Caucasian hairs until approaching the quite constant level of the ratio in East Asian hairs in the elderly individuals. Our results evidence differences in the content and composition of eumelanin in human hair among African-American, Caucasian and East Asian individuals. Furthermore, we show evidence of age-dependent changes in the quantity and quality of eumelanin in pigmented human hairs. In particular, the age-dependent modification of the PDCA/PTCA ratio, a marker for 5,6-dihydroxyindole units in eumelanin, suggests a chronological evolution of hair follicle melanocyte phenotype (e.g. decrease in dopachrome tautomerase expression).

Challenging the scalp with 'dry' wash shampooing (DW) on Chinese men: an in vivo study.

Cosmetic habits show a strong diversity worldwide. The same applies to procedures that are considered as basic hygiene acts such as shampooing. Apart from common traditional shampoo routine, a special shampoo procedure, 'dry' wash is daily performed in Chinese hair salons. Without pre-wetting step, hairdresser applies the shampoo directly to dry hair with small quantity of water to create foam. Hand massaging is then offered on the scalp. This shampooing process is distinguished from traditional 'regular wash' by slightly diluted shampoo, and longer contact with the scalp and scalp massaging, all of which could be possibly considered as challenges to the structure and barrier function of the scalp. In this study, the effects of 'dry' wash on Chinese scalp have been investigated, in vivo, using regular wash as a control. A total of 67 Chinese males in two cohorts, non-dandruff and with dandruff, were included. In each cohort, volunteers were split into two groups, one with 'dry' wash and the other one with regular wash. Both shampoo methods were performed daily for five consecutive days using the same shampoo formulation. Volunteers' self assessment, clinical assessment by an expert and instrumental measurements were carried out. The results showed that Chinese 'dry' wash did not induce any negative consequence on the scalp physiology under such experimental conditions.

[Circadian chronophysiology of the scalp]. / Chronophysiologie circadienne du cuir chevelu.

Biological rhythms are numerous and vary according to periodicity and amplitude. Variations over the course of a day, particularly circadian rhythms have been reported in the skin of the face and forearms. However, little information is available about daily biorhythms of the scalp. The aim of the present study was to explore the chronophysiology of the scalp. We report a series of diurnal rhythms in 8 Caucasian men using the plexogram method at 4-h intervals over a 5-day period. Statistically significant biorhythms were found for the colour, hydration, barrier function and pH of the skin, and for the sebum excretion as well. The early afternoon was the time of maximum increase in redness, hydration, pH and sebum output, combined with decreased skin barrier function. The amplitude of some of these biorhythms was so discrete that their clinical relevance remained uncertain even when they appeared statistically significant. In view of the apparent synchronization between them, it is likely that a central oscillator exerts a major influence. However, only rare subjects exhibited biorhythms for all the considered physiological parameters. Hence, peripheral reactivity and/or environmental synchronizers also play an important role to define the good and bad respondors to the potential biorhythms of each individual parameter.

Dandruff-associated smouldering alopecia: a chronobiological assessment over 5 years.

BACKGROUND: In some individuals, dandruff may be recurrent and even chronic. This represents a difficult-to-treat condition. Excessive hair shedding and diffuse alopecia may develop. OBJECTIVES: To evaluate the chronobiological aspects of dandruff, hair shedding and alopecia. METHODS: This study was performed in eight men suffering from such a scalp condition. They were examined 40 times at regular intervals for five consecutive years. Hair-density assessments and trichograms were performed at least twice per trimester on the parietal region of the scalp, corresponding to an area where dandruff was present. RESULTS: A positive correlation was found between the percentage of telogen hairs and dandruff severity as assessed by the squamometry index. Bimodal yearly biorhythms were found for dandruff severity, telogen counts and hair loss between two consecutive visits. The chronobiological fluctuations were prominent in some study participants, but remained inconspicuous in others. The biorhythms were almost synchronized in all participants, showing variations over the year. CONCLUSIONS: The biorhythms on the hair cycle in dandruff are not fundamentally different from those previously reported in subjects without dandruff. The periodicity may be an intrinsic feature of human skin.

