We examine the importance of thermal expansion on relative stabilities and properties of crystalline polymorphs. We present a large-scale study of the temperature-dependence of structures, free energy differences and properties of polymorphic molecular organic crystals. Lattice-vibrational Gibbs free energy differences between 475 pairs of polymorphs of organic molecular crystals have been calculated at 0 K and at their respective melting points, using a highly accurate anisotropic multipole-based force field and including thermal expansion through the use of a (negative) thermal pressure. Re-ranking of the relative thermodynamic stability of the polymorphs in each pair indicates the possibility of an enantiotropic phase transition between the crystal structures, which occurs in 21% of the studied systems. While vibrational contributions to free energies can have a significant effect on thermodynamic stability, the impact of thermal expansion on polymorph free energy differences is generally very small. We also calculate thermal expansion coefficients for the 864 crystal structures and investigate the temperature-dependence of mechanical properties, and pairwise differences in these properties between polymorphs. It is now well established that polymorphism is common, occurring in about half of all studied compounds with no strong correlation to the size or flexibility of the molecule.10Polymorphism, 14 Kinetic factors affecting the crystallization of different polymorphs, and the mechanisms by which their phase transitions are facilitated, are less well understood.15Polymorphism occurs because different crystalline packing arrangements can result in nearly the same free energy.i.e. the higher energy polymorph is always thermodynamically unstable and will eventually transform to the more stable form. Consequently, monotropic transitions are irreversible and exothermic. Once a transition has occurred, it is sometimes nearly impossible to obtain the less stable form again.17At any given pressure, each polymorph pair is either enantiotropic or monotropic. In monotropic pairs, one polymorph has a larger free energy than the other at all temperatures (below the melting point), 18 and concomitant crystallisation.19 Enantiotropic transitions are in principle reversible. The transition is endothermic when going from the low-temperature form to the high-temperature polymorph. However, there are often significant deviations from ideal thermodynamic behaviour, caused by lattice defects, impurities and kinetic barriers.20 Crystals can be kinetically stable for apparently indefinite time and reversible transitions usually display significant hysteresis.In enantiotropic pairs, both polymorphs can be thermodynamically stable, but in different temperature intervals. At a particular temperature, both polymorphs have exactly the same free energy, which can manifest as solid-to-solid phase transitions, iso-energetic polymorphism22 The simulation can be augmented with metadynamics to allow a global exploration of free energy minima and transition barriers.24 The lattice vibrations, in the form of a phonon density of states g(ω) can be obtained from the velocity autocorrelation function over a molecular dynamics trajectory,25 and this has the advantage of directly including anharmonic effects, such as thermal expansion. The main difficulties lie in the sampling of long wavelength phonons, requiring large simulation cells, and in performing molecular dynamics with an energy model that is accurate enough. Lattice dynamics offers an alternative to MD simulations that can be much more efficient, but in addition to the problem of sampling low-frequency modes,26 difficulties arise in the treatment of anharmonic vibrations and thermal expansion.It is possible to computationally study the relative stability of, and transitions between, polymorphs through molecular dynamics simulations (MD).27 fragment based QM/MM methods29 or anisotropic force fields based on distributed multipoles.32To calculate realistic free energy rankings of molecular crystals it is usually necessary to employ periodic dispersion-corrected density functional approximations (DFT-D),–1 at room temperature and that in 9% of cases this is large enough to reverse their relative stability.14 This article is to a large extent a follow-up study of our earlier results, focusing on the temperature-dependence of polymorphic relative stabilities.We have recently demonstrated that the difference in lattice vibrational energy between polymorphs is typically very small but can be as large as 4 kJ mol33 To meet the challenge, we have improved our methods in several ways. We have benchmarked our force fields to verify their accuracy. The force field used in this study is comparable to periodic PBE with Grimme's D2 or the Tkatchenko–Scheffler dispersion corrections and similar methods.32 Our previously described method for Brillouin zone sampling by linear supercells has been supplemented with a Debye model to treat dispersion of acoustic phonons. In addition, we use a kernel density method to model dispersion of optic phonons, which improves the convergence of the calculated thermodynamic properties.34 In this study we also consider some effects of the anharmonicity in the lattice vibrations; the thermal expansion is modelled in the quasi-harmonic approximation (QHA),35 using the thermal pressure method.36The small energy differences between polymorphs makes it challenging to correctly rank their relative stabilities, making for instance crystal structure prediction (CSP) difficult.37 is a rare exception. In examining their influence on the ranking of predicted structures, it was found that the harmonic vibrational zero point and thermal vibrational energy can have an important effect, but that the energetic impact of thermal expansion generally does not significantly affect rankings. Because of the computational cost and the small size of the effect it was concluded that it may be preferable to neglect thermal expansion in CSP. More recently, Heit and Beran applied high level fragment-based quantum chemical methods to investigate the importance of thermal expansion in modelling the thermodynamic properties of four small molecule crystal structures.38 The errors in free energies that they associated with ignoring thermal expansion were small, due to cancellation of opposing effects in enthalpy and entropy.Few studies have been made of the anharmonic vibrational contribution to polymorph free energy ranking. The CSP study of glycol and glycerol by van EijckThe number of molecular crystals for which the importance of thermal expansion has been assessed is currently very small. Therefore, we feel that a larger sample of polymorph pairs is needed to estimate its importance in modelling the energetics and properties of organic molecular crystals. The large sample studied here should provide good estimates of general trends among polymorphs.The influence of thermal expansion on polymorph relative stabilities will depend on how different the volumetric thermal expansion coefficients are between polymorphs, and this in turn depends on differences in elastic properties of the materials. Since elastic properties are also of interest in the formulation and production of pharmaceutical tablets, we examine the shear and bulk moduli, and their temperature-dependence.i.e. we are estimating how common enantiotropism is, and how large lattice energy differences can be overcome by thermal contributions to the free energy.In this study, we present results of rigid-body lattice-vibrational free energy differences at 0 K and at the respective melting point temperature for a large number of organic molecular polymorphic crystals. In doing so, we hope to be able to estimate how common it is that relative stabilities are re-ranked by temperature, 2Tm. We determine how often temperature causes a re-ranking of the thermodynamic stability, and obtain an estimate for how commonly polymorph pairs are enantiotropically related.The main objective of this study is to use a large selection of known polymorph pairs and for each pair calculate the difference in lattice-vibrational free energy and other properties at 0 K and at the melting point temperature Below we describe the set of polymorph pairs and the calculations we have performed on them. We describe how the structures were energy-minimised on a realistic potential energy surface and how rigid-body harmonic (HA) and quasi-harmonic approximation (QHA) lattice dynamics calculations were carried out. Finally, we explain how melting point temperatures were obtained for all the crystal structures.Although the main focus is on general trends in the large set of polymorph pairs, we also performed more detailed studies of a selected set of crystals. The calculated properties of individual systems are compared to experimental literature data, allowing us to estimate the overall accuracy of our results.2.114 We have used the best experimentally determined structure of each polymorph.39 These are polymorphs of compounds containing the elements H, C, N, O, F, S and Cl in any combination up to ca. 55 atoms per asymmetric unit. The methods presented here could be applied to molecular salts or solvates, but we choose to focus on single-component systems in this study. The compounds and crystal structures are referred to by their Cambridge Structural Database (CSD) reference codes.40 Crystals with a non-integer number of molecules in the asymmetric unit (Z′) have had their symmetries lowered to obtain whole molecules in the asymmetric unit.We started from a set of 1061 single-component organic molecular packing polymorphs of 508 compounds that we have described previously.ormat,41 ignoring hydrogen positions.From this set we had to exclude a number of structures for various reasons; see the ESI.42 InChI identifiers and the chemical formula of the compounds, calculated thermal pressures, experimental and predicted melting points, elastic moduli, volume thermal expansion coefficients, unit cell volumes, Z and Z′-values. We offer this data, the Nyman Polymorph Library (NPL2016), in the hope it will facilitate further computational studies of polymorphs.The final structure set is included in the ESI2.2i and k are atoms of type ι and κ belonging to molecules M and N, respectively and separated by the distance Rik. The first two terms model the short range repulsive and attractive non-electrostatic intermolecular interactions, with force field parameters Aικ, Bικ and Cικ.The lattice energy consists of the intermolecular cohesive energy and a quantum chemical evaluation of the intramolecular energy, calculated with dispersion-corrected density functional theory (DFT-D).43 supplemented with parameters for sulfur,44 fluorine and chlorine. Halogen atoms were modelled with an anisotropic repulsion,45 using parameters provided in the ESI.We have used the W99rev6311 and W99rev6311P force fields,aussian09 at the B3LYP/6-311G level of theory, with a Becke–Johnson damped version of Grimme's D3 dispersion correction.47The intramolecular electronic structure and energy is calculated with GEelec, describing electrostatic interactions, was calculated from atom-centred multipoles up to rank 4 (hexadecapole) on all atoms,31 obtained from a distributed multipole analysis48 of the molecular electron density using Gdma 2.2.06.49 The multipoles may be polarized by performing the DFT calculation in a polarizable continuum model (PCM).51 Charge–charge, charge–dipole and dipole–dipole interactions were calculated with Ewald summation. All other interactions were calculated between whole molecules with a centre of mass distance less than 20 Å.The final term 32 Furthermore, calculations of polymorph energy differences benefit from an important cancellation of systematic errors in the force field.The type of hybrid force field/DFT energy model used here is highly accurate and may even be competitive with GGA DFT-D methods, such as PBE-D2, in reproducing absolute lattice energies.2.3rystalOptimizer53 version 2.4.2. CrystalOptimizer minimises the lattice energy (eqn (1)) while allowing a set of selected intramolecular degrees of freedom  and unit cell dimensions to change in response to intermolecular forces. All other intramolecular degrees of freedom are optimised to their DFT-D equilibrium values. The