The family of cobalamin class-III dependent enzymes is composed of the reductive dehalogenases (RDases) and related epoxyqueuosine reductases. RDases are crucial for the energy conserving process of organohalide respiration. These enzymes have the ability to reductively cleave carbon-halogen bonds, present in a number of environmentally hazardous pollutants, making them of significant interest for bioremediation applications. Unfortunately, it is difficult to obtain sufficient yields of pure RDase isolated from organohalide respiring bacteria for biochemical studies. Hence, robust heterologous expression systems are required that yield the active holo-enzyme which requires both iron-sulphur cluster and cobalamin incorporation.Bacillus megaterium, Escherichia coli HMS174(DE3), Shimwellia blattae and a commercial strain of Vibrio natrigenes, for cobalamin class-III dependent enzymes expression. The Nitratireductor pacificus pht-3B reductive dehalogenase (NpRdhA) and the epoxyqueuosine reductase from Streptococcus thermophilus (StoQ) were used as model enzymes. We also analysed whether co-expression of the cobalamin transporter BtuB, supports increased cobalamin incorporation into these enzymes in E. coli. We conclude that while expression in Bacillus megaterium resulted in the highest levels of cofactor incorporation, co-expression of BtuB in E. coli presents an appropriate balance between cofactor incorporation and protein yield in both cases.We present a comparative study of the heterologous expression strains  •Reductive dehalogenases and epoxyqueuosine reductases are cobalamin-dependent.•Cofactor incorporation presents a challenge for heterologous expression of these enzymes.•A range of bacterial hosts were assessed as expression host for these enzymes.•An inverse correlation between protein yield and cofactor content was observed.•E. coli yielded best results.Co-expression with the BtuB B12 transporter in  However34U35N36-3′ anticodon sequence , that contain a central cobalamin cofactor bound by a nitroreductase-like fold and 2 [4Fe–4S] clusters ,7. Unliksequence . The hettitution .Fig. 1S.E. coli, resulted in soluble but catalytically inactive protein . Be. Be17]. Bmeg (90%), presumably due to the ability of B. megaterium to synthesize cobalamin de novo. StoQHMS with and without co-expression of BtuB contain the next highest cobalamin incorporation, with the co-expressing BtuB strain resulting in a slightly higher cobalamin content than HMS174(DE3) alone (70 and 62% respectively). The cobalamin incorporation in StoQHMS is higher than expected, showing the high efficiency of BtuB and its ability to transport cobalamin, even while under translational control  clusters ,17. DeteVmax−TB, NpRdhAVmax−YT and NpRdhABmeg have high iron:protein ratios of 7.7 ± 0.1, 6.3 ± 0.2 and 6.5 ± 0.1 respectively, indicating the presence of 1.9 ± 0.1, 1.6 ± 0.1 and 1.6 ± 0.1 [4Fe–4S] clusters per NpRdhA, respectively. In contrast, NpRdhAShim has a low iron:protein ratio of 4.1 ± 0.1, suggesting only 1 [4Fe–4S] cluster per NpRdhA. As observed with StoQ, NpRdhAHMS (both with and without BtuB) has a low iron:protein ratio of 5.4 ± 0.2 and 3.6 ± 0.5 respectively. The [4Fe–4S] cluster content for NpRdhA is increased when co-expressed with BtuB . The cobalamin incorporation has previously been implicated in NpRdhA iron-sulphur cluster maturation, due to the close contact between cobalamin and the proximal [4Fe–4S] [The iron-content for purified NpRdhA appears generally lower compared to StoQ . NpRdhAV[4Fe–4S] .3.4E. coli flavodoxin reductase and spinach ferredoxin) to support the transfer of electrons from NADPH to the NpRdhA  cluster and cobalamin incorporation.Although  require . AnotherE. coli HMS174(DE3) may provide an acceptable compromise between protein yield and specific activity. Not only does this strain result in high protein yields, but also leads to relatively high cobalamin incorporation for both StoQ and NpRdhA . Unfortunately, the iron-sulphur cluster incorporation appears substantially lower when compared to other strains, which will require further study to improve incorporation further, potentially by co-expression of the iron-sulphur cluster pathway operon [Co-expression of class-III cobalamin-dependent enzymes with the cobalamin transporter BtuB in y operon ,40.V. natrigenes, therefore potentially explaining both the increased purified StoQ and NpRdhA obtained under these conditions, as well as cobalamin incorporation due to increased expression of salvage systems [S. blattae (data not shown), under conditions previously successful for expression of other RDases [In an attempt to standardize expression conditions, Vmax™ cells were grown in suboptimal conditions. Upon expression in a recommended enhanced 2xYT medium, both quantities of purified StoQ and NpRdhA and cobalamin incorporation increased in comparison to when grown in TB, therefore showing the importance of not only expression strain utilized, but also conditions under which they are expressed. A potential explanation for the increased protein obtained and cobalamin incorporation could be due to the large quantity of NaCl present in the enhanced 2xYT medium. NaCl has proved essential for optimal growth and also protein synthesis in  systems . The impr RDases .5B. megaterium offers the highest specific activity/level of cobalamin incorporation.In this study we have used two model class-III cobalamin-dependent enzymes, StoQ and NpRdhA, to test a range of heterologous expression hosts for their ability to produce the corresponding holo-enzymes in high yield. Neither isolation directly from the OHRB host, or reconstitution of the apo-protein have been reported as efficient procedures that can yield the required protein amounts to support detailed biochemical and biophysical studies on the mechanism and structure of these enzymes. When keeping most variables, in terms of protein purification and cell growth conditions, as similar as possible, we find that the cobalamin-producing E. coli strain is the best candidate heterologous host for the expression of class-III cobalamin-dependent enzymes, on the basis of the two model enzymes tested.However, protein yield is modest compared to other strains tested under these conditions, and when striking a balance between yield and levels of cofactor incorporation, the BtuB expressing Future work will need to focus on the study of additional members of the class-III enzymes to verify our findings. The NpRdhA enzyme is arguably distinct from respiratory reductive dehalogenases and appears not associated with the membrane. Furthermore, there are the so-called “self-sufficient” reductive dehalogenases, that are fused to the redox module that provides reducing equivalents from NAD(P)H, that provide an attractive target for future bioremediation. To date there are very few examples of these proteins having been expressed successfully and purified to homogeneity ,45.The authors declare that they have no conflict of interest with the contents of this article.T.H. and K.F. grew cells, purified protein and performed experiments. K. F. and S. E. J. R. carried out EPR spectra and analysed the EPR spectroscopic data. K.A.P.P. helped with molecular biology. D.L. conceived and coordinated the study. All authors wrote the paper, reviewed the results and approved the final version of the manuscript.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Optokinetic nystagmus (OKN) is an involuntary eye movement induced by motion of a large proportion of the visual field. It consists of a “slow phase (SP)” with eye movements in the same direction as the movement of the pattern and a “fast phase (FP)” with saccadic eye movements in the opposite direction. Study of OKN can reveal valuable information in ophthalmology, neurology and psychology. However, the current commercially available high-resolution and research-grade eye tracker is usually expensive. Methods & Results: We developed a novel fast and effective system combined with a low-cost eye tracking device to accurately quantitatively measure OKN eye movement. Conclusions: The experimental results indicate that the proposed method achieves fast and promising results in comparisons with several traditional approaches. Research on eye movement has provided a valuable source of information to vision science, psychology, and neurobiology ,2,3. TheOptokinetic nystagmus (OKN)  is an inIn this study, a novel fast and effective system is developed and combined with a low-cost eye tracking device to accurately quantitatively measure OKN eye movement. During eye movement recording, the human eyes often show unwanted noise or baseline drift due to fatigue or distraction, which interfere with the analysis of signals. Our algorithm can detect the position of FP and optimize the result by iterative signal modification. To validate the performance of the proposed system, the results of FP detection were comparable with those of manual reading by experienced physician and of several traditional approaches.In this study, we designed a dynamic stripe simulation program for OKN stimulation that is easy to adjust. We used the low-cost eye tracking device GP3 to collect OKN response data in a simple simulation environment and simply fixed the head to ensure the collected data would be free from interference. The GP3 eye tracker, which is produced by the Gazepoint company, is an easy-to-use, high-performance eye tracker. Gazepoint company produces the eye tracking system technology for everyone. The eye tracker comes with a software suite, Gaze