Molecular basis of inward rectification: structural features of the blocker defined by extended polyamine analogs.

Polyamines cause inward rectification of Kir K(+) channels by blocking deep within the channel pore. We investigated structural constraints of polyamine block of strongly rectifying mutant K(ATP) channels (Kir6.2[L164C,N160D,C166S] + SUR1). We studied three groups of polyamine analogs: 1) conformationally restricted linear tetra-amines with a cycloalkyl or alkene group between the second and third amines (CGC-11047, CGC-11093, CGC-11099, and CGC-11098), 2) conformationally restricted linear deca-amines with a cycloalkyl or alkene group between the fifth and sixth amines (CGC-11150, CGC-11179, and CGC-11241), and 3) cyclic tetra-amines (CGC-11174, CGC-11197, CGC-11199, and CGC-11254). All linear analogs cause a voltage-dependent block similar to that of spermine, but slightly weaker (at 1 microM, V(1/2) for spermine block = -10 +/- 1 mV, Z = 2.9 +/- 0.1, n = 19; V(1/2) for analogs varies from polyamine -7 to +10 mV, Z = 2.6-3.9). These data indicate tolerance for conformational restriction and an upper limit to the voltage dependence of the blocking process. There was no voltage-dependent block by the cyclic compounds; instead, they induce irreversible rundown of the current. Structural models of Kir channels suggest that a narrow entry at the top of the cytoplasmic pore may exclude cyclic analogs from the inner cavity, thereby explaining the structure-activity relationship that we observe.

Phosphatidylinositol-4,5-bisphosphate, PIP2, controls KCNQ1/KCNE1 voltage-gated potassium channels: a functional homology between voltage-gated and inward rectifier K+ channels.

Phosphatidylinositol-4,5-bisphosphate (PIP(2)) is a major signaling molecule implicated in the regulation of various ion transporters and channels. Here we show that PIP(2) and intracellular MgATP control the activity of the KCNQ1/KCNE1 potassium channel complex. In excised patch-clamp recordings, the KCNQ1/KCNE1 current decreased spontaneously with time. This rundown was markedly slowed by cytosolic application of PIP(2) and fully prevented by application of PIP(2) plus MgATP. PIP(2)-dependent rundown was accompanied by acceleration in the current deactivation kinetics, whereas the MgATP-dependent rundown was not. Cytosolic application of PIP(2) slowed deactivation kinetics and also shifted the voltage dependency of the channel activation toward negative potentials. Complex changes in the current characteristics induced by membrane PIP(2) was fully restituted by a model originally elaborated for ATP-regulated two transmembrane-domain potassium channels. The model is consistent with stabilization by PIP(2) of KCNQ1/KCNE1 channels in the open state. Our data suggest a striking functional homology between a six transmembrane-domain voltage-gated channel and a two transmembrane-domain ATP-gated channel.

Effect of residence time on the efficacy of antidandruff shampoos.

Dandruff is known to be controlled by fungistatic shampoos active against Malassezia spp. These products also remove the loosely attached scales. This study was performed to assess the effect of a 5-min residence time on the efficacy of antidandruff shampoos. Two commercially available shampoos were used in two groups of 21 panelists with severe dandruff. They contained either 1% ketoconazole or 1% piroctone olamine. In each group, intraindividual comparisons were made by a split-scalp design between the effect of a 5-min residence time versus no residence time. Both shampoos induced significant reductions in scaliness and yeast colonization. The beneficial effects were obvious immediately after one single shampooing and 3 days later as well. The improvement was greater with a 5-min residence time. The piroctone olamine treatment benefited more than the ketoconazole treatment from the extension of shampoo-exposure time. In conclusion, the benefit of a residence time in treating dandruff is documented. The level of improvement in efficacy may vary according to the nature of the shampoo.

The hair eclipse phenomenon: sharpening the focus on the hair cycle chronobiology.