optimisation is performed numerically by iterating between inter- and intramolecular calculations. Intermolecular packing forces were calculated with Dmacrys 2.0.4, using the W99rev6311 force field and distributed multipoles, as described above.Crystal geometry optimisations were performed with CZ-matrix generated by CrystalOptimizer, as previously described,14 although for this study we have improved the selectivity for 3-, 4- and 5-membered rings . A relative permittivity of εr = 5.0 was used in this study.We have re-optimised the crystal structures obtained from C32 These calculations make use of a Debye approximation for the acoustic phonon dispersion near the Brillouin zone centre, and a Gaussian kernel density estimate (KDE) of dispersion around all calculated phonon frequencies.For all crystal structures, we calculated the lattice-vibrational free energy at 0 K and at the respective melting point for each structure with both the harmonic and quasi-harmonic methods, as described in the ESIAHA(T), as the sum of the lattice energy and the vibrational energy, FHAvib(T), from the phonon frequency density of states calculated at the fully relaxed structureAt non-zero temperatures we calculate the free energy in the harmonic approximation, 14 have shown that vibrational contributions can alter polymorph relative stabilities by up to about 2 kJ mol–1, which is mostly due to entropy differences. These contributions are significant since the static energy difference between pairs of polymorphs is found to be less than 2 kJ mol–1 in over half of the molecules investigated. However, at temperatures near the melting point the harmonic model could lead to unacceptable errors. We have previously described our implementation of a quasi-harmonic method to account for thermal expansion and the temperature-dependence of lattice vibrations.32The harmonic approximation is based on an assumption that phonon frequencies and the crystal structure do not change with temperature. Our previous results using the harmonic model36 where a thermal pressure is calculated from the change in vibrational energy with volume, Pth(T) = –∂Fvib(T)/∂V. Geometry optimisation in the rigid body approximation at –Pth results in a thermally expanded crystal structure close to the free energy minimum at constant pressure. The quasi-harmonic free energy AQHA can then be calculated for the thermally expanded structure asThermal expansion and free energies in the quasi-harmonic approximation are calculated using the thermal pressure method,macrys 2.0.4,31 using algorithms that have been described elsewhere.56 Full details of the free energy calculations are provided in the ESI.All lattice dynamics calculations were performed in DFor a number of structures it was not possible to obtain a valid energy minimum. The structures MALOAM01, SIRMIQ and SALIAZ02 drastically changed structure when the thermal pressure was applied and were excluded. In addition, 42 polymorph families were excluded because one or more of the structures became unstable at negative pressures, exhibiting imaginary phonon frequencies and indefinite elastic tensors.57 The thermal expansion is expected to lead to significant softening, lowering the bulk and shear moduli as the temperature is raised. This effect is investigated between 0 K and the melting point. Due to the importance of mechanical properties for processing at room temperature, thermal expansion calculations were repeated for the entire structure set at 298 K, to also determine room temperature bulk and shear moduli distributions.Elastic moduli were calculated for a crystalline aggregate of each structure by averaging over the calculated elastic tensors, for which we used the Hill average.2.5Melting points are known for many of the polymorphs in the set. The melting temperature differences between polymorphs are smaller than the required accuracy for our purposes. Therefore, we have only attempted to determine one melting point per polymorph family. We have used experimentally determined melting temperatures whenever this was reported in the CSD. This was the case for 225 (44%) of the 508 initially selected polymorph families.tbtnt program in the EPASuite58 software by the US Environmental Protection Agency. The program internally uses two algorithms for estimating the melting temperature and returns a weighted average of these. The first algorithm is based on Joback's method.59 The second algorithm estimates the melting point as Tm = 0.5839·Tb as suggested by Gold and Ogle,60 where Tb is the boiling point, which is also calculated from fragment contributions using a method by Stein and Brown.61 The Mtbtnt program takes SMILES strings42 as input. These were obtained from atomic coordinates via the Open Babel software.62For all other polymorph families, we have predicted the melting points using two different methods. The first method is based on molecular fragment contributions, and is implemented in the M58 We have therefore attempted to improve the predicted temperatures by using a second method relying on the correlation between lattice enthalpies and melting points. A straight proportionality is sometimes assumed between these quantities,63 but we do not find this to be correct. A good fit can be obtained after a variance-stabilising Anscombe transformation in the form of a square root.64 A non-linear regression analysis in Minitab 17 between the 225 previously obtained experimental melting points and the crystals' intermolecular energies Einter yields the following model for the melting points:14 The residual standard deviation for this model is 54.5 K, and we note that this very simple regression model yields more accurate melting point predictions than the molecular fragment method. Details of the regression and residual analysis are included in the ESI.However, molecular fragment based methods are notoriously imprecise and the standard deviation of the error in estimated melting points is 63.9 K.We have used the mean of these two methods as our predicted melting temperatures. We estimate the mean absolute error of this average to be 39 K, or less than 10%. Predicted and experimental melting temperatures are included as ESI.2.6We performed a more in-depth study of a few selected systems. These were chosen primarily as families with several experimentally well characterized polymorphs.66 1,2-ethanediamine (ETDIAM),68m-nitrophenol (MNPHOL),71 acetaminophen/paracetamol (HXACAN),73 glutaric acid (GLURAC),76 octa-sulfur (FURHUV),80 adipic acid (ADIPAC),82 2,2′-dipyridylamine (DPYRAM),83 theophylline (BAPLOT),89 and pyrazine-2-carboxamide (PYRZIN).90Plenty of experimental data have been reported for polymorphs of the following compounds: acridine (ACRDIN),Polymorphs of these compounds were studied computationally in greater detail. We have calculated free energy curves, thermal expansion rates and phonon densities of states that can be compared to published experimental data.91 using both the free energy and analytic first derivatives (entropies) at several temperatures.The lattice energy was calculated relative to the energy of the gas phase molecular conformer. Continuous free energy curves were obtained by hermite piecewise polynomial interpolation,3We first present the results of studies of selected crystal structures. Comparisons to experimental data allows us to estimate the overall accuracy of the calculations. We then show general trends in the whole set of polymorph pairs. We direct the reader to the ESI3.1For polymorphs of selected compounds we calculated free energy curves, the thermal expansion and phonon densities of states which can be compared to published experimental data. Only a few results are shown here, most results are included in the ESI.m-nitrophenol, glutaric acid, sulfur and 1,2-ethanediamine are included in the ESI80 In all cases we see a realistic softening of the vibrational frequencies with increasing temperature, often in quantitative agreement with experiment.In the harmonic approximation, the phonon frequencies are assumed not to change with temperature. In reality, the vibrations soften as the molecules move apart due to thermal expansion, resulting in a shift toward lower frequencies. This frequency shift is reproduced well with the quasi-harmonic approximation. In 43 The mean absolute relative error in calculated thermal expansion coefficients is estimated to be 15% based on data for 9 crystal structures, see In m-nitrophenol  and for 244 families we have used predicted melting temperatures. We find no differences in the trends between the sets with experimentally known and predicted melting points.14Most crystal structures in this study have melting points between 350 and 470 K, see 3.2.1–6 K–1, corresponding to an expansion of 0.8 and 2.4% per 100 K, which can be used as a rule of thumb for organic molecular crystals in general. The largest thermal expansions are seen for weakly bound crystals of organic solvents, such as acetonitrile , chloroethane , cyclobutanol  and 2,5-dimethylpyrazine .The volumetric thermal expansion coefficient of each crystal structure was calculated as a finite difference between the 0 K and the thermally expanded structure. –6 K–1 for 50% of pairs, and rarely differ by more than 50 × 10–6 K–1. So, while thermal expansion tends to be similar between polymorphs, the differences can sometimes be relatively large.The pairwise differences in volumetric thermal expansion coefficient between polymorphs are shown in The thermal expansion of a crystal is related to its elastic properties and differences in bulk and shear moduli are often of interest, for example in the manufacturing of drug tablets. The Hill averaged bulk and shear moduli calculated for all structures at 0 K are displayed in 94Bulk modulus differences between polymorphs exceed 2 GPa in only 10% of polymorph pairs and are less than 0.5 GPa in 41% of pairwise comparisons, see Pairwise absolute differences in elastic moduli between polymorphs at 0 K are shown in 3.2.2AQHA and the harmonic approximation free energy AHA. This difference, calculated at the melting point of each structure, is displayed in –1 and never more than 4 kJ mol–1 to the total free energy of organic molecular crystals.The thermal expansion leads to a softening of vibrational modes and hence an increase in vibrational entropy. This is counteracted by a less favourable lattice energy with increasing volume. The resulting total energetic effect of thermal expansion can be calculated as the difference between the quasi-harmonic free energy –1.38 We find that the effect is often larger than 1 kJ mol–1, which is likely due to our set containing larger molecules, for which the absolute energies are larger.Our results are consistent with those of Heit and Beran's fragment-based quantum chemical study of four crystals of small molecules , where the neglect of thermal expansion led to free energy errors of up to about 1 kJ mol–1. Hence, we do not see a trend in the thermal expansion contribution that tends to reduce the free energy difference between polymorphs, as is the case for the harmonic contribution.14 The thermal expansion rarely changes the relative stability between polymorphs by more than a small fraction of 1 kJ mol–1 and never by more than 2 kJ mol–1. For this reason, our harmonic approximation results presented previously14 are an accurate description of room temperature polymorph free energy differences.The thermal expansion contributions to the free energy difference between polymorph pairs is shown in Thermal expansion also leads to a softening of the crystal structure. The bulk and shear moduli decrease with temperature so that near the melting point, the bulk modulus has reduced by on average 50%, see 14In 14 which is expected since the melting points are substantially higher than 300 K for the vast majority of the crystals studied.The shaded green region in vs. temperature curves of polymorphs can either diverge with increasing temperature or converge. Diverging free energy vs. temperature curves have previously been described as extremely rare,12 because the higher energy polymorph is generally expected to be less well packed, leading to lower vibrational frequencies and a higher entropy. Indeed, only a few polymorph pairs with possibly diverging curves have been studied experimentally.96 Our results, on the other hand, predict that diverging free energy curves should be common, occurring in 38% of cases. At least some of this discrepancy is most probably caused by incorrect lattice energy rankings for some of the polymorphs with our energy model, which might lead to an overestimation of diverging polymorph pairs. For the same reason, the fraction of enantiotropic polymorph pairs (21%) may be underestimated. We have previously shown that trends in polymorph lattice energy and vibrational entropy differences with packing efficiency (or volume) exist, but are very weak.14 We believe that the larger proportion of polymorph pairs with diverging free energy vs. temperature curves than was previously expected are due to the weakness of these trends and the fact that both lattice energy and entropy have a more complex relationship with crystal structure than simply to packing efficiency. If the prediction in our study is true, it is an important finding that a significant proportion of polymorphic systems have diverging free energy vs temperature curves, and this prediction should be revisited as the models for lattice energies and vibrational energy contributions are further improved.The free energy AQHA(Tm) vs. AQHA(0) re-ranking (AQHA(Tm) vs. Elatt re-ranking  – is included in the ESI.