point Control and Gaze point Analysis, which allows you to calibrate a participant, build an experiment, collect data, and analyze data. Finally, the collected data were used to get the position of the scanning moment by the FP filtering method. Six healthy male adults were recruited in the study. The participants had no neurological, ophthalmological, or vestibular impairments. The six subjects underwent complicated tests, and the measured signals collected obvious OKN characteristics for these six subjects. Only participants with best-corrected visual acuity of 6/6 or higher on the Snellen scale were included in this study. The study was approved by the Institutional Review Board at the Ditmanson Medical Foundation Chiayi Christian Hospital and complied with the Declaration of Helsinki . ParticiParticipants sat in a chair in front of the middle of a 23-inch LCD  monitor . Distance between the head of the participant and the LCD monitor was set at 50 cm. The ophthalmologist recommended that the distance between the human eye and the computer screen should be 45–60 cm. Therefore, the setting of our environment uses a distance of 50 cm as a fixed value, and the width of the stripe animation is reversed according to this distance. To reduce the signal noise from vertical movement, the participants’ head was placed on a chin rest and a horizontal black bar of 0.5 cm height was placed at 3 cm below the vertical midline of the LCD monitor. The OKN stimulus was generated by showing moving black and white stripes on the LCD monitor at a frame rate of 60 Hz. Eye movements were recorded using a video-based eye tracker  that recorded eye movements with a maximum resolution of 30 arcmin at a sampling rate of 150 Hz, where arcmin is a unit of angular measurement equal to 1/60 of one degree.The eye tracker was calibrated for each participant at the start of each procedure. Calibration was performed by fixating at five different positions on the monitor. The OKN was elicited with a square-wave grating alternative black and white vertical stripes of 4.6 cm width moving horizontally at fixed 20 cm/s speed. The participant was instructed to look at the moving patterns binocularly. Each test lasts for 30 s and was repeated three times. Measurements were recorded on a computer and stored for offline analysis. All programs and algorithms for analyzing data were developed in MATLAB .After completing a series of tests and data collection, the dynamic SlopeThreshold and MoveThreshold were set up to screen out the eligible FP sections based on the data pre-processing and feature determination. The specific slope and length were used as the threshold, where SlopeThreshold represents the filtering range of the slope, which is used to control the OKN state of the eyeball according to the movement direction of the fringe, usually set positive or negative, and MoveThreshold stands for the distance the pupil moves, because the distance the pupil moves in the Fast Phase (FP) is larger, which is our index for screening. Finally, iterative corrections were made to fine-tune each eligible signal to the correct position.Using the slope determination method, we narrowed the selection range of FP in accordance with the occurrence conditions of FP formulated by Kanari et al. 2017) [ [17]. WeAlgorithm 1. Proposed FP position detection.1for i = 1: length (I)2 if (I’s slope meet S)3  Put Up/Down point to matrix;3 end4end5for j = 1: IC6 for i = length(M)7  gather points around peak (±1)9  find maximum/minimum of points10 end11endI:is the input dataS:is the slope thresholdM:is the matrix after select matching data from inputIC:is the iteration Counts (interationCount), through multiple iterations, the signal identified as the FP interval gradually moves to the highest and lowest points of FPSince the GP3 tracking device can only take a maximum signal of 150 Hz and the data received are not stable and complete, even in the SP stage, involuntary eye movement can be incorrectly read as an FP, resulting in the need to dynamically determine the SlopeThreshold and dynamically set the MoveThreshold to filter FPs that are incorrectly read when nystagmus occurs in SP. The detailed processing steps (Algorithm 1) are as follows:We narrowed the selection range of the signal according to the FP occurrence condition formulated by Kanari et al. 2017) [ [17]: ThFor accuracy strategy, generally 2 to 5 saccades occur in an OKN test per second. It varies with different subjects. We performed OKN tests on the left and right three times, respectively (each time about 30 s) for six subjects. According to the definition of FP formulated by Kanari et al. (2017), the highest point and the lowest point of each FP are manually marked as the position where FP occurred correctly. The formula for accuracy is defined as:The detected method is used to detect the FP position and is then compared with the ground truth.The above condition for signal filtering by slopes is relaxed, but it can be dynamically adjusted. In this study, the subjects received the OKN test with a stripe that shifted to the right. For the convenience of sampling, we only sampled at a negative slope.After collecting all the points at which FP occurred, we set a certain length (the maximum of FP between two points in SP) to filter uncontrolled nystagmus. Based on the displacement of length as the threshold, we used an FP length greater than Thr to filter the unsmooth signal caused by nystagmus (Thr (threshold) refers to the setting of MoveThreshold). After multiple tests by several subjects, it was found that the X-coordinate movement of the eyeball in GP3 exceeds Thr = 0.0006, which has a very high possibility for the FP interval. It can be adjusted slightly . It can As shown p-values using paired t-test between each intermediate process of our method and ground truth. More specifically, the paired t-test was used to estimate whether the differences between the performance of each intermediate process of our method and ground truth were significant or not. The results showed that 97% of the FP occurrence points of OKN could be found through our method  at which FP occurred. High-pass and band-pass FIR filtering were adopted for peak detection. After smoothing, an adjustable threshold was set for the screen, and the filtered peak was found. The adjacent maximum/minimum values were found according to the signal position  19](3)(3)19] filtering after the Fourier transform. The reason was that the slope of FP would not affect the threshold judgment with the involuntary eye shift of the subject. Our method could eliminate the need for complex calculations and make the judging simpler and more accurate.p-values using paired t-test between the three traditional methods and the proposed method. More specifically, the paired t-test was used to estimate whether the differences between the performance of three traditional methods and that of the proposed method were significant or not. At first, we thought that we should only use Peak finding [In order to evaluate the performance of the proposed method, the three traditional methods ,8 were i finding  to find  finding  depends Regardless of the normal OKN identification rate, the uncontrolled noise produced by the subjects affected the determination of the correct FP during the prolonged test time. The OKN with noise includes dullness, fixation position deviation, and other phenomena, which further deepen the difficulty of filter setting and greatly decrease the accuracy. Our method simply filtered by the slope and moving distance, which solved many judgment problems, and screened the FP position from an intuitive angle. It reduced the influence of fixation deviation, leaving only the stagnant fixation problem . In addition to the normal FP judgment of OKN and FFT combined with a high-pass filter that can be used well, there were good results on the correct FP judgment of OKN signals with noise.Clinical detection of OKN often requires costly instruments, which poses considerable difficulties in clinical applications. Our main contributions were as follows: (1) low-cost instruments could be used to detect OKN, and (2) simple conditional screening and iteration could be used to obtain the accurate FP position, which was conducive to the calculation and analysis of the subject’s eye disease status.Our method could be used to completely identify the FP position under stable conditions without large eyeball stagnation. However, the reaction of the eyeball cannot be controlled, and abnormal nystagmus and dullness are randomly generated and not included in FP/SP for reference . Our metIn the field of neuro-ophthalmology, many scholars have tried to use OKR to study the physiological and pathological phenomena of eye movements . In the 1940s, scholars studied the relationship between OKR and VOR . Sangi et al. (2015) tested and analyzed the data of children , findingThe data collected by the six subjects in this experiment will be used as a reference index for future clinical eye disease patients. In future work, we will increase the screen distance, stripe animation changes, and other variables to test the patients next to the bed. With diversified tests, OKN factors that may affect patients with eye diseases will have the opportunity to be discovered. The high-accuracy FP recognition rate is of great help to the subsequent calculation of mean slow-phase velocity (MSPV) and other eye movement analysis parameters, and is beneficial to use as a reference index for patients with strabismus and other eye diseases.