Chronobiology governing the hair cycle is a fascinating and complex process. Both the hair growth cycle and the hair shaft growth are coordinated and depend on the interplay of different biological signals and various exogenous stimuli. A latency period may occur between hair shedding (teloptosis, exogen phase) and the early emergence of the next anagen VI stage. This lag time referred to on the hair eclipse phenomenon likely depends on the influence of a series of distinct synchronizers, and does not represent per se a peculiar hair cycle phase. It is the result of some dysregulations of the hair cycling, involving early teloptosis, delayed anagen I initiation or stunted hair growth at any stage between the anagen I and anagen V phases. As such, the hair eclipse phenomenon may be an erratic process occurring in physiopathological conditions affecting hair follicles singly or in focal to generalized patterns. It may be more frequent when it follows synchronized teloptosis occurring in telogen effluvium (newborn alopecia, post-partum alopecia, seasonal alopecia and alopecia areata). It may also be prominent when microinflammation is abutted on the permanent portion of the hair follicle as in dandruff, seborrhoeic dermatitis, androgenic alopecia and photoageing baldness. Local synchronizers such as growth factors and other mediators may eventually be lacking or involved in the hair eclipse phenomenon. Their identification and characterization might drive new corrective or preventive applications.

Structural basis of inward rectifying potassium channel gating.

The last 10 years have seen rapid advances in the understanding of potassium channel function. Since the first inward rectifying (Kir) channels were cloned in 1994, the structural basis of channel function has been significantly elucidated, and determination of the crystal structure of a bacterial K channel (KcsA) in 1998 provided an atomic resolution of the permeation pathway. This review considers recent experimental studies aimed at uncovering the structural basis of Kir channel activity, and the applicability of comparative models based on KcsA to illuminate Kir channel pore structure and opening and closing processes.

The follicular automaton model: effect of stochasticity and of synchronization of hair cycles.

Human scalp hair consists of a set of about 10(5)follicles which progress independently through developmental cycles. Each hair follicle successively goes through the anagen (A), catagen (C), telogen (T) and latency (L) phases that correspond, respectively, to growth, arrest and hair shedding before a new anagen phase is initiated. Long-term experimental observations in a group of ten male, alopecic and non-alopecic volunteers allowed determination of the characteristics of hair follicle cycles. On the basis of these observations, we previously proposed a follicular automaton model to simulate the dynamics of human hair cycles and the development of different patterns of alopecia [Halloy et al. (2000) Proc. Natl Acad. Sci. U.S.A.97, 8328-8333]. The automaton model is defined by a set of rules that govern the stochastic transitions of each follicle between the successive states A, T, L and the subsequent return to A. These transitions occur independently for each follicle, after time intervals given stochastically by a distribution characterized by a mean and a standard deviation. The follicular automaton model was shown to account both for the dynamical transitions observed in a single follicle, and for the behaviour of an ensemble of independently cycling follicles. Here, we extend these results and investigate additional properties of the model. We present a deterministic version of the follicular automaton. We show that numerical simulations of the stochastic version of the automaton yield steady-state level of follicles in the different phases which approach the levels predicted by the deterministic equations as the number of follicles progressively increases. Only the stochastic version can successfully reproduce the fluctuations of the fractions of follicles in each of the three phases, observed in small follicle populations. When the standard deviation is reduced or when the follicles become otherwise synchronized, e.g. by a periodic external signal inducing the transition of anagen follicles into telogen phase, large-amplitude oscillations occur in the fractions of follicles in the three phases. These oscillations are not observed in humans but are reminiscent of the phenomenon of moulting observed in a number of mammalian species.

African hair growth parameters.