Young men who have sex with men (YMSM) and transgender people in the Detroit Metro Area are the only risk group for whom the incidence of HIV and sexually transmitted infections (STI) has increased since 2000, with HIV incidence nearly doubling among youth. Substance use , which is relatively frequent among YMSM and transgender people, creates barriers to the optimal delivery of HIV prevention and care services. Standard HIV counseling, testing, and referral (CTR) is limited in providing strategies to identify and address substance use. Hence, in its current form, CTR may not be serving the prevention needs of substance-using YMSM and transgender people. Brief counseling interventions, grounded in principles of motivational interviewing, may offer a mechanism to meet the HIV prevention and care needs of substance-using YMSM and transgender people.This prospective, 4-arm, factorial randomized controlled trial aims to examine the efficacy of an motivational interviewing–based substance use brief intervention (SUBI) on participants’ substance use and engagement in HIV prevention.The research implements a prospective randomized controlled trial (Project Swerve) of 600 YMSM and transgender people recruited both online and in person. Eligibility criteria include participants who (1) are between the ages of 15 to 29 years, (2) live in the Detroit Metro Area, (3) self-identify as a man or transgender man or woman, (4) have had sexual contact with a man in the 6 months before enrollment, (5) self-report binge drinking or any substance use in the 3 months before enrollment, and (6) self-report an unknown or negative HIV status upon enrollment. Participants are randomized to receive, 3-months apart starting at baseline, 2 individual sessions. Sessions are CTR-only, SUBI-only, CTR followed by SUBI, or SUBI followed by CTR.Project Swerve was launched in April 2017 and enrollment is ongoing.Incorporating a SUBI that utilizes the principles of motivational interviewing into HIV CTR provides an opportunity to tailor counseling services for YMSM and transgender people to address additional client barriers to HIV and STI testing.ClinicalTrials.gov NCT02945436; http://clinicaltrials.gov/ct2/show/NCT02945436 (Archived by WebCite at http://www.webcitation.org/6yFyOK57w) Young men who have sex with men (YMSM) and transgender people  are at heightened risk for HIV and other sexually transmitted infections (STI) ,2. The 3The Centers for Disease Control and Prevention (CDC) have recommended that HIV and STI testing be repeated frequently  for high-risk YMSMTG  . Recent Changes in the use of alcohol, tobacco, and other drugs (ATOD) during adolescence and young adulthood are developmentally noteworthy because they can have short- and long-term consequences that affect one’s adult life trajectory including HIV/STI acquisition , developAIDS Service Organizations (ASOs) often serve and are sensitive to the HIV-related needs of underserved YMSMTG. Delivery of HIV services through ASOs has been an efficient rollout mechanism because they reach and affect large numbers of people efficiently; create and establish grassroots policies and procedures that maximize the adoption and diffusion of interventions while considering the community’s social context; increase program sustainability and advocacy; and incorporate the needs of specific communities into tailored services. At present, however, HIV test counselors situated in ASOs are not trained to comprehensively and systematically screen for and address ATOD use in counseling, testing, and referral (CTR) sessions—the routine procedure used to test for HIV. Preliminary data from our community partners indicate that lack of AOD screening and counseling within CTR is a missed opportunity. The authors of this study  and otheConsistent with the National HIV/AIDS Strategy’s call  to reducThe prospective, 4-arm factorial randomized controlled trial (RCT) aims to examine the efficacy of Project Swerve, an MI-based SUBI (intervention) compared with the current standard of care CTR (control) on participants’ substance use and engagement in HIV prevention.The research and ethics presented in this study has been reviewed and approved by the University of Michigan Institutional Review Board (HUM00105125), in addition to the Data Safety Monitoring Board. The study is also registered on ClinicalTrials.gov (NCT02945436).The research activities involve a 4-arm factorial RCT of approximately 600 AOD-using YMSMTG aged 15-29 years in the DMA. We will follow participants for 18 months, with follow-up assessments collected every 3 months.The intervention comprises 2 visits separated by 3 months. Participants are randomized to receive either CTR (control) or a SUBI-adapted version of CTR  in each visit. To examine how the sequencing and dosing of interventions impacts efficacy, we randomize at baseline into a factorial RCT. The control arm will receive CTR-only at both study visits 1 and 2. Experimental arm 1 (CTR+SUBI) will receive CTR at visit 1 and SUBI at visit 2. Experimental arm 2 (SUBI+CTR) will receive SUBI at visit 1 and CTR at visit 2. Experimental arm 3 (SUBI+SUBI) will receive the intervention condition at visits 1 and 2. Individuals who test HIV-positive at study visits 1 or 2 will receive case management and linkage to care, as offered routinely by each ASO where study activities take place.This 4-arm factorial randomized design will help answer 3 important questions: (1) what is the impact of adapting current CTR to include SUBI on HIV engagement in care and sexual- and substance-related risk-taking behaviors among high-risk YMSMTG; (2) what combination of services  has the greatest impact on engagement in HIV prevention (where engagement in care is defined as routine HIV testing for seronegative YMSMTG and linkage and retention in care for seropositive YMSMTG); and (3) if effective, what are the costs of delivering SUBI compared with those of delivering CTR?Eligible participants are: (1) between 15 and 29 years old, (2) identify as a cisgender man or as transgender man or woman, (3) have had sexual contact with a man  in the last 6 months, (4) live in the DMA, (5) have unknown or negative HIV status, (6) and report binge drinking or using any illicit substance or nonmedical use of prescription drugs in the prior 3 months. The ATOD eligibility criteria are measured using the Alcohol, Smoking and Substance Involvement Screening Test (ASSIST)  to assesParticipants are recruited using Web-based advertisements on social media websites , which will be tailored to target YMSMTG in the DMA. Recruitment will also occur in person at local venues and community outreach events in the region.Eligible participants will be invited for their baseline visit at the ASO closest to them: offices are available in Ann Arbor, Ypsilanti, Detroit, and Flint, allowing access by participants from across the DMA. Participants who present for participation in the trial will have already set up a study account online and completed an online baseline survey with informed consent given online. When they arrive for their first visit, they will be reconsented verbally and offered a physical copy of the consent form. A study counselor will be available to answer any questions that the participants may have about the study before they decide to participate. Participants who do not consent to participate in the trial will be offered free HIV testing and counseling. Resources will be readily available for providing tools to avoid ATOD-related risks , and referrals to community resources as needed .Participants will be randomly assigned to 1 of the 4 conditions using a 1:1:1:1 treatment allocation. The treatment assignments will be generated with the use of a pseudo-random-number generator with permutated blocks that will be used to ensure balance between the numbers of YMSMTG assigned to each treatment. Assignments to the control or intervention arms will use concealment of allocation techniques designed to minimize assignment bias including generating, in advance, the sequence of assignment in sealed envelopes, which will be opened by the counselor at the time of randomization. When YMSM are randomized to receive CTR-only at a visit, they will receive standard CTR (30 min). YMSM randomized to SUBI at a study visit will receive the CTR that has been adapted to include the SUBI. On average, both conditions will last approximately 30 min.Participants will receive US $25 at each ASO visit and US $30 for each follow-up, making the total potential incentives  US $200 per participant.Study interventionists will be trained to deliver SUBI and will offer YMSMTG the opportunity to explore and strengthen motivations for changing their ATOD use during session. Interventionists are trained in CTR and typically have a Master’s degree in Public Health, Social Work, or a health-related discipline. The SUBI intervention consists of 2 components. Similar in style to other MI-based brief interventions for substance use and related risk behaviors -54, CompIn Step 1, counselors focus on MI-based engagement strategies to explore areas of strength and aspirations that the participant holds. Affirming these areas allows counselors to build rapport with participants and begin to uncover potential sources of motivation to change risky behaviors.Step 2 invokes the MI process of focusing by reviewing participants’ recent substance use. Counselors explore participants’ frequency of current substance use, their motivations for use, and elicit any consequences to using substances. Possible links between substance use and risky sexual behaviors are examined by querying the potential role of substance use in having sex or hooking up and use of condoms. Counselors are trained to listen for, elaborate on, and evoke change talk as it begins to occur in Step 2 and throughout the remainder of the session. In Step 2, the counselor conducts the rapid HIV test.In Step 3, counselors provide basic psychoeducation about how substance use and/or risky sex can impact risk for HIV infection, tailored to the participant’s own high-risk behaviors.Step 4 seeks to elicit from participants any potential benefits to changing their substance use , with a specific emphasis on how changing use can impact the risk for HIV transmission.In Step 5, counselors reflect on participants’ perceived benefits and assess the importance of and readiness to change using the visual of a ruler in order to elicit their current stage of change. If participants are interested in changing, the counselor uses evocative questions to elicit a potential first step; for those not interested in making changes currently, the counselor elicits participants’ views on what might prompt them to consider a change in the future.In Step 6, the counselor provides a strategic summary of what was discussed during Component 1. Here, counselors are beginning to transition into disclosing the HIV results from the test that was conducted in Step 2 and it is important for participants to think about what was discussed in each step as a whole.Finally, in Step 7, counselors elicit and