We use a low-temperature catalytic-spillover process involving the post-growth incorporation or removal of hydrogen from α-Fe2O3 thin films. Hydrogenation drives pronounced changes in its magnetic anisotropy, Néel vector orientation and canted magnetism via electron injection and local distortions. We explain these effects with a detailed magnetic anisotropy model and first-principles calculations. Tailoring our work for future applications, we demonstrate reversible control of the room-temperature spin-state by doping/expelling hydrogen in Rh-substituted α-Fe2O3.Antiferromagnetic insulators are a ubiquitous class of magnetic materials, holding the promise of low-dissipation spin-based computing devices that can display ultra-fast switching and are robust against stray fields. However, their imperviousness to magnetic fields also makes them difficult to control in a reversible and scalable manner. Here we demonstrate a novel proof-of-principle ionic approach to control the spin reorientation (Morin) transition reversibly in the common antiferromagnetic insulator α-Fe One major challenge for antiferromagnetic spintronics is how to control the antiferromagnetic state. Here Jani et al. demonstrate the reversible ionic control of the room-temperature magnetic anisotropy and spin reorientation transition in haematite, via the incorporation and removal of hydrogen. Among them, insulators promise efficient magnon-based spin transport with the absence of charge-related Joule losses6. They also host attractive functionalities like spin colossal-magnetoresistance7, ultra-fast dynamics11, high domain-wall propagation velocities13 and spin-superfluidity14. Despite all these advantages, they have so far played only an auxiliary role as pinning or exchange bias layers in thin film spintronic devices as their magnetic order is difficult to detect and hard to control15. Although many recent reports demonstrating clear detection of the AFM-state in insulators by spin-Hall or anisotropic magneto-transport have emerged18, control of the ground-state continues to be a challenge15.Antiferromagnets (AFM) exhibit long-range magnetic order with two or more sublattices aligned so that they produce no net magnetization. There has recently been a surge of interest2O3, which is a well-known antiferromagnetic insulator with a bulk Dzyaloshinskii–Moriya interaction (DMI) that undergoes a spin-reorientation Morin transition between the in-plane and out-of-plane states 21, sizable spin-Hall magnetoresistance18 (when combined with a Pt overlayer) and is also the only reported natural AFM to date that hosts a wide family of topological AFM textures24 at room temperature. Consequently, reversible engineering of the anisotropy, and thereby the Morin transition, is crucial as it would allow control over magnon-transport21 and topological texture dimensions22. Previous reports have shown that strain25, chemical doping30 and meso-structure31 can modify the antiferromagnetism in α-Fe2O3. However, lack of reversibility and scalability makes these approaches difficult to exploit in practical applications.In this work, we focus on α-Fe9, but they require table-top lasers and large interaction volumes. Secondly, electric-field control of the AFM-state, which is possible in multiferroic32 and magnetoelectric33 oxides, cannot be replicated in a vast majority of functional materials, where magnetic and electrical degrees of freedom are not coupled. Alternatively, electric-field control via strain coupling to piezoelectric substrates, which has been quite successful in tuning antiferromagnetic metals36, require epitaxial matching or texturing constraints to be satisfied for crystalline systems37. Thirdly, electric-current control of the AFM-state via Néel spin–orbit torques is also quite useful39, but is limited to materials with strong spin–orbit coupling that break local inversion-symmetry in a specific manner. Finally, spin-Hall effect based spin–orbit torques injected from a heavy-metal overlayer are also quite promising in controlling the AFM-state of insulators43. However, studies in α-Fe2O3 have so far shown in-plane tuning43, which is insufficient to directly control the Morin transition. Hence, there is a need for developing alternative pathways to directly tune antiferromagnetic properties and spin reorientation in α-Fe2O3 thin films.In the literature, coherent optical techniques have enjoyed success in manipulating antiferromagnetic insulators2O3 thin films by using spillover. We draw inspiration from pioneering ionic studies, where doping light ions  was used to control anisotropy in ferromagnetic systems45, or transport in strongly correlated-oxides48 and conventional insulators49, although the crystal physics and chemistry underpinning our implementation is completely different. By incorporating or removing hydrogen, we demonstrate a hitherto unexplored ionic approach to control a wide range of antiferromagnetic properties in a reversible and stable manner. H-doping can drive these pronounced changes by delicately tuning the competing contributions to the magnetic anisotropy, as a result of local electron injection and ensuing local distortions. Lastly, we show reversible control of the room-temperature Morin transition by hydrogenation of α-Fe1.97Rh0.03O3 films.Here, we present a new method of hydrogen (H)-doping to tailor antiferromagnetism in epitaxial α-Fe2O3 has the trigonal corundum structure, with antiparallel sublattices  stacked alternately along the hexagonal c-axis, as shown in Fig. c-axis. Presence of the bulk DMI causes canting of the sublattices within the basal planes19, resulting in a weak macroscopic in-plane canting -oriented α-Al2O3 single crystals by pulsed laser deposition , with hysteretic appearance/disappearance of the in-plane canted moment, Fig. 25. To confirm the magnetic origin of the transition, we performed X-ray linear dichroism (XLD) spectroscopy at the Fe L2 edge, which is a good element-specific approach to identify the direction of the sublattice magnetization in α-Fe2O3 (‘Methods’)51. The dichroism reverses sign across L to the basal plane from the c-axis. We also performed X-ray magnetic circular dichroism (XMCD) spectroscopy but saw no signal from the weak canted moment (Supplementary-M(H), revealed a hysteretic evolution due to in-plane realignment of antiferromagnetic domains driven by the magnetic field coupling to the canted moment, at Highly-oriented epitaxial thin films of α-FeTM0 Fig. , due to 2O3, catalytic spillover53 of hydrogen into our films was performed through sputtered platinum nano-structures  at low temperatures  was used (see ‘Methods’). The hydrogen concentration in the H-doped films was found to be in the range ~1.57 ± 0.14 at.% to 2.47 ± 0.14 at.% depending on the H-spillover temperature  performed in tandem with ERDA, which is resonantly sensitive to the O-content of films  curves of the H-doped films revealed a multi-hysteretic in-plane evolution of the antiferromagnetic domains possibly due to the enhancement of local DMI after H incorporation  had little effect on the Morin transition  in a bulk-sensitive fluorescence mode at the Fe K edge. A systematic chemical shift of the Fe K edge and shoulder to lower binding energies was evident in the normalized spectrum and its derivative, of the H-doped samples with respect to the undoped counterparts Fig. . Howeverons Fig. , unequiv2O3 containing different types of point defects , see ‘Methods’. In addition, we manually introduced or removed electrons to or from this defect-containing super-cell, to mimic charge-doped regimes of α-Fe2O3. We found, firstly, that H-dopants prefer to be interstitials, with the formation energy of Hi markedly lower than HO/VO in all charge-doped regimes  spectroscopy of H-doped α-Fe2O3  calculations of defect formation and charge transfer in a 2 × 2 × 1 super-cell  of α-FeHi corresponds to the Hi-driven charge transfer. In the neutral and negative-charged cases, we observed that only a small fraction of Bader-charge is transferred from Hi to the nearby O-sites  the classical magnetic-dipolar anisotropy, following a 2O3, these interactions are opposite in sign but similar in magnitude (differing by only ~2% in bulk at 0 K), with KSI (KMD) dominating below (above) 50. Hence, the total anisotropy, To understand the observed change of anisotropy and Morin transition, we built a phenomenological model describing their evolution as a function of temperature and H-doping. Based on the discussion in the previous section, the model consists of a matrix of Fesee Fig. . EssentiK would evolve approximately linearly26 with the H-concentration, x, as per the relation  concentration (x) into the ratio of Fe2+-species per total Fe cations. The first term in Eq. , and Zi(T) are cation-dependent functions. The latter are obtained from a modified two-sublattice mean-field model , calculated from our phenomenological model for different x values, is shown in Fig. x, Hence, the overall effect of hydrogenation is to reduce the single-ion contribution more than the magnetic dipolar counterpart, thereby suppressing the Morin transition. The temperature dependence of the total anisotropy Hi-defects in α-Fe2O3 . The evolution of T = 0 K. We find that the evolution of 2O3 under H-doping.To test the validity of our model, we also performed DFT calculations of the total anisotropy change, 2O3 with a heavy transition metal increases its total anisotropy, thereby increasing the Morin transition temperature30. Here, we used α-Fe1.97Rh0.03O3 films which have an elevated Morin transition near room temperature  from stoichiometric targets on single crystalline α-Al2O3 substrates (CrysTec GmbH), using KrF excimer laser (248 nm). Substrates were cleaned by ultra-sonication in high purity acetone, alcohol and DI Water prior to deposition. The growth was performed at 700 °C, in oxygen partial pressure of 2/20 mTorr with a laser repetition rate of 3 Hz. Subsequently, in situ high oxygen pressure annealing was performed to minimize oxygen vacancies formed during growth. The films were cooled at 10 °C/min.