BACKGROUND: Hair growth parameters have been studied mostly in caucasian hair, whereas few data on African hair have been reported in the literature. OBJECTIVES: To evaluate hair growth characteristics of African volunteers born in Africa. METHODS: Thirty-eight young adults (19 women, 19 men, mean +/- SD age 27 +/- 10 years), native of central and western Africa, took part in the study. Phototrichograms were performed in order to record three parameters of hair growth: hair density, telogen percentage and rate of growth. For each volunteer, three regions of the scalp, namely vertex, temporal and occipital areas, were assessed. RESULTS: Hair density varied from 90 to 290 hairs cm(-2), with higher counts on the vertex. No significant difference between men and women was recorded. Telogen percentage showed wide variations, from 2 to 46%, with higher levels on the temporal area and in men. The rate of growth fluctuated from 150 to 363 microm day(-1) with no difference related either to gender or to scalp region. These data were compared with those previously obtained in caucasian volunteers of comparable age, and showed significant differences between the two ethnic groups in all three parameters studied. Hair density in African volunteers was lower than that in caucasians (mean +/- SD 190 +/- 40 and 227 +/- 55 hairs cm(-2), respectively). African hair grew at a much slower rate than caucasian hair (mean +/- SD 256 +/- 44 vs. 396 +/- 55 microm day(-1)), and telogen counts were frequently higher in African hair (mean +/- SD 18 +/- 9% vs. 14 +/- 11%). CONCLUSIONS: This study demonstrated significant differences between African and caucasian hair growth parameters, which might suggest a trend towards increased hair loss in Africans, even though it contrasts with a lower and slower incidence of the development of alopecia in Africans.

Dynamic sensitivity of ATP-sensitive K(+) channels to ATP.

ATP and MgADP regulate K(ATP) channel activity and hence potentially couple cellular metabolism to membrane electrical activity in various cell types. Using recombinant K(ATP) channels that lack sensitivity to MgADP, expressed in COSm6 cells, we demonstrate that similar on-cell activity can be observed with widely varying apparent submembrane [ATP] ([ATP](sub)). Metabolic inhibition leads to a biphasic change in the channel activity; activity first increases, presumably in response to a fast decrease in [ATP](sub), and then declines. The secondary decrease in channel activity reflects a marked increase in ATP sensitivity and is correlated with a fall in polyphosphoinositides (PPIs), including phosphatidylinositol 4,5-bisphosphate, probed using equilibrium labeling of cells with [(3)H]myo-inositol. Both ATP sensitivity and PPIs rapidly recover following removal of metabolic inhibition, and in both cases recovery is blocked by wortmannin. These data are consistent with metabolism having a dual effect on K(ATP) channel activity: rapid activation of channels because of relief of ATP inhibition and much slower reduction of channel activity mediated by a fall in PPIs. These two mechanisms constitute a feedback system that will tend to render K(ATP) channel activity transiently responsive to a change in [ATP](sub) over a wide range of steady state concentrations.

Flexibility of the Kir6.2 inward rectifier K(+) channel pore.

Interactions of sulfhydryl reagents with introduced cysteines in the pore-forming (Kir6.2) subunits of the K(ATP) channel were examined. 2-Aminoethyl methanethiosulfonate (MTSEA(+)) failed to modify Cd(2+)-insensitive control-Kir6.2 channels, but rapidly and irreversibly modified Kir6.2[L164C] (L164C) channels. Although a single Cd(2+) ion is coordinated by L164C, four MTSEA(+) "hits" can occur, each sequentially reducing the single-channel current. A dimeric fusion of control-Kir6.2 and L164C subunits generates Cd(2+)-insensitive channels, confirming that at least three cysteines are required for coordination, but MTSEA(+) modification of the dimer occurs in two hits. L164C channels were not modified by bromotrimethyl ammoniumbimane (qBBr(+)), even though qBBr(+) caused voltage-dependent block (as opposed to modification) that was comparable to that of MTSEA(+) or 3-(triethylammonium)propyl methanethiosulfonate (MTSPTrEA(+)), implying that qBBr(+) can also enter the inner cavity but does not modify L164C residues. The Kir channel pore structure was modeled by homology with the KcsA crystal structure. A stable conformation optimally places the four L164C side chains for coordination of a single Cd(2+) ion. Modification of these cysteines by up to four MTSEA(+) (or three MTSPTrEA(+), or two qBBr(+)) does not require widening of the cavity to accommodate the derivatives within it. However, like the KcsA crystal structure, the energy-minimized model shows a narrowing at the inner entrance, and in the Kir6.2 model this narrowing excludes all ions. To allow entry of ions as large as MTSPTrEA(+) or qBBr(+), the entrance must widen to >8 A, but this widening is readily accomplished by minimal M2 helix motion and side-chain rearrangement.