reflect how participants would react to a positive (reactive) or negative (nonreactive) result before disclosing the HIV results.Component 2 varies based on the HIV test results see , with thIf participants’ results are nonreactive, repeat HIV testing and pre-exposure prophylaxis (PrEP) are the focal points of Steps 8 through 12. In Step 8, counselors elicit and reflect participants’ responses to receiving a nonreactive result.Step 9 explores the benefits of repeat testing. Counselors discuss the window period, recommendation for testing every 3-6 months, and how participants feel about repeat testing, particularly eliciting concerns regarding the window period. As with change talk regarding substance use, counselors are trained to reflect selectively and affirm statements favoring repeat testing.PrEP referrals are discussed in Step 10. Participants are asked what they know about PrEP, and counselors provide additional information and/or referrals to PrEP providers.In Step 11, counselors tie the goals and strengths from Component 1 into encouraging repeat testing and PrEP evaluation. Possible barriers to repeat testing and PrEP evaluation are discussed along with strategies to overcome these barriers.Step 12 summarizes what was talked about during Component 2 while affirming the strengths and goals from Component 1. Here, counselors elicit goals with regard to repeat testing and/or PrEP uptake and elicit steps to achieve these goals that are achievable, clear, and have a distinguishable end point. Barriers to achieving the goal are elicited and problem-solved, and strengths are affirmed as a means of supporting follow-through with the goals established. Counselors then thank the participants for their time and end the session. Alternatively, if participants’ HIV test results are preliminary reactive, linkage to HIV care is encouraged in Steps 8 through 11. Step 8 focuses on participants’ reactions to the test result. Counselors allow the participants to process their emotions and use empathic reflections in response and offering statements of hope.With permission, counselors begin to discuss the importance of linkage to HIV care in Step 9. Counselors explain how people with HIV can live healthy lives provided that they attend medical appointments and take medications while also exploring the participant’s perceived benefits of seeing a HIV medical provider.Step 10 links the strengths and goals from Component 1 as a tool to continue to encourage linkage to care. Possible barriers to linkage to care are explored, with an emphasis on the potential impact of substance use.Step 11 reflects on the participants’ goals for next steps toward linkage to care with an emphasis on eliciting goal-setting with goals that are achievable, clear, and have a defined end point. Counselors provide support to participants by affirming their strengths to meet these goals. Counselors thank the participants for their time and end the session.For those who received a nonreactive HIV test result at visit 1 and who are randomized to receive SUBI at study visit 2, the same intervention components and steps are delivered, as described above. For those who received a reactive test result at visit 1, the SUBI session focuses employing MI skills to address adherence with HIV care and the role of substance use.P<.05 altered by the number of comparisons using a Bonferroni adjustment (significance level is P=.02 for 3 comparisons). To have 80% power to compare each active treatment to the control group in a 4-arm trial, we require at least 500 participants to find a 20% difference between each treatment and control and 228 participants to find a 30% difference. To allow for 20% loss to follow-up , we estimate a sample of 600 YMSMTG to be enrolled.The primary outcome for the proposed trial is successful engagement in care. For those who test seronegative at baseline, we define engagement in care as participation in routine HIV testing. For those who test seropositive at baseline, we define engagement in HIV care as linkage and retention in HIV care  and achievement of viral suppression. We define power as correctly identifying the difference in the proportions of YMSM with serospecific engagement in HIV care within 15 months of each active treatment condition  to the control arm (CTR-CTR), thus powering for 3 independent hypothesis tests. Our sample size calculations are based on a 2-sample test of proportions using a 2-sided significance level of Participants may continue the study even if they miss follow-ups or visits intermittently over the data collection period. We will compare those who completed different follow-up interviews with those who did not on key predictors from the baseline assessment to check for possible sampling bias due to missing data. Missing data will be minimized by the computer-based entry for all measures. The use of Expectation-Maximization algorithm and multiple imputation approach in longitudinal analyses will help overcome missing data concerns when appropriate.The trial focuses on 3 sets of outcomes: engagement in HIV prevention and care services; AOD use; and sexual risk. The trial will also measure satisfaction with the intervention.We will ask YMSMTG having unknown or negative HIV status to indicate the date of any recent HIV and STI tests. At baseline, we will ask participants to note if they have ever been medically diagnosed as having one or more STIs. Among those diagnosed, we will ask if the STI diagnosis occurred in the prior 12 months. In follow-up surveys, we will ask participants to report if they tested for STIs in the prior 3 months and whether any of their tests were reactive.At each follow-up session (visits 3-7), we will measure the primary outcome of repeat HIV testing. For anyone who tested in each intersurvey period, we will assess the test result and motivations for testing . For seropositives, the survey will assess the incidence of HIV-related physician visits, including whether CD4 counts and viral load tests were conducted and prescriptions for antiretroviral therapy (ART) were given. We will also ask participants to self-report their adherence to ART using an abbreviated 6-item questionnaire based on the AIDS Clinical Trial Group assessment. Participants are asked to note, using a scale ranging from “never” to “often,” if they missed their HIV medication over the past month for one of the listed reasons. Seropositives will also provide a blood sample for a viral load test. Viral load testing will be done every 6 months. We prioritize viral load tests over a biomarker of adherence, given that adherence is the primary pathway to viral suppression.In addition to self-reported behavioral measures, the study will also collect biomarkers to test for HIV and STIs. At the intervention visits, HIV testing will be performed as part of CTR. STI screening: For syphilis screening, participants will undergo a blood draw for subsequent unheated serum regain test and, for chlamydia and gonorrhea screening, participants will provide a urine sample and pharyngeal, rectal, and/or vaginal swab cultures. Hepatitis C will be assessed using the OraQuick rapid test. Although we expect prevalence of hepatitis C to be low in an AOD population that has low levels of injection drug use (and thus hepatitis C will not be a secondary outcome), we will include hepatitis C testing to assess prevalence in this population. Those who are asymptomatic and test positive will be referred by study staff to local health care providers for further evaluation and treatment. All other STI tests will be processed by the Michigan Department of Health and Human Services. As required by state law, all new positive STI results will be reported to the State Health Department for the purposes of disease surveillance. The requirement for reporting of confidential test results to the health department will be explained in the informed consent. Condoms and water-based lubricant will be provided to all participants. We will screen for STIs at the final study assessment.We use the ASSIST  to assesIn addition to self-reported behavioral measures of drug and alcohol use, the study will also collect biomarkers of drug and alcohol use. Participants will provide urine samples for on-site toxicology screening using an EZ split key test kit . In our prior clinical research with methamphetamine-using MSM, urine samples were obtained for toxicology testing in 98% of study visits. The on-site urine screening kits are designed to test for recent amphetamine, barbiturates, benzodiazepines, buprenorphine, cocaine, marijuana, methadone, methamphetamine, oxycodone, propoxyphene, morphine, and ecstasy. Urine toxicology screening will be used to provide information regarding recent substance use and will be conducted each study visit. Alcohol screening will be used where ethyl glucuronide (EtG) a direct metabolite of ethanol indicates that ethanol has been ingested within the last 3-4 days (80 hours). EtG will be analyzed using the 1-step EtG test dip card. Urine will be collected in a urine specimen cup and the tip of the EtG dip card will be submerged into the urine sample for 15 seconds. EtG will allow us to adjust for under-reporting of recent unhealthy drinking on participants’ surveys and will also be screened at each study visit.Sexual behaviors will be assessed using a modified version of the Sexual Practices Assessment Schedule (SPAS) used in previous studies with YMSMTG  to exploYMSMTG will report data on the acceptability of the intervention after completing each intervention session. We will use 2 different assessments: (1) Self-Evaluation Forms (SEF) and (2) Client Satisfaction Questionnaires (CSQ-8). The SEF is a brief 12-item questionnaire that elicits information about the experience with the intervention . The CSQ-8 will be used at the completion of the intervention to assess satisfaction with the intervention. The CSQ-8 has demonstrated high internal consistency across a large number of studies . The SEFt tests or Wilcoxon rank sum tests for continuous variables and chi-square tests for categorical variables. We will conduct primary analyses of our pooled successful engagement measure using logistic regression analyses to compare each active treatment group with the control in pairwise comparison tests at an adjusted significance level of .017 to reduce Type-I errors in our 4-arm trial. We will then conduct exploratory logistic regression analyses by sero-status. For seronegative YMSMTG, the proportion of participants who obtain at least 2 tests at least 3 months apart within 15 months will be calculated and presented with corresponding 95% exact binomial CIs. Among seropositive YMSMTG, we will examine how intervention conditions influence HIV linkage and retention in care per IOM guidelines. We will also examine for all participants the pursuit of substance use treatment services (if necessary). Hence, for seronegative participants, the outcome will be repeat HIV testing at regular intervals, and for seropositive participants, the outcome will be linkage and retention in care. We will not consider viral suppression in the regression analysis due to the short time period available to achieve suppression. However, we will present descriptive statistics on viral load and suppression across the 4 treatment arms. The regression will be run with group assignment in the model while controlling for patient, structural, and agency characteristics. Interactions between group assignment and these characteristics will be tested to explore potential moderators of treatment effect. We will repeat these analyses for STI/ATOD biomarkers conducted during the trial. Additional analyses include comparing groups in (1) the average number of tests obtained using Poisson regression and (2) time to getting tested using repeated events survival analysis.Descriptive statistics of the clinical and demographic characteristics of the participants will described for all and by treatment group. These will be compared between treatment groups using β0i are assumed to be normally distributed with a common variance and they account for within-person correlation. We will also explore if we need a subject-specific random slope corresponding to visit in the above model. Maximum likelihood estimation will be used for fixed effect parameters. To ensure robustness, we will also