α-Fe2/Ar ratio of 5%/95%) at annealing temperatures in the range 150–270 °C. Control annealing experiments were carried in high purity 100% Ar or O2 atmospheres under the same temperature conditions.Nano-sized Pt islands used for catalytic H-spillover were grown by sputtering in an Ar atmosphere from a high purity Pt target. The deposition current was 10 mA and duration was 5 s. HAADF mode in STEM identified that Pt formed small and discontinuous nano-structures (NSs) (see Supplementary-ω-scans), reciprocal space mapping (RSM) and ϕ-scans, and thickness measurements via X-ray reflectivity (XRR) at a four-circle diffractometer in the XDD beamline at the Singapore Synchrotron Light Source (SSLS), see Supplementary-Structural characterization of epitaxial films included high-resolution synchrotron X-ray diffractometry (HR-XRD) involving Atomic positions in the films and the quality of the film-substrate interface were determined through atomic-resolution STEM in a JEOL-ARM200F (with ASCOR aberration corrector and cold-field emission at 200 kV), in HAADF and annular bright field (ABF) acquisition modes. The HAADF and ABF images were acquired with a probe‐forming aperture of 30 mrad and collection inner and outer semi-angles in the range 68–280 mrad and 12–24 mrad, respectively. While HAADF shows the positions of the heavier elements Fe and Al, ABF shows the positions of O better.2O3 (H-doped and undoped) fine powder samples were placed directly on the crystal for data collection. The spectra were obtained by averaging multiple curves collected using a DTGS detector, with a spectral resolution of 4 cm−1, scanner velocity 10 kHz and sampling time of 1 min (68 scans). The bench of the spectrometer was evacuated down to ~1.5 mbar for all experiments. The FTIR spectra were fitted by Lorentzian functions to identify the bond peaks.For the Fourier transform infrared (FTIR) spectroscopy experiments, MIRacle ATR sampling accessory for analysis of solids was used. MIRacle was placed in the sample chamber of Vertex 80 v Bruker spectrometer. α-Fe67 and molecular bonding changes in α-Fe2O3, before and after H-doping, were measured by employing Fe K edge X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS), respectively, at the XAFCA beamline in SSLS. The measurement was performed in fluorescence geometry and was sensitive to the Fe-signal throughout the films. Normalization, background correction, and analysis were performed in the Athena software. Following the approach in iron-oxide literature, the fraction of reduced Fe2+ species with respect to Fe3+ species in H-doped and control Ar-annealed samples relative to undoped α-Fe2O3 samples, was estimated by analyzing the chemical shifts of Fe K XANES  and circularly (XMCD) were carried out at the Fe L edge at BL4.0.2 of the Advanced Light Source (ALS). The measurement was performed in a transmission geometry with the detection in the luminesce-yield mode (which is a transmission measurement using sapphire as a scintillator and is thus sensitive to X-ray absorption through the entire film) in an open-flow liquid nitrogen cryostat which allowed dichroism measurements in the range 80–325 K. X-rays were incident with a grazing angle of 30° relative to the surface. For the XLD asymmetry, defined as M(T) curves were measured after field-cooling the samples with an applied in-plane field of 500 or 5000 Oe, and a measurement field of 500 Oe applied during the subsequent warming and cooling scans. Changing the cooling-field magnitude had no discernible effect on the transition temperature. M(H) curves were carried out at a series of temperatures. The diamagnetic α-Al2O3 substrates contribute a background in the raw M data. This background is removed to extract the final magnetometry curves of the thin films.Magnetic characterization was performed in a superconducting quantum interference device (SQUID) magnetometer of Quantum Design MPMS. 16O16O, to accurately measure the O-content in the α-Fe2O3 films (see Supplementary-−6 Torr. The He ions were incident at an angle of 75°. Backscattered He (for RRBS) and recoiled H (for ERDA) were collected in surface-barrier detectors placed at 165° and 30° with respect to the incident ions, respectively. A 14-μm-thick Al absorber-foil was used to stop the forward-scattered He and only allow recoiled H toward the ERDA detector. Both ERDA and RRBS spectra were fit using the SIMNRA software. For RRBS experiments, the energy calibration was performed by using a standard tungsten silicide sample. To calibrate the yield and energy of ERDA measurements, a quantitative analysis of H density was obtained by using an H-implanted Si standard and by performing ERDA measurements at two energies in the range ~2.80-3.04 MeV.The quantitative analysis of H-dopant concentration in samples was performed using elastic recoil detection analysis (ERDA) wherein He ions incident on the sample lead to selective forward recoil scattering of H atoms from the sample. ERDA experiments were performed in tandem with oxygen resonant Rutherford backscattering spectrometry (RRBS) at Inter-University Accelerator Centre (IUAC), using 1.7 MV pelletron Tandem accelerator. The ERDA and RRBS measurements facilitated a direct quantitative determination of Fe/O/H concentrations simultaneously in the samples. RRBS experiments were performed with a resonant energy for oxygen reaction 68 was used for the d-electrons of Fe atoms, and the value of U was set to 4.5 eV, which brings the indirect electronic bandgap of α-Fe2O3 from 0.56 eV (PBE result) to 2.0 eV, consistent with our experimental bandgap value from UV-Vis-NIR absorption spectroscopy. In addition, spin–orbit coupling (SOC) was also involved in all the calculations to accurately obtain information related to magnetic anisotropy.Density functional theory (DFT) based first-principles calculations were performed through the Vienna Ab-initio simulation package (VASP). The projector-augmented-wave and the Perdew–Burke–Ernzerhof (PBE) method were used for the pseudo-potentials and the exchange-correlation functionals, respectively. The plane-wave cut-off energy was set at 600 eV. The GGA+U approachα-Fe2O3, respectively, to mimic different doping concentrations. The Monkhorst–Pack grid with 7 × 7 × 3, 7 × 3 × 3, and 3 × 3 × 3, k-point meshes were used, respectively. For each of these cases, defects both of the interstitial (Hi) and substitutional (HO) type were considered. In addition, eight different initial sites for Hi were considered, shown in Supplementary-Etot(X) and Etot(bulk) are the calculated total energies of the system with and without defect X, respectively. nj is the number of atoms of type j (either host atoms or dopants) that have been added to (nj > 0) or removed from (nj < 0) the system to form that defect, and μj is the corresponding chemical potential of the species. Here, the chemical potential of H and O are obtained by considering hydrogen and oxygen molecules as their sources, respectively.For hydrogen-doping calculations, one H atom was incorporated into 1 × 1 × 1, 2 × 1 × 1, and 2 × 2 × 1 unit-/super-cells  of 69,q indicates the charged state, EF is the Fermi energy, EVBM the reference valence band maximum (VBM) position, and ΔV is a correction term added to align the VBM level of the cell containing the defect (in different charge states) to that in undoped α-Fe2O3, as discussed in ref. 69.Moreover, defects/host atoms can further acquire or lose electrons, forming charged states. This effect is simulated by performing the calculation with excess electron(s) added to or removed from the cell, respectively. The relative stability of each charged state of the cell containing the defect can be measured by modifying the above formation energy in Eq.  as folloα-Fe2O3, we adopted the Bader-charge calculations as it considers not only the core charge but also the valence charge, serving as a good approximation of the total electronic charge.Lastly, to analyse charge transfer between atoms in H-doped Supplementary Information


Bartonella. The aim of this study was to investigate the presence of Bartonella spp. in African giant pouched rats (Cricetomys gambianus) and their ectoparasites in Dakar, Senegal. In 2012, 20 rats were caught, and their fleas were identified. DNA was extracted from 170 selected fleas and qPCR was carried out to detect Bartonella spp. Subsequently, a Bartonella culture was performed from the rat blood samples and the isolated strains  were genotyped. A total of 1117 fleas were collected from 19 rats and identified as Xenopsylla cheopis, the tropical rat flea. Bartonella DNA was detected in 148 of 170 selected fleas (87.1%). In addition, Bartonella strains were isolated from the blood of 17 rats (85%). According to Bartonella gene-sequence-based criteria for species definition, the isolated strains were identified as B. massiliensis (four strains) and two potential new species related to the zoonotic B. elizabethae. In this paper, these potentially new species are provisionally called Candidatus Bartonella militaris (11 strains) and Candidatus Bartonella affinis (two strains) until their description has been completed. Cricetomys gambianus and its fleas could constitute a public health risk in Dakar due to the high prevalence of Bartonella infection reported.Bartonellae are bacteria associated with mammals and their ectoparasites. Rodents often host different species of  Bartonella is the only genus of bacteria belonging to the family Bartonellaceae. These microorganisms are Gram-negative and facultative intracellular bacteria. Currently, more than 40 Bartonella species have been described. Many of them can infect a large range of different mammal species, often rodents, which are regarded as the main reservoir hosts of many species of these bacteria. At least 20 species of Bartonella are rodent-associated [Bartonella bacilliformis, Bartonella quintana and Bartonella henselae; these species are not associated with Rodentia. However, many small rodent-associated bartonellae have been reported to cause human diseases. Rattus-associated B. elizabethae, B. washoensis and B. vinsonii arupensis may cause culture-negative endocarditis, B. grahamii is suspected to induce neuroretinitis and endocarditis and B. tribocorum may cause chronic bacteriaemia [Bartonella species [sociated  and moreeriaemia ,4. Blood species .Cricetomys gambianus), also known as the Gambian pouched rat, is a large rodent in the exclusively African family Nesomyidae. This is one of the largest rodents in the world. It weighs between 1.0 and 1.4 kg and grows to approximately 0.9 m long, including the tail, which makes up half of its total length [Cricetomys gambianus is widespread in different natural biotopes and can frequently be found in semi-urban places, near human habitations. This species is nocturnal and eats fruit, seeds and small animal-like insects. In many African countries, especially in West Africa, giant pouched rats are considered as an important source of meat and are hunted by local populations [C. gambianus is a commensal animal and one of the most common rodents encountered in the city of Dakar.The African giant rat , a residential quarter of Dakar, Senegal. Metal cages were placed outdoors, in areas surrounding homesteads, in deep gutters and rainwater harvesting systems. These traps were checked for the presence of rodents every day, early in the morning. Each cage containing a rat was transported to the French military veterinarian at the camp, who identified the rat and collected the ectoparasites.®, Boehringer Ingelheim, Lyon, France). Whole blood was collected by cardiac puncture in an EDTA-coated tube.A system was used to restrain the animal. After restraining it, each rodent was anesthetised by an intramuscular ketamine injection  were stored  and transported two weeks later to the IHU Méditerranée Infection, Marseille, France. In the IHU, ectoparasites were stored at −20 °C and blood samples at −80 °C until analysis.Within the framework of the Nagoya accord application, we have obtained authorization to access and use, in France, genetic resources from captured rodents .18S rRNA gene [All ectoparasites were morphologically identified using identification keys. Each one was photographed using a Stereo Discovery V20 stereomicroscope. The ectoparasites’ genus and species were then confirmed for some specimens, selected randomly, by using primers targeting the RNA gene .®DNA tissue kit,  and was performed on a BIOROBOT EZ1  per the manufacturer’s instructions. Prior to DNA extraction, ectoparasites or 200 μL of rat blood was digested with proteinase K and incubated at 56 °C overnight.DNA was extracted individually from selected ectoparasites, as well as from all rat blood samples. Extraction was performed using the commercial EZ1Bartonella DNA) and negative (only mixture) controls were added in each reaction.DNA was eluted in 200 μL of distilled water and stored at −20 °C until analysis. The qPCRs were prepared and performed in 20 μL final volume containing 10 μL of Master Mix Roche , 0.5 μL (of 20 μM concentration) of each primer, 0.5 μL of the probe (of 5 μM concentration), 0.5 μL UDG, 3 μL of distilled DNAse- and RNAse-free water and 5 μL of the DNA sample. The assay was performed in a CFX96 Real-Time system  using the protocol: one incubation step at 50 °C for two minutes and an initial denaturation at 95 °C for five minutes, followed by 40 cycles of denaturation at 95 °C for ten seconds and annealing and extension at 60 °C for 30 s. Positive  region for the selected ectoparasites and all rat blood samples. Positive samples were screened again using the ITS2 qPCR . Plates were incubated at 37 °C with 5% COal genes . In paraBartonella isolates were tested by PCRs for species identification using the primers in https://blast.ncbi.nlm.nih.gov/Blast.cgi; accessed on 28 December 2021). The phylogenetic analyses, based on 16S rRNA, rpoB, FtsZ and ITS regions of Bartonella spp., were inferred using neighbour-joining methods, and tree reconstructions were performed using MEGA software version 7 [All of the selected fleas were suggested to the 18S-based standard PCR. ersion 7 . BootstrCricetomys gambianus species, the African giant pouched rat. Overall, 19 of the 20 (95%) rats carried fleas (18S rRNA) confirmed the morphological identification of the fleas. All of the specimens belonged to the same species: Xenopsylla cheopis, the tropical rat flea, also known as the Oriental rat flea.Twenty rodents were captured during the study period. They were all formally identified as ed fleas . The numBartonella DNA by qPCR. Bartonella spp. DNA was confirmed in 148 of the 170 fleas (87.1%) that were analysed. Overall, 17 Bartonella isolates were isolated from the blood of 17 of the 20 captured rats . Using MALDI-TOF MS, four isolates were identified as Bartonella massiliensis with scores > 1.7. The other isolates were not identified to the species level, although their MS spectra undoubtedly clustered within pools of other Bartonella spectra.Regarding the 1117 tropical rat fleas collected, we selected 170 fleas  for screening for 16S rRNA (1400 bp) showed that four almost similar sequences, also identified by MALDITOF-MS, exhibited a 99.9% to 100% identity with Bartonella massiliensis OS09 (HM636440). These strains also showed a 99–99.8%, 97.8–98.9% and 99–99.3% identity in the FtsZ, ITS and RpoB regions with B. massiliensis, respectively. All of the other 16S rRNA sequences of the other isolates were close to each other and showed a 99.57% to 99.64% identity with Bartonella elizabethae (LR134527) and Bartonella kosoyi strain Tel Aviv (MN627780). They had a similarity of 95.3–96.9%, 86.7–94.5% and 92.8–96.8% with these two species at the FtsZ, ITS and RpoB regions, respectively. Some sequences were deleted from analysis because of their low sequencing quality  and in tropical rat fleas (Xenopsylla cheopis) in this country.This study reports the isolation, molecular detection and genetic characterisation of Bartonella strains have been detected in rodents in several countries in Africa: Algeria, Tunisia, Egypt, Democratic Republic of Congo, Ethiopia, Tanzania, Kenya, Uganda, South Africa, Nigeria, Benin and Senegal. The infection rates reported ranged from 4% to 67% [Cricetomys gambianus trapped and examined by PCR detection and culture were Bartonella-positive. A genotype related to Bartonella elizabethae was found in one of these Cricetomys gambianus. Bartonellae, similar to Bartonella massiliensis isolated from a soft tick species Ornithodorus sonrai in Senegal [C. gambianus [Bartonella sp. was detected in 34.7% (69/199) of X. cheopis fleas collected in Cotonou, Benin [Bartonella species were also detected in both C. gambianus blood and X. cheopis carried by the rats, in Nigeria [Bartonella species had already been detected in ectoparasites collected from humans and animals. Bartonella quintana was identified in lice found on the heads of patients in Dakar [Bartonella were isolated from rodents trapped in the Sine-Saloum region of Senegal: B. saheliensis sp. nov from the gerbil Gerbilliscus gambianus [B. mastomydis sp. nov. from the Guinea multimammate mouse Mastomys erythroleucus [B. senegalensis sp. nov and B. massiliensis sp. nov) were also found in the soft tick Ornithodoros sonrai, the vector of relapsing fever [% to 67% ,15,16,17 Senegal , were foambianus . In Westu, Benin . Bartone Nigeria . In Senein Dakar . Two newambianus  and B. mroleucus . Other nng fever .Bartonella is very high, even regarding previous reports on rats and their ectoparasites. In 2017, a study reported a collection of 407 fleas from small mammals trapped around homes in five villages, in north-western Uganda. Among these fleas, 202 (49.6%) were identified as X. cheopis, mainly from Rattus rattus (n = 148), the black rat. The Bartonella prevalence in oriental rat fleas collected from all of the rodents was relatively low . The phylogenetic relationships of Bartonella genetic variants, identified from sequences obtained by analysing fleas, showed the infection by variants partly belonging to the B. elizabethae species complex [Bartonella DNA in 27.5% (53/193) of X. cheopis fleas collected from Rattus rattus specimens. The genotypes were closely related to different Bartonella species, including B. elizabethae [In this study, the prevalence of n = 1117) of Xenopsylla cheopis collected from only 20 C. gambianus suggests a large infestation of the African giant rats by fleas in the location studied and, potentially, in the surroundings. The flea X. cheopis is a well-known parasite of rodents, primarily of the genus Rattus, but can live on many warm-blooded mammals. Moreover, this flea species is a vector for bubonic plague, caused by Yersinia pestis, and can transmit Rickettisa felis, the aetiological agent of murine typhus [Bartonella prevalence in this study can, first, confirm the role of C. gambianus and X. cheopis as reservoirs and vectors, respectively. Second, this high prevalence can be due to the infestation rate of rats (around 55.8 fleas per rat): this facilitates the transmission to and between rats and the reinfection of fleas in return. It seems that the high infection rate in rats conduces to a high detection rate in fleas feeding on these rats. Other arguments are needed to demonstrate that fleas can maintain these Bartonella spp. (such as the persistence of flea infection).In our study, the high number  Candidatus Bartonella affinis (16S, RpoB and ITS, whereas FtsZ did not show this difference, which required the use of more than one target to identify bacteria belonged to the bartonella genus. The high Bartonella infection rate in rodents and their ectoparasites, observed here and in the previous studies, could explain this emergence of new species. However, the role and/or pathogeny of this species in humans or animals remain to be explored. Since our team began studying Bartonella in rodents over the past decade, at least seven species have been isolated and described, not counting these two species: B. florenciae, B. mastomydis, B. massiliensis, B. senegalensis, B. saheliensis, B. gabonensis and B. raoultii [The isolated strains from rats are, in general, close to the finition . Here, w affinis . The genraoultii ,27,28,29Bartonella sp. found in this study may alert local sanitary authorities to the public health risk for hunters or breeders, but also for inhabitants, given that rat holes have been observed in residential areas, especially in semi-urban locations.In addition, due to its size and weight, the African giant rat is considered as a natural resource of animal protein by the local population . The preB. elizabethae, a species that is currently recognised as a zoonotic agent responsible for endocarditis [B. quintana was described in a Senegalese patient [B. quintana in febrile patients visiting a health facility in rural Senegal [The potential new species described in this study are related genetically to carditis , neurorecarditis ,31 and bcarditis  in humancarditis . In Sene patient  in 2002. Senegal .X. cheopis commonly carry the bacteria of the genus Bartonella and potentially play an important role in the transmission of potential zoonotic infections among Gambian pouched rats and from rats to humans. Due to the nature of the Bartonella species detected, this study shows the importance, from a public health point of view, of leading further investigations into the potential zoonotic Bartonella species in Senegal and, more generally, in Africa. Future research may include more epidemiological studies on pests and farm animals due to the close and increasing interaction between humans, animals and the environment. The new potential species merit a complete description, including their genomic features, due to their microbiological, epidemiological and pathological characteristics being unknown to date.This study helps to confirm that


Their expression profiles were different in the basal and superficial layers. Most of the processes influenced by the UPA in the basal layer were connected to the cell cycle and immune regulation. (4) Conclusion: Specific changes were observed in both layers of the endometrium in the UPA group. However, the miRNA expression in the basal layer was not consistent with that in the superficial layer. Other large studies analysing the long-term impact of SPRM on endometrial miRNA expression are necessary.(1) Background: Ulipristal acetate (UPA) is a selective progesterone receptor modulator (SPRM) widely used for emergency contraception and mid- to long-term leiomyoma treatment. The aim of this study was to identify modifications of miRNA expression in superficial and basal layers of the human endometrium at the end of the UPA treatment for at least 3 months. (2) Methods: Microarray miRNA analysis of formalin-fixed, paraffin-embedded hysterectomy tissue samples was conducted, followed by an Ingenuity Pathway Analysis. Samples were divided into three groups: women having had 3 months of UPA treatment ( Ulipristal acetate (UPA) is a selective progesterone receptor modulator (SPRM). It binds to the major progesterone receptor (PR) isoforms and exerts a wide spectrum of actions, from antagonistic to agonistic effects, depending on the target tissue . It is wUPA, like other SPRMs, has a reversible impact on the morphological aspect of the whole endometrium . EndometAt a molecular level in the myoma tissue, UPA has been shown to modify a number of processes including a decrease in inflammation and induction of cell apoptosis ,10. SpecMicroRNA (miRNA) are small non-coding RNAs. They modify the expression of around 60% of proteins at the posttranscriptional level and promote mRNA destruction. The miRNAs are involved in a broad variety of physiological mechanisms such as morphogenesis, differentiation, apoptosis or cellular metabolism .Some miRNAs have been associated with endometrial receptivity  and flucThere are no data about the miRNA expression regulation in UPA exposed endometrium. Furthermore, comparing miRNA expression in the superficial and basal layers of the endometrium could provide information about the potential persistence of UPA impact on the endometrium. In fact, the superficial layer sheds every month while the basal layer persists.The aim of this study was to identify modifications of miRNA expression, and thus the potential target pathways, in both superficial and basal layers of the human endometrium after 3 months of UPA treatment, before treatment discontinuation, in women treated for symptomatic myomas.Endometrium-containing blocks of hysterectomy samples from non-menopausal women aged 40–52 years with moderate to severe symptoms of myomas requiring hysterectomy between 2014 and 2017 were retrospectively collected in three university hospital surgical gynaecology units in Paris, France . Women who had hormonal treatment for at least 3 months before surgery or a history of cancer were excluded. The clinical variables of the women included age, parity and body mass index . The localisation of the myoma according to the FIGO classification  and withThe menstrual phase was assessed on a histological basis and on evaluation of the ER and PR expressions ,18. The The Obstetrics and Gynecology Research Ethics Committee (CEROG—Comité d’Ethique de la Recherche en Obstétrique et Gynécologie) allowed retrospective sample analysis and approved the study . All women gave pre-operative written informed consent for the participation in the study.Formalin-fixed, paraffin-embedded (FFPE) tissue sample sections of 3 μm were processed and stained with Haematoxylin and Eosin by standard methods. Immunohistochemistry of the ER and PR were performed on the Ventana Ultra platform using the manufacturer’s recommended settings.Whole tissue sections (4-µm) were deparaffinized, rehydrated, treated and incubated for 16 min with the pre-diluted anti-ER antibody  and the pre-diluted anti-PR antibody , and then visualized using the ultraView Universal DAB Detection Kit .All evaluations were performed under blinded conditions by a single expert pathologist (CB). Endometrial differentiation was assessed by histological dating according to the Noyes criteria , and oesFFPE tissues were obtained from hysterectomy specimens. Macro-dissections were undertaken with a surgical scalpel on 20 μm sections under a light microscope to separate the superficial and the basal layers of the endometrium. The superficial layer sampling included the superficial endometrial epithelium and the first row of endometrial glands and scattered stroma. The basal layer sampling included the juxta-myometrial portion of the endometrium with dense stroma. The intermediate layer of the endometrium was excluded from analysis. The samples were stored at 4 °C and RNA extraction was performed within 2 h.Total RNA was extracted using the miRNeasy FFPE Kit  according to the manufacturer’s instructions. The purity, quality and concentration of all RNA samples were examined by using a microspectrophotometer Bioanalyzer 2100  . The ana®, Santa Clara, CA, USA) was conducted at the genomic platform of the Institut Cochin, Paris. Each sample was tested once as the microarray hybridization is a robust technology with a broadband analysis. Specific miRNA analysis was performed using Partek® Flow® software version 3.0 . CEL files were imported and normalized using robust multi-array averaging (RMA) [p-value ≤ 0.05 were considered to be differentially expressed. The miRNA showing a 2-fold change or more were considered for further analysis. The results were synthetically presented using Venn diagrams. Microarray analysis was conducted on seven samples from seven women treated for at least 3 months with UPA and who were still using it (UPA group), eight samples from eight control women in the proliferative phase (proliferative phase group) and six samples from six women in the secretory phase (secretory phase group). Microarray hybridization on the miRNA 4.0 chips (Affymetrixng (RMA) . Those mhttp://www.ingenuity.com, accessed on 30 April 2018).Evaluation of the impact of UPA on bio-functions and canonical pathways associated with endometrial receptivity was conducted using Ingenuity Pathway Analysis software (IPA: https://www.r-project.org/ (accessed on 19 March 2018).Unless otherwise specified, data were managed using an Excel database and analysed using R version 3.1.3 , available online at n = 7) (n = 8) or the secretory phase . The menstrual phase was assessed on a histological basis and on ER and PR expression evaluations [Three groups of patients were included in the study. The UPA group included women operated on after a 3-month course of UPA, before treatment discontinuation  (n = 7) . Two disluations ,18.The baseline characteristics of the three study groups—the UPA group, and the proliferative phase and secretory phase groups—were comparable . The FIGIn the UPA group, focal and not pronounced PAEC were observed in two cases (29%). The proliferative aspect of the endometrium in the UPA group was observed in six samples and one endometrium was characterised by an early secretory aspect.Endometrial miRNA profiles in the UPA, proliferative phase and secretory phase groups were analysed in each endometrial layer separately using microarray hybridization.The median ratio of the RNA integrity number (RIN) of the analysed samples was 2.3 (interquartile range (IQR) 2.2–2.4) and the median concentration of RNA was 66.0 ng/μL (IQR 36.5–102.8).The expression of miRNA was differentially regulated in the UPA group as compared to the proliferative and secretory phase groups in both the basal and superficial layers of the endometrium .p < 0.05 and fold change > |2| identified two up-regulated and 17 down-regulated miRNAs in the UPA group compared to the proliferative phase group. According to the IPA analysis they were experimentally proven to regulate three and 269 mRNAs, respectively. In the superficial layer the cut-off values for Two miRNAs were up-regulated and 22 down-regulated in the UPA group when compared to the secretory phase group. They were experimentally proven to regulate 23 and 125 mRNAs, respectively. Among these miRNAs, miR-184 was differentially expressed in the UPA group as compared to both the proliferative and secretory phase groups a.In the basal layer, 21 miRNAs were down-regulated and none up-regulated in the UPA group as compared to the proliferative phase group. They were experimentally proven to regulate 444 mRNAs. In comparison to the secretory phase group, three miRNAs were up-regulated and 12 down-regulated in the UPA group. These miRNAs regulate the expression of 11 and 103 mRNA, respectively. Among these miRNAs, miR-1246 and miR-3197 were differentially expressed in the UPA group compared to both the proliferative and secretory phase groups b. Among the 59 miRNAs identified as being modified by the UPA treatment, only 16 showed the same UPA-related profile in both layers of the endometrium. Among these, eight miRNAs were differentially expressed in the UPA group as compared to the proliferative phase group  c and eigUsing IPA, we conducted a functional analysis of the mRNAs regulated by the miRNA previously described in the miRNA expression section. The top 10 statistically most significant biological processes potentially involved in the physiopathology of the human endometrium for each analysis group are presented in IPA analysis of the list of mRNAs of interest  identified the top ten canonical pathways involved. The results are presented in The most significantly influenced canonical pathways were connected to the cell cycle, cell signalling linked to cancer development and inflammation. The miRNAs involved in these processes were down-regulated.The aim of this study was to analyse the effect of the UPA on the endometrial expression of miRNA as compared to the proliferative and secretory phases in superficial and basal layers of the human endometrium. The analysis revealed 59 miRNAs differentially expressed in the UPA group. The identified miRNAs showed differential expression profiles in the basal and superficial layers.Among the identified miRNAs, miR-449a, miR-449b-3p, miR-449b-5p and miR-449c-5p showed the biggest fold change in both endometrial layers. They were down-regulated in the UPA group as compared to the proliferative phase group. These miRNAs are known to be up-regulated in the endometrium of women with repeated embryo implantation failure (RIF) during the implantation window . The miRAmong the identified miRNAs, miR-188-5p has been described to be up-regulated in endometrial cancer as compared to normal endometrium . In our The literature data on the involvement of identified miRNA in embryo implantation and endometrial cancer are resumed in Our IPA analysis of the top ten statistically most significant biological processes regulated by the identified miRNA showed that UPA might potentially indirectly modify the expression of ESR1 (ERα) and PTEN mRNA. The expression of these mRNAs has previously been found unchanged in endometrium exposed to UPA . HoweverUPA binds to both major isoforms of progesterone receptors and, together with transcription co-regulators, induces or inhibits the signal transduction ,27. Its The impact of UPA on the basal layer of the endometrium suggests a potential long-term impact since the superficial functional layer sheds with menstrual bleeding, while the basal layer persists. The endometrial basal layer may be considered as the ‘memory’ of the endometrium, as it could be a source of regeneration of the functional layer among others mechanisms, by harboring endometrial stem cells . HoweverThe presence of PAEC in two out of seven samples was observed. This ratio is higher than that described in the literature. However, the changes were focal and not pronounced.The major strength of our study was the large number of patients drawn from a homogeneous population of women with symptomatic myomas included in the analysis. Furthermore, as we analysed hysterectomy samples, we were able to evaluate both endometrial layers. The miRNA analysis was performed using a robust technology with a broadband analysis. The quality control was undertaken for all samples before the analysis.The limitations of this study included its retrospective design and the absence of hormonal assessment of the menstrual cycle phase. However, as symptomatic myomas usually require hormonal treatment, the number of control samples was limited. Another limitation was that all the included patients were experiencing severe myoma-related symptoms and our data cannot be extrapolated to other clinical situations. UPA licence has been discontinued since March 2020 due to severe adverse events and more particularly to liver cell damage, therefore larger studies on UPA in the indication of uterine leiomyoma are impossible in the current circumstances. However, the data may be of scientific interest for other SPRM molecules with mixed agonist and antagonist effects. Finally, the use of paraffin-embedded samples reduced the quality of total RNA (RIN around two). RIN is poorly informative on the miRNA quality obtained from FFPE tissue according to the literature ,31 and mOur results show that UPA specifically modifies the expression of 59 miRNAs, the majority of which have been described as being involved in processes connected to embryo implantation and cancerogenesis. The changes in miRNA expression observed in the basal layer, from which the functional layer renews every month, are different from the molecular changes observed in the superficial layer.While miRNA can act as promoters or inhibitors of cancerogenesis, the regulation of miRNA observed in the UPA exposed endometria was opposite to the modifications described in the literature for tumoral endometria in all but two miRNA. Other large studies analysing the role of miRNAs in the endometrium and the long-term impact of SPRMs on endometrial miRNA expression are necessary.miRNAs are involved in the inflammatory processes playing essential roles in embryo implantation. All but four miRNA were oppositely expressed in the UPA exposed endometrium compared to the published data on the endometria of RIF women.These results deserve further confirmation but suggest that UPA might be used in women willing to conceive without impairing the chances of pregnancy or increasing the risks of developing endometrial malignancies.


Important information about medical laboratory providers is not readily available to all patients, clinicians nor regulators in Kenya. This study was conducted as part of a wider project aiming to improve access to high quality diagnostics by addressing information asymmetries in the Kenyan market for laboratory services.The purpose of this study was to: 1) Gather pricing information for 49 common laboratory tests from medical laboratories in Nairobi, Kenya, noting where these prices were publicly available or withheld. 2) Assess patients’ knowledge of testing information including: turnaround time, price, and test availability.This was a cross-sectional study where a mystery caller approach was used to survey 49 tests for turnaround time, price, and availability across 13 laboratories selected purposively. The mystery shopper survey was complemented by 251 patient exit interviews at two Kenyan hospitals to understand whether patients seeking laboratory tests in Nairobi had access to such information. All 251 patients were selected by convenience sampling.We noted that 85% of the private laboratories did not disclose test prices and turnaround times to their patients. There was a wide range of prices on several key tests, with private in-facility laboratories charging an average test price of 468% of the average test price in public laboratories across all the 49 tests. We also found that many patients lacked key information regarding the tests they needed: 65% did not know the purpose of the test while 41% did not know the test price at all.Under the current system, patients have limited access to information regarding the key criteria required to make a rational decision. This has a significant impact on the quality, price, and turnaround time (TAT) offered by the medical laboratories that operate in this dysfunctional market. Timely access to laboratory diagnosis is crucial to the provision of high quality health care . This isIn some contexts, where patients are not covered by insurance and are paying out of pocket , they tePatients, like their clinicians, also expect high quality diagnostic laboratory services. According to WHO, laboratory quality is defined by the accuracy, reliability, and timeliness of the reported test results . To achiIn summary, research to date suggest that patients make choices based on test price, convenience and their own perception of quality . These pThis study was approved by the Aga Khan university Ethics Committee (Ref No: 2017/REC-103 (V6)).It is regrettable that we were obliged to use a mystery shopper approach to gather test information for many of the laboratories. Such approaches do not allow for informed consent, and it would have been preferable to have communicated openly with the laboratory providers, but this option was not possible unfortunately. Anecdotal evidence would suggest that the laboratories withhold pricing information because they do not wish to compete on price. This issue should be a focus of further study, but for practical reasons during this preliminary research, we adopted the mystery shopper approach.This study was conducted in Nairobi, the capital city of Kenya. According to the 2019 census report, Nairobi has a population size of about 4.4 million people . The medWe collected primary data both on test prices and availability from laboratories as well as from patients seeking tests.A list of all the licensed medical laboratories in Nairobi was obtained from the KMLTTB and purposive sampling was used to generate a list of the top medical laboratory service providers. In the purposive sampling approach, priority was given to laboratories that have a large share of the private clinical laboratory market, operated as chains and had operational licenses as at the time of the survey. We also included an academic (not-for-profit) laboratory, faith-based and public clinical laboratories. Our study sample was made up of 13 medical laboratories consisting of seven private laboratories, two faith-based, three public and one academic (not-for-profit) laboratory. The private laboratories were later categorized into stand-alone and ‘in-facility’ laboratories. One of the three public laboratories was attached to the National Referral Hospital (NRH). All seven private laboratories operated as part of large commercial chains. The seven chains represented by these seven laboratories have a total of 91 laboratories in their networks within Nairobi. This represents about 18% of the total number of the licensed private laboratories.The cross-sectional survey was conducted between March and June 2019 to obtain testing information for each of the listed 13 laboratories. The information sought for every laboratory included test availability, test prices and the respective TAT. Most of the tests used in this survey were obtained from a list of the top 25 tests that Horton et al found to be the most common by volume across six different international hospital sites .Save for one private stand-alone laboratory, most of the private and faith-based medical laboratories did not display their test price and TAT publicly to their customers. Although test prices and TATs among some public hospitals were not displayed publicly, patients who inquired were informed how much the test would cost and the stipulated TAT. The mystery caller approach was used to collect this information from the laboratories that did not display the information publicly . Two traWe sought to understand if patients had access to relevant information about the tests they needed and how the information they had informed their choices. To achieve this, convenience sampling was used to recruit patients seeking laboratory tests for face to face interviews as we did not want to cause long queues and unnecessary delays . A semi-We set up a station at the entrance of each of the two laboratories for two days at each facility and patients exiting all clinics including antenatal care with test request forms were directed to us by the clinicians. Data were collected in two sessions every day, the morning session and the afternoon session. At least 60 study participants of 18 years and above were targeted on each day of data collection. This was approximately 30% of all the patients seeking laboratory tests in their respective hospitals. Patients under 18 years of age were only recruited if they were accompanied by an adult who became the study respondent.A semi-structured questionnaire was then used to obtain information from the patients and their guardians. For example, the patients were asked whether they knew the price of the test they wanted to request, its availability and the TAT. The tests requested were confirmed from the patients’ test request forms. Additionally, we also asked them if they considered test quality when seeking for laboratory tests, how they identified good quality laboratories and if they knew that poor quality tests could result in misdiagnosis. We later informed them of the exact test price, TAT and whether the laboratory in the same public facility was offering the test at that time. All the responses provided by the participants were entered into REDCap and sent to the server of KEMRI Wellcome Trust.Although data were collected from 251 patients seeking laboratory tests, a total of 6 respondents were excluded from the analysis due to incomplete data. The findings reported are therefore based on the responses provided by 245 study participants. The data were analysed both in Microsoft excel 2016 and R statistical software version 4.0.5 [Pricing Survey: The study collected information on 49 tests within 13 central laboratories (described above). We noted that 85.7% of the private facilities and the two faith-based laboratories did not provide information on test price and TAT publicly to the patients. None of the three public facilities displayed their TATs publicly to their clients. However, two of these public laboratories displayed their test prices for their clients to refer and consider. Test availability was highest in stand-alone laboratories as they offered 95.9% of the tests on average, compared with 91.8% in private-in-facility laboratories and 51% in public laboratories. Test availability within public laboratories 8, 9 and 10 was 65.3%, 40.8% and 46.9% respectively . In termThere was great variation in test prices among the laboratories. Mean prices across all 49 tests in stand-alone private laboratories were 300% of the mean in public laboratories and 468% in private in-facility laboratories compared to public. Public laboratories have the lowest mean test prices, with some tests such as HIV viral load and other tests for children under 5 years being free of charge. Malaria smear test had the largest price discrepancy with the private-in-facility laboratories charging up to 6 times more on average as compared to the public laboratories .Figure A total of 251 patients or their guardians were interviewed from the two public hospitals although our analysis was based on 245 of them who had complete data. Of the 245 respondents, 70.6% were female while 29.4% were male. The average age of the respondents was 33.7 years and 60.8% of them were employed. The overall number of tests requested was 371 as observed from the 245 test request forms. The highest number of tests noted on a single test request form were six and these included: Liver Function Tests, full haemogram (Complete Blood Count plus a smear), sickling test, Urine chemistry, Oral Glucose Tolerance Test and Syphilis test (VDRL). The full haemogram test, Urine chemistry and Malaria smears were the most frequently ordered tests at 86, 43 and 31 respectively as shown in Of all the 245 study respondents, 64.9% did not know the purpose of the laboratory tests requested by the clinician. Only 86 participants attempted to state exactly what the laboratory test was for and out of these, 75.6% correctly determined the use of the laboratory test requested while 24.4% made an incorrect statement about the purpose of the test.The majority of patients had no information on the test price. Of the total respondents, 41.2% said that they did not know the price of the tests at all while a further 34.7% who attempted to estimate the test price underestimated it. On average, patients that attempted to guess the actual test price underestimated it by 41.5%. Although 59.6% of all the respondents mentioned that they valued quality when it comes to laboratory testing, 45.2% of those who valued quality agreed that it was difficult to identify a good quality laboratory. Generally, 38% of all the study respondents found it difficult distinguishing good quality laboratories from poor quality ones. Nonetheless, some patients (13.1%) trusted government laboratories as good quality laboratories while 15.1% others mentioned that they relied on reviews from other people such as friends and relatives as indicators of quality in medical laboratories. Almost 10% of the respondents trusted that doctors/clinicians always referred them to good quality laboratories .Figure Based on the actual TAT information provided by the laboratories, the average TAT of the 371 tests requested by patients was two hours with a range of 1–72 hours. The longest turnaround time was 72 hours for a test requiring bacterial culture . At least, 28% of the interviewed patients did not know how long they would wait to get their laboratory results. Almost half of the respondents underestimated TAT while 14.7% overestimated the TATs. Only 11.4% of the laboratory seeking patients correctly identified the TATs of their tests .Figure Our preliminary data revealed a significant lack of transparency in the medical laboratory market in Nairobi, especially in the