apply an exchangeable working correlation structure to its corresponding generalized estimating equation model [We will test for intervention effects over time on sexual risk  and drug use  outcomes. We will use the general framework of generalized linear mixed models -60 to moon model .The study team balanced the clinical and ecological validity of the design and procedures of project Swerve as the intervention was developed for YMSMTG in the DMA. First, we recognize that the CTR and SUBI treatments may have different amounts of contact time with participants. Although it is possible that contact time may influence intervention effects across CTR and SUBI conditions, the control sessions may actually last longer than often expected during CTR, given the high-risk characteristics of our study participants. In our analyses, we will also examine whether length of sessions differ statistically across treatment arms and include time spent in each session as a covariate due to its potential confounder. Second, we recognize that there may be variability in how counselors deliver CTR and SUBI sessions, which could confound our ability to measure the intervention’s strength. We have put in place several procedures to minimize potential biases including training counselors using 2-day training with boosters and ongoing group and individual supervision, allowing counselors at each site—who are not trained in the intervention—to deliver CTR, and monitoring and addressing treatment fidelity for both conditions throughout the trial using the Motivational Interviewing Treatment Integrity-4 rating system .The study team will collect information on: (1) time spent by study staff for training, supervision, and technical assistance of counselors; (2) time participants spent in a counseling session; and (3) costs associated with test counselor delivery of the intervention. Capital equipment cost  and facility cost  at the study sites that are attributable to our intervention will be obtained from each site’s accounting records. Capital equipment cost will be distributed over a 4-year period with a 3% discount rate. Cost items that are not directly divisible between participants will be spread across relevant individuals . A flat rate covering the cost of CTR materials for each intervention group will be estimated. No costs associated with research data collection will be included. These components of cost will be summed over the 15-month study period for each participant to generate an estimated per person cost. Effectiveness will be measured by examining relevant substance use and HIV-related outcomes reported by each YMSMTG over the 15-month period. Incremental cost effectiveness ratio (ICER) across treatment arms will be defined as ΔC/ΔE, where ΔC denotes the estimated difference in mean cost per intervention and ΔE reflects the estimated difference in mean effectiveness between the intervention and control group. ICER indicates the additional costs associated with the intervention for each new HIV infection avoided. Nonparametric bootstrap resampling will be used to estimate the 95% CI of ICER . PrimaryProject Swerve launched in April 2017 and is currently recruiting YMSMTG into the trial. Current recruitment strategies combine online and in-person (venue-based sampling) approaches. As of February 22, 2018, 5183 people began the screener survey, of which 3178 (61.32%) completed it. In total, 594 (18.70%), people successfully screened were eligible to participate in the study, of which 378 (63.6%) provided consent and 223 of these (58.9%) enrolled into the study. Of these, 160 (71.7%) have completed the baseline survey and 18 dropped from the study; the remaining 142 participants have been randomized into study arms as follows: 36 SUBI-SUBI; 36 SUBI-CTR; 34 CTR-SUBI; and 36 CTR-CTR.SUBI is a promising approach to address AOD use as part of HIV prevention and care services for YMSMTG. Efficacious SUBI approaches are typically delivered on-site to clients and have the advantage of being reimbursable  and costAt present, few RCTs have examined the efficacy of brief interventions targeting ATOD use as a strategy to reduce sexual-risk taking behavior or to increase engagement in HIV care and prevention among high-risk YMSMTG who, compared with heterosexual adults or older MSM, may not have yet developed sustained drug use and abuse patterns. Furthermore, distinct developmental considerations including the use of ATOD before the legal age for use, the high prevalence and visibility of ATOD within YMSMTG’s social networks, and the influence of these social networks’ norms on YMSMTG’s behavior may require particular attention when developing a SUBI for YMSMTG -80. In a


Hair cells are specialized sensors located in the inner ear that enable the transduction of sound, motion, and gravity into neuronal impulses. In birds some hair cells contain an iron-rich organelle, the cuticulosome, that has been implicated in the magnetic sense. Here, we exploit histological, transcriptomic, and tomographic methods to investigate the development of cuticulosomes, as well as the molecular and subcellular architecture of cuticulosome positive hair cells. We show that this organelle forms rapidly after hatching in a process that involves vesicle fusion and nucleation of ferritin nanoparticles. We further report that transcripts involved in endocytosis, extracellular exosomes, and metal ion binding are differentially expressed in cuticulosome positive hair cells. These data suggest that the cuticulosome and the associated molecular machinery regulate the concentration of iron within the labyrinth of the inner ear, which might indirectly tune a magnetic sensor that relies on electromagnetic induction. Hair cells are located in defined sensory epithelia within the vertebrate inner ear, enabling the transduction of sound, motion, and gravity into neuronal impulses . This isFollowing the studies of Dickman and colleagues, who reported that neurons within the vestibular nuclei of pigeons are responsive to weak magnetic stimuli, we conducted a histological analysis of the inner ear of birds employing the Prussian blue stain which labels ferric iron , 2012. TAccordingly, we set out to investigate the developmental maturation of cuticulosomes in pigeons, as well as the molecular and subcellular architecture of cuticulosome positive hair cells. Exploiting electron microscopy and histological analysis we show that cuticulosomes develop rapidly after hatching, in a process that involves vesicle fusion and nucleation of ferritin nanoparticles within the cuticular plate. Coupled with a comparative transcriptomic analysis of hair cells with and without cuticulosomes our results emphasize the importance of vesicular function in the formation and maintenance of this iron-rich structure.3+) bright blue in color  which stains ferric iron  on basilar papillae isolated from pigeons aged 1 day, 8 days, 16 days, 30 days and 1 year. At 1 day of age we observed that 100% of organelles were in the process of formation , which gradually decreased to 34.6% at 8 days , 22.2% at 16 days  and 17.6% at 30 days  . In contTo investigate this further we performed electron tomography on 8 day old birds, preparing 200–250 nm thick resin embedded sections of the basilar papilla that were analyzed on a Polara transmission electron microscope with a −60° to +60° tilt series. Segmentation of the ferritin-like nanoparticles, vesicular structures, and the cell membrane allowed the generation of 3D tomographic models of the developing cuticulosome  –4. This 2+Fe3+2O4) and maghemite (Fe2O3) (2+) on sections from the basilar papilla and lagenar macula at hatching, 8 days, 16 days, 30 days and 1 year (n = 3 birds per timepoint). As the staining was very light, indicative of low levels of Fe2+, we post stained sections with the chromogen 3,3'-Diaminobenzidine (DAB) which is known to amplify the Turnbull Blue reaction  . We therreaction . We obsereaction . These dColumba livia genome  with 10,277 (90.1%) having an annotated function . To confNext, transcripts were subject to differential expression analysis with DESeq2  Figure . We founIn this study we investigated the developmental, subcellular, and molecular architecture of cuticulosome positive hair cells. We report that when pigeons hatch only a few cuticulosomes are present, followed by a striking increase in their prevalence at 8 and 16 days of age, with the highest number observable at 30 days. Our electron microscopy studies have further demonstrated that the cuticulosome is formed within the cuticular plate itself. The development of the cuticular plate, which is closely associated with the formation of stereocilia, begins at embryonic day 8 in chickens. At this time it is primarily occupied by vesicles, mitochondria, and lysosomes, before growing basally until embryonic day 14 when it resembles that present in the adult cochlea . Post haOur data indicate that the formation of the cuticulosome is initiated at the apical cellular border opposite the kinocilium. It appears that the cuticulosome is formed by the fusion of vesicles, some containing ferritin, that result in an organelle that increases in size and density . It appeOur results have also yielded insight into the molecular architecture of cuticulosome positive hair cells. Employing immunohistochemical methods we have shown that the cuticulosome contains the ferritin heavy chain protein. Moreover, given our RNA sequencing results, which have revealed that the ferritin light chain is expressed at very low levels, we would expect that the cuticulosome is dominated by the heavy chain isoform. Our transcriptomic studies have further implicated molecules associated with vesicular trafficking, endosomal sorting, extracellular exosomes and metal binding with cuticulosome maintenance. Of particular interest are RAB5B and RNF128. RNF128, which was significantly downregulated in cuticulosome containing hair cells, is a Ring finger E3 ubiquitin ligase. Drosophila and human family members of this protein class have been associated with the formation of enlarged Rab5 positive endosomes both in knockdown and overexpression studies . NotablyThe critical question that remains is: What is the function of the cuticulosome? And might it be associated with the magnetic sense? This is a tempting proposition given that pigeons begin to fly at approximately one month of age, a time when the cuticulosome is on the cusp of maturation . HoweverThe data presented in this manuscript strongly suggest that the formation of the cuticulosome is not the product of late-onset iron accumulation, and is more likely the result of a genetically encoded developmental or homeostatic program. Could the cuticulosome therefore serve as a torque based magnetoreceptor that would exert a direct force on a mechanosensitive channel located on the apical membrane? This would seem unlikely. Firstly, based on our current understanding of the magnetic properties of ferritin, the cuticulosome lacks sufficient magnetic susceptibility in an Earth strength magnetic field to open a mechanosensitive channel, and, secondly, there seems to be no direct physical connection with the apical membrane that would mediate the transfer of a force . Indeed Birds were maintained on a 12:12 light-dark cycle at 25°C in a custom built aviary. All experiments were performed in accordance with an existing ethical framework (GZ: 214635/2015/20) granted by the City of Vienna (Magistratsabteilung 58).Columba livia) of different ages were dissected with ceramic-coated tools and drop fixed in 4% phosphate buffered formaldehyde (PFA) (pH 7.4). After dehydration in a series of ethanol concentrations, the cochlear ducts were embedded in paraffin and sectioned on a rotary microtome (10 μm) with Dura Edge High Profile ceramic-coated blades . For Prussian blue (PB) stainings, slides were incubated for 20 min in 5% potassium hexacyanoferrate (II)  with 10% HCl. The slides were washed 3 times for 5 min in PBS and then counterstained with Nuclear Fast Red  for 1 min, followed by another washing step, dehydration and cover-slipping. All hair cells and PB-positive hair cells on every fourth section were counted and multiplied by a factor of 4 to obtain estimates of total hair cell number and total PB-positive hair cell number in each cochlear duct. For whole mount experiments, the tegmentum vasculosum and the tectorial membrane were removed, the cochlear ducts were then fixed in 4% PFA (pH 7.4), washed in PBS, incubated in 5% potassium hexacyanoferrate  with 10% HCl for 20 min, followed by another wash in PBS and counterstained in Nuclear Fast Red for 1 min. The whole mounts were embedded in 87% glycerol  and coverslipped. For Turnbull blue (TB) stainings, 5–10 sections per bird were incubated for 20 min in 5% potassium hexacyanoferrate (III)  with 10% HCl (n = 3 per timepoint). The slides were washed 3 times for 5 min in PBS after which endogenous peroxidase was blocked by 30 min of incubation in 0.3% H2O2 in methanol. After three further PBS washes, the TB staining was intensified by incubating with 3,3’ Diaminobenzidine . Another three washes with PBS were followed by counterstaining with Nuclear Fast Red  for one minute, another washing step, dehydration and coverslipping. All hair cells and TB-positive hair cells on at least five sections per bird and region (lagena and basillar papilla) were counted.The cochlear ducts from pigeons (http://imagej.net). We defined a cuticulosome as being in formation if its size was less than 250 nm, and/or the periphery of the cuticulosome was amorphous. Density plots were generated by drawing lines (200 nm each) radially from the edge of cuticulosomes in 45° increments (http://imagej.net). The mean intensity of every increment was calculated for eight cuticulosomes individually (n = 3 birds aged 8 days).Cochlear ducts from pigeons of different ages were dissected before fixing overnight in 2.5% glutaraldehyde supplemented with 2% paraformaldehyde (PFA) in Soerensen buffer (pH 7.4). Tissues were then incubated in osmium tetroxide  for 1 hr, dehydrated in increasing ethanol solutions, embedded in epoxy resin and polymerized at 60°C for 48 hr. Ultra-thin (70–120 nm) sections were prepared and analyzed with a Morgagni 268D transmission electron microscope operated at 80kV . The size of cuticulosomes was measured on electron micrographs of cuticulosomes using the ImageJ software (crements . The denEpon embedded sections (200–250 nm) were collected on slot grids, poststained with 2% aqueous uranyl-acetate followed by Reynold's lead citrate. For image alignment during tomogram reconstruction 10 nm gold beads Aurion  were added as fiducial markers. Room temperature tilt series were acquired at a Tecnai G2 20 microscope (FEI) operated at 200kV and equipped with an Eagle 4 k HS CCD camera (FEI). Dual axis tilt series were collected using the SerialEM acquisition software with a tilt range from −60° to +60° with one degree increments . TomograC. Slides were cooled down at room temperature for 30 min afterwards, washed three times in PBS for 5 min and incubated with a ferritin heavy chain primary antibody  over night at 4°C at a concentration of 1:500 in 0.3% Triton/PBS (pH 7.4) supplemented with 2% donkey serum. Slides were washed three times in PBS for 5 min, incubated with donkey anti-goat Alexa Fluor-568 secondary antibody  at a concentration of 1:500 for 1 hr at 4°C. This was followed by another washing step, coverslipping and imaging using a confocal microscope . Whole mounts were fixed in 4% PFA for 10 min, washed in PBS following antigen retrieval as described above and incubated with primary antibodies for heavy chain ferritin (1:500) and actin  overnight at 4°C in 0.3% Triton/PBS (pH 7.4). This was followed by incubation with secondary antibodies donkey anti-goat Alexa Fluor-568  and donkey anti-mouse Alexa Fluor-488  at a concentration of 1:500 for 1 hr at 4°C, before washing and coverslipping. Z-stacks were acquired using a confocal microscope and z-projections with maximum intensity were generated using the ImageJ software.The cochlear ducts of pigeons were dissected as described above and fixed in 4% phosphate buffered formaldehyde (PFA) (pH 7.4) for 10 min. After fixation, the tissue was dehydrated in 30% Sucrose/PBS overnight, embedded in Neg-50 Frozen Section Medium  and sectioned (12 μm) on a cryostat . Slides were dried for 3 hr at room temperature, washed three times in PBS for 5 min followed by incubation in antigen unmasking solution  in a waterbath that was heated up to 90°2, 1 mM MgCl2, 5 mM Glucose, 20 mM HEPES, DEPC treated Mono Q H2O, pH 7.4). The lagenar maculae were removed from the cochlear ducts with a ceramic scalpel blade. The remaining parts of the cochleae were incubated in 0.05% Trypsin:EDTA  at 37°C for 5 min and cut into small pieces. Trypsinization was stopped by adding trypsin inhibitor  and recombinant RNAse inhibitor was added to prevent RNA degradation . Hair cells were screened and isolated by employing a modified, inverted microscope , using anmicromanipulator  equipped with glass micropipettes . RNA extractions of 30 isolated hair cells with and without cuticulosomes from nine birds respectively  were performed using the TRIZOL/Chloroform method and the RNA Clean and Concentrator-5 Kit . A total of 21 cDNA libraries (18 samples plus three negative controls) were prepared employing a full length RNA-seq from single cells protocol (The cochlear ducts from pigeons (1 year of age) were dissected with titanium tools and transferred to Bird Ringer solution . A maximum of 6 miss matches and introns between 20–150000 bp were allowed. Maximum multihits were set to one and InDels and Microexon-search was permitted. Furthermore, a gene model was implemented as GTF (Shapiro). Aligned reads were subjected to FPKM estimation with Cufflinks (v1.3.0)  and bias detection and correction was performed. Only fragments compatible with annotation were accepted and counted towards the number of mapped hits used in the FPKM denominator. The aligned reads were counted with HTSeq (0.6.1p1) and the transcripts were subjected to differential expression analysis with DESeq2 that incorporates a Benjamini and Hochberg procedure for multiple testing . The sequencing data was analyzed using an adapted published protocol . Briefly testing . Transcrhttp://primer3plus.com/cgi-bin/dev/primer3plus). The efficiency of each primer pair was assessed and only primers with efficiency between 90% and 100% were selected for the experiments. Reactions were performed in technical triplicates together with GAPDH and HPRT as control genes and no template controls on a Bio-Rad CFX Connect cycler  using the SsoAdvance Universal SYBR Green Supermix . Gene expression levels of individual genes were calculated relative to the geometric mean of GAPDH and HPRT. To confirm our transciptomic results qPCR was performed on cDNA libraries prepared from cuticulosome positive and cuticulosome negative cells as described above (n = 8 birds). Statistical analysis was performed using paired one-tailed t-tests followed by Bonferroni correction for multiple comparison.To assess the levels of ferritin heavy chain microdissections of the cochlear duct were performed on the distal region of the basilar papillar isolated from birds at hatching, 8 days, 16 days, 30 days and 1 year (n = 3 birds per timepoint). Total RNA from hair cell epithelia was extracted using the RNeasy Mini Kit  and cDNA was prepared using the QuantiTect Reverse Transcription Kit . Intron-spanning qPCR primers for each gene of interest were designed using the Primer3Plus program , form the basis of this letter. The handling Senior Editor was Andrew King. As you will see, all of the reviewers were impressed with the importance and novelty of your work.Thank you for submitting your manuscript "Subcellular analysis of pigeon hair cells implicates vesicular trafficking in cuticulosome formation and maintenance" to I am including the three reviews at the end of this letter, as there are a variety of specific and useful suggestions in them. We appreciate that the reviewers' comments cover a broad range of suggestions for improving the manuscript. Given these disparate concerns, we feel it would be best for you to evaluate the suggestions and write back to us with a prioritized list of experiments that could be completed in a reasonable period of time. We will share your plans with the reviewers and then respond with advice on how best to proceed.Reviewer #1:eLife.The cuticulosome is a newly discovered, orphan organelle in search of a function and a developmental understanding. In this manuscript, Nimpf et al. describe the distribution and formation of ferritin-containing cuticulosomes in hair cells from the basilar papilla of pigeons with high-resolution transmission electron micrographs taken at different developmental stages and observe that the cuticulosomes are surrounded by multiple vesicles with or without ferritin. In addition, single cell transcriptome analysis conducted in this manuscript reveals the potential importance of vesicle fusion in formation of cuticulosomes. And it also provides a rich resource in the form of a gene-candidate list that could now be used to understand the formation or the function of cuticulosomes in the future. This is a well-written and interesting manuscript, which could be improved with the below suggestions, for suitability in Major concerns1) The models put forth of cuticulosome development are interesting. I feel that to make the study more complete the author should also explore the cuticular plate actin structure immediately around the cuticulosome. How is the actin altered by the vesicles? Are there actin-free tunnels (or pockets) in which these vesicles may travel (or reside)? The answers to these questions should be addressed with tomography and added to 2) Can the authors localize RAB5B to the cuticulosome or any proteins from their list (preferred) that are differentially expressed? If not, can the authors perform in situ hybridization to show differential expression. The data needs validation. As a last resort, can they do single cell RT-PCR?3) Why is there a very wide range of sizes of cuticulosomes in one-year-old pigeons? Since birds can regenerate hair cells, could this be that some of the cuticulosomes measured are in newly born hair cells? Or is there a big variation of cuticulosomes' sizes in a one-year-old pigeon. If so, can the authors further explain the criteria of selecting a single dissociated hair cell?4) If, as the authors proposed, vesicles surrounding the developing cuticulosome help the cuticulosome to grow in size, is fusion between the vesicle and the cuticulosome ever observed by electron microscopy?5) 6) When is the onset of hearing and flying ability in pigeons? Additional information on the stages of hair cell and ear development represented by the different ages chosen for analysis would be helpful in interpreting the results. Could the organized ferritin particle be used as a marker for maturation of the cuticulosome?Reviewer #2:This manuscript is a developmental and transcriptomic characterization of the pigeon hair cells that contain cuticulosomes, an organelle that was originally described by the Keays Lab. The current manuscript suggests that this organelle is tightly regulated and therefore must have an important function. However, what that function might be is very speculative.The authors have performed a morphological analysis of this organelle using Prussian blue (PB) staining across post hatching developmental stages. Their novel finding in this section is that cuticulosomes form rapidly, increase in number through development and their localization changes from a lateral-apical to a medio-basal position as they mature. The characterization of hair cells using TEM reveals that hair cells from recently hatched animals contain cuticulosomes that are "diffuse" and around 16 days after hatching they become larger and paracrystalline in structure.Q1) Is it possible that the observed change in size and structure is due to the TEM sample preparation and for some reason younger hair cell cuticulosomes are more susceptible to degradation?Next, the authors demonstrate that the cuticulosomes are composed of nanoparticles of ferritin by performing ferritin heavy chain IHC.Q2) Although the ferritin heavy chain-rich puncta resemble the cuticulosomes there is no quantification to demonstrate that these structures develop similar to PB-positive cuticulosomes and hence they are likely the same organelle.The transcriptomic analysis of hair cells with and without cuticulosomes is done with 1-year-old animals. One of the most highly expressed transcripts is the ferritin heavy chain in cuticulosome positive and negative hair cells.Q3) The ferritin IHC data in Differential expression analysis of the transcriptomic data reveals that "genes associated with vesicle trafficking, endocytosis, the formation of membrane bound organelles, and metal binding are differentially expressed in cuticulosome positive hair cells."Q4) It is necessary to confirm at least some of these gene expression profiles with in situ hybridization.Q5) In their abstract the authors state that "These data suggest that the cuticulosome and the associated molecular machinery regulate the concentration of Fe3+ within the labyrinth of the inner ear, which might indirectly tune a magnetic sensor that relies on electromagnetic induction." However, in their previous paper they have stated that "There is no evidence that sensory neurons play a role in iron homeostasis, and it is not clear why a role in iron storage would necessitate the conspicuous positioning of a single organelle in the cuticular plate." In addition, it is not clear why these structures would need to be composed of ferritin particles that are arranged in a paracrystalline array, heavily regulated developmentally in size/position and appear only in a percent of cells to perform the proposed homeostatic function.Reviewer #3:The manuscript by Keays and colleagues is the first study that investigates the formation and trafficking of cuticulosomes in pigeon hair cells during early development after hatching. The data revealed in this detailed manuscript are important for understanding the development of magnetoreception in young pigeons. However, a few questions should be addressed before the manuscript is suitable for publication:1) One of the main concerns is the low-number animal cohorts (3-6) and few (e.g. 3) repetitions of each experiment. Was the power analysis ever performed, or what was the rationale for cohort size selection? (There's only one RNASeq experiment for which authors discuss their sample size selection.)2) Subection “Cuticulosomes form rapidly after birds hatch”, percent averages of cuticulosome positive cells are provided with no standard deviation/error. Given the small cohort sizes it would be valuable to see the deviations.3) As authors indicate that the ferritin clusters cuticulosomes may be contributing to magnetoreception, it would be interesting to identify what is the iron content of these structures – that is spacing between individual iron atoms.4) Following up on the previous point, the magnetic properties of the cuticulosome are of great interest. Have the authors performed vibrating sample or SQUID magnetometry on these structures? Before magnetoreception can be discussed, the structures need to be proven magnetic or conductive (if the inductive mechanism is considered).5) The authors suggest that the cuticulosome and the associated molecular machinery regulate the concentration of Fe3+ within the labyrinth of the inner ear. However, whether the concentration of Fe3+ changes during development is still unknown. The correlation between Fe3+ concentration in the labyrinth and the expression of cuticulosomes is also not clear. The authors need to provide stronger evidence or supporting references to show the relation between Fe3+ concentration in the labyrinth and the number of cuticulosomes in hair cells.6) It is surprising that the number of cuticulosomes is the highest in 30-day old pigeons. The authors, however, do not show the localization or the size distribution of cuticulosomes in 30-day old pigeons as compared to 1-year old pigeons. The authors should demonstrate whether the cuticulosomes are fully developed in 30-day old pigeons.7) In 8) According to the results presented in this manuscript, cuticulosomes develop within a few days after hatching, and the number of cuticulosomes in the basilar papilla peaks in 30-day old pigeons. This is at odds with prior work that demonstrated that young pigeons (<12 weeks old) are incapable of perceiving magnetic field in the dark . The authors should discuss this in their manuscript.References: Wiltschko, W. and Wiltschko, R., (1981) Disorientation of inexperienced young pigeons after transportation in total darkness. Nature 291: 433 – 434. Reviewer #1:The cuticulosome is a newly discovered, orphan organelle in search of a function and a developmental understanding. In this manuscript, Nimpf et al. describe the distribution and formation of ferritin-containing cuticulosomes in hair cells from the basilar papilla of pigeons with high-resolution transmission electron micrographs taken at different developmental stages and observe that the cuticulosomes are surrounded by multiple vesicles with or without ferritin. In addition, single cell transcriptome analysis conducted in this manuscript reveals the potential importance of vesicle fusion in formation of cuticulosomes. And it also provides a rich resource in the form of a gene-candidate list that could now be used to understand the formation or the function of cuticulosomes in the future. This is a well-written and interesting manuscript, which could be improved with the below suggestions, for suitability in eLife.We thank the reviewer for his/her kind remarks and constructive criticism.Major concerns1) The models put forth of cuticulosome development are interesting. I feel that to make the study more complete the author should also explore the cuticular plate actin structure immediately around the cuticulosome. How is the actin altered by the vesicles? Are there actin-free tunnels (or pockets) in which these vesicles may travel (or reside)? The answers to these questions should be addressed with tomography and added to The reviewer has raised an interesting issue regarding the architecture of the actin rich meshwork surrounding the cuticulosome. Specifically, the reviewer has asked whether there are any actin-free tunnels or pockets. To investigate this we have examined high-resolution electron micrographs of developing cuticulosomes (8 days post hatching). This has not revealed any obvious tunnels (or pockets) in which vesicles travel. To display this data quantitatively we measured the intensity of osmium staining along 200nm lines that extend radially from the edge of cuticulosomes . This result suggests that there are no "vesicle highways", but rather vesicles squeeze through the actin meshwork which presumably involves some remodeling of the structure. These data are shown in 2) Can the authors localize RAB5B to the cuticulosome or any proteins from their list (preferred) that are differentially expressed? If not, can the authors perform in situ hybridization to show differential expression. The data needs validation. As a last resort, can they do single cell RT-PCR?in situ hybridization on the pigeon adult inner ear. Nevertheless, we agree with the reviewer that validation of our transcriptomic data is important. We have therefore undertaken qPCR analysis of our best candidates RAB5B and RNF128 on cuticulosome positive and negative cells (n=8 birds). These data which are now shown in We have found that few commercially available antibodies work well on pigeon tissue . We have likewise had limited success employing 3) Why is there a very wide range of sizes of cuticulosomes in one-year-old pigeons? Since birds can regenerate hair cells, could this be that some of the cuticulosomes measured are in newly born hair cells? Or is there a big variation of cuticulosomes' sizes in a one-year-old pigeon.The average size of cuticulosomes in 1 year old birds is 545 ± 31.4 nm (n = 18 cuticulosomes), which is similar to that in 30 day old birds (514.3 ± 31.4 nm (n = 17 cuticulosomes). It does seem that the variation in cuticulosome size is particularly pronounced at 1 year of age. However, as the reviewer suggests the smaller cuticulosomes at this time could reflect hair cell regeneration which is known to occur in Avian species. Those hair cells with smaller cuticulosomes may represent newly born hair cells. Indeed, this could account for the 7% of cuticulosomes that we observe at 1 year of age that appear to be in the process of formation See .If so, can the authors further explain the criteria of selecting a single dissociated hair cell?When selecting single hair cells for the transcriptomic analysis, a visual inspection of individual hair cells was made by moving through the focal plane in the Z-direction. Those hair cells with a round opaque structure within the cuticular plate were determined to be cuticulosome positive.4) If, as the authors proposed, vesicles surrounding the developing cuticulosome help the cuticulosome to grow in size, is fusion between the vesicle and the cuticulosome ever observed by electron microscopy?In our electron tomograms we observed vesicular structures that looked like they were about to fuse with the cuticulosome see  but none5) This has now been rectified and the relevant table inserted. See 6) When is the onset of hearing and flying ability in pigeons? Additional information on the stages of hair cell and ear development represented by the different ages chosen for analysis would be helpful in interpreting the results.Flying in pigeons occurs at approximately one month of age. It is therefore tempting to correlate this behavioural milestone with the formation of the cuticulosome. However, we recommend caution on this front as it is unclear whether young pigeons have a magnetic sense.We now state in the Discussion section:"The critical question that remains is: What is the function of the cuticulosome? And might it be associated with the magnetic sense? This is a tempting proposition given that pigeons begin to fly at approximately one month of age, a time when the cuticulosome is on the cusp of maturation . However, this correlation should be viewed cautiously as there is conflicting behavioural data supporting the presence of a magnetic sense in young birds. Wiltschko and colleagues have reported that chickens aged 10 days can be conditioned to a magnetic anomaly  and juvenile European Robins are known to possess a magnetic sense , whereas pigeons aged 12 weeks are disorientated if they are transported in the dark . An experiment that assessed whether magnetic stimuli results in c-fos associated neuronal activation in pigeons of different ages would be valuable ."We are not aware of any specific study in pigeons that have assessed their hearing at different ages, however, for chickens it is known that the tonotopic map of the cochlear develops during embryogenesis, and at 21 days of age the auditory system is mature structurally and functionally .Could the organized ferritin particle be used as a marker for maturation of the cuticulosome?As only 16% of cuticulosomes are ordered in a paracrystalline array in the adult , we do not think this particular attribute is best for defining whether or not a cuticulosome is mature.Reviewer #2:This manuscript is a developmental and transcriptomic characterization of the pigeon hair cells that contain cuticulosomes, an organelle that was originally described by the Keays Lab. The current manuscript suggests that this organelle is tightly regulated and therefore must have an important function. However, what that function might be is very speculative.The authors have performed a morphological analysis of this organelle using Prussian blue (PB) staining across post hatching developmental stages. Their novel finding in this section is that cuticulosomes form rapidly, increase in number through development and their localization changes from a lateral-apical to a medio-basal position as they mature. The characterization of hair cells using TEM reveals that hair cells from recently hatched animals contain cuticulosomes that are "diffuse" and around 16 days after hatching they become larger and paracrystalline in structure.Q1) Is it possible that the observed change in size and structure is due to the TEM sample preparation and for some reason younger hair cell cuticulosomes are more susceptible to degradation?While we cannot exclude this possibility, we think it is very unlikely. For all developmental time points the samples were prepared the same way. Cochlear ducts were dissected from the inner ear, before drop fixing overnight in 2.5% glutaraldehyde supplemented with 2% paraformaldehyde in Soerensen buffer (pH 7.4). Tissues were then incubated in osmium tetroxide for 1 hour, dehydrated in increasing ethanol solutions and embedded in epoxy resin. Moreover, when analysing the samples by TEM there was no evidence of tissue degradation in younger birds.Next, the authors demonstrate that the cuticulosomes are composed of nanoparticles of ferritin by performing ferritin heavy chain IHC.Q2) Although the ferritin heavy chain-rich puncta resemble the cuticulosomes there is no quantification to demonstrate that these structures develop similar to PB-positive cuticulosomes and hence they are likely the same organelle.We thank the reviewer for this comment, but we think an additional developmental time series  is beyond the scope of this 2-month revision. We are, however, very confident that the ferritin positive structures we report in The transcriptomic analysis of hair cells with and without cuticulosomes is done with 1-year-old animals. One of the most highly expressed transcripts is the ferritin heavy chain in cuticulosome positive and negative hair cells.Q3) The ferritin IHC data in Our ability to perform single cell qPCR on cuticulosome positive cells is dependent on our ability to pick hair cells with cuticulosomes. As newly hatched birds have few cuticulosomes this experiment is problematic. Moreover, the smaller cuticulosomes at hatching are challenging to identify with transmitted light. As an alternative we have microdissected the distal region of the basilar papillar from multiple developmental stages , extracted mRNA, generated cDNA, and performed qPCR on heavy chain ferritin transcripts. These data are now shown in Differential expression analysis of the transcriptomic data reveals that "genes associated with vesicle trafficking, endocytosis, the formation of membrane bound organelles, and metal binding are differentially expressed in cuticulosome positive hair cells."Q4) It is necessary to confirm at least some of these gene expression profiles with in situ hybridization.in situ hybridisation in the adult inner ear very challenging. As an alternative we assessed the levels of RAB5B and RNF128 in cuticulosome positive and cuticulosome negative hair cells (n=8 birds) by qPCR. These data which are now shown in As indicated above (reviewer 1), we have found Q5) In their abstract the authors state that "These data suggest that the cuticulosome and the associated molecular machinery regulate the concentration of Fe3+ within the labyrinth of the inner ear, which might indirectly tune a magnetic sensor that relies on electromagnetic induction." However, in their previous paper they have stated that "There is no evidence that sensory neurons play a role in iron homeostasis, and it is not clear why a role in iron storage would necessitate the conspicuous positioning of a single organelle in the cuticular plate." In addition, it is not clear why these structures would need to be composed of ferritin particles that are arranged in a paracrystalline array, heavily regulated developmentally in size/position and appear only in a percent of cells to perform the proposed homeostatic function.In this manuscript we present data which supports a role of vesicular trafficking in the development and maintenance of the cuticulosome. We agree with the reviewer that it is unclear why hair cells nucleate iron within the cuticular plate in paracrystalline arrays. We cannot (at this stage) make any definitive statements with regards to the function of this organelle, but we think our proposition that it may play a role in the regulation of cation concentrations within the inner ear labyrinth is not unreasonable.Reviewer #3:The manuscript by Keays and colleagues is the first study that investigates the formation and trafficking of cuticulosomes in pigeon hair cells during early development after hatching. The data revealed in this detailed manuscript are important for understanding the development of magnetoreception in young pigeons. However, a few questions should be addressed before the manuscript is suitable for publication:1) One of the main concerns is the low-number animal cohorts (3-6) and few (e.g. 3) repetitions of each experiment. Was the power analysis ever performed, or what was the rationale for cohort size selection? (There's only one RNASeq experiment for which authors discuss their sample size selection.)For our histological experiments we did not perform a power analysis. In choosing a n=3-6 for our experiments we were guided by our previous developmental studies on mice  and histological work on pigeons . Moreover, this reflects the standard in the field, where n's of 3-5 are routinely employed to study the development of the avian inner ear .2) Subection “Cuticulosomes form rapidly after birds hatch”, percent averages of cuticulosome positive cells are provided with no standard deviation/error. Given the small cohort sizes it would be valuable to see the deviations.This data is displayed graphically in 3) As authors indicate that the ferritin clusters cuticulosomes may be contributing to magnetoreception, it would be interesting to identify what is the iron content of these structures – that is spacing between individual iron atoms.3+)2O3•0.5H2O) . Consistent with this finding our Prussian Blue staining has revealed the presence of Fe3+. However, it has been reported that ferritin also contains magnetite . While a specific stain for magnetite (Fe2+Fe3+2O4) is not available, Ferric iron (Fe2+) (which is a component of magnetite) can be stained with the Turnbull Blue method . We have now performed this experiment on our birds at hatching, 8 days, 16 day, 30 days and 1 year. As the staining was very light, indicative of low levels of Fe2+, we post stained sections with the chromogen 3,3'-Diaminobenzidine (DAB) which is known to amplify the Turnbull Blue reaction . We observed a sharp increase in Turnbull blue staining in the basilar papilla and lagena from hatching to one month of age, mirroring the results of our Prussian Blue staining.We have previously undertaken selected area electron diffraction experiments on cuticulosomes in epon embedded sections. This has yielded diffraction patterns indicative of ferrihydrite  in the basilar papilla and 0.4% of hair cells are TB positive  in the lagena macula.· 8-day-old birds 17.5% of hair cells are TB positive  in the basilar papilla and 1.3% of hair cells are TB positive  in the lagena macula.· 16-day-old birds 29.12% of hair cells are TB positive  in the basilar papilla and 1.64% of hair cells are TB positive  in the lagena macula.· 30-day-old birds are 23.9% of hair cells are TB positive  in the basilar papilla and 1.12% of hair cells are TB positive  in the lagena macula.· 1-year-old birds 19.2%, of hair cells are TB positive  in the basilar papilla and 1.16% of hair cells are TB positive  in the lagena macula.2+ at all developmental stages, which is consistent with presence of low levels of magnetite. These data are now shown in This experiment reveals that the cuticulosome contains Fe4) Following up on the previous point, the magnetic properties of the cuticulosome are of great interest. Have the authors performed vibrating sample or SQUID magnetometry on these structures? Before magnetoreception can be discussed, the structures need to be proven magnetic or conductive (if the inductive mechanism is considered).We have made a number of attempts to purify cuticulosomes , as well as undertaking SQUID analysis on the basilar papilla itself. Despite the pooling of large numbers of samples we have not been able to detect any magnetic signature above background for these experiments – indicating that SQUID lacks the sensitivity we require. As a consequence we are currently in the process of undertaking nanoscale magnetic imaging employing nitrogen valence centers. This method relies on the exchange of magnetisation between nitrogen valence centers on a diamond slide and the magnetic domains of the cuticulosome . We have recently encountered some technical issues regarding the preparation of the diamonds. We are therefore not in a position to make any conclusions regarding the magnetic properties of the cuticulosome at this stage. We will aim to publish these findings at a later time.We wish to emphasize, however, that the inductive mechanism that we propose in the discussion is not dependent on the magnetic properties of the cuticulosome. It is rather dependent on the conductive properties of the endolymph and the presence of a highly sensitive electroreceptor in a population of sensory cells.5) The authors suggest that the cuticulosome and the associated molecular machinery regulate the concentration of Fe3+ within the labyrinth of the inner ear. However, whether the concentration of Fe3+ changes during development is still unknown. The correlation between Fe3+ concentration in the labyrinth and the expression of cuticulosomes is also not clear. The authors need to provide stronger evidence or supporting references to show the relation between Fe3+ concentration in the labyrinth and the number of cuticulosomes in hair cells.The reviewer has suggested an experiment that we are currently attempting to undertake. We wish to sample the endolymph at various developmental stages, followed by analysis of its ionic composition. As far as we are aware this experiment has not previously been performed – it is technically very very challenging. It requires that we pierce the bony labyrinth of the inner ear with a glass pipette or needle, sample microliters of the endolymph (while avoiding contamination from the perilymph), followed by analysis by inductively coupled plasma-mass spectrometry. We hope that we will be able to master this protocol, in birds of various ages, in the next 12-24 months.6) It is surprising that the number of cuticulosomes is the highest in 30-day old pigeons. The authors, however, do not show the localization or the size distribution of cuticulosomes in 30-day old pigeons as compared to 1-year old pigeons. The authors should demonstrate whether the cuticulosomes are fully developed in 30-day old pigeons.We have undertaken a subcellular analysis of cuticulosomes at 30 days of age, quantitating their size, association with vesicles, presence of membranes and paracrystalline organisation . These data are now shown in an updated version of 7) In We have rectified this as requested.8) According to the results presented in this manuscript, cuticulosomes develop within a few days after hatching, and the number of cuticulosomes in the basilar papilla peaks in 30-day old pigeons. This is at odds with prior work that demonstrated that young pigeons (<12 weeks old) are incapable of perceiving magnetic field in the dark . The authors should discuss this in their manuscript.References: Wiltschko, W. and Wiltschko, R., (1981) Disorientation of inexperienced young pigeons after transportation in total darkness. Nature 291: 433 – 434.We think it is best to exercise caution when assessing behavioural studies in light of the timeline of cuticulosome formation. It should also be noted that Wiltschko and Wiltschko, 1981 did not directly assess the magnetic sense in 12-week-old pigeons, but rather the orientation of outward homing when pigeons were transported in darkness. They note in this manuscript that "we do not know the exact cause of the disorientated behavior of our experimental birds […] as homing experiments with birds is a highly complex behaviour". Nonetheless, we think the reviewer has raised an interesting point and have therefore modified the Discussion as follows:"The critical question that remains is: What is the function of the cuticulosome? And might it be associated with the magnetic sense? This is a tempting proposition given that pigeons begin to fly at approximately one month of age, a time when the cuticulosome is on the cusp of maturation . However, this correlation should be viewed cautiously as there is conflicting behavioural data supporting the presence of a magnetic sense in young birds. Wiltschko and colleagues have reported that chickens aged 10 days can be conditioned to a magnetic anomaly  and juvenile European Robins are known to possess a magnetic sense , whereas pigeons aged 12 weeks are disorientated if they are transported in the dark . An experiment that assessed whether magnetic stimuli results in c-fos associated neuronal activation in pigeons of different ages would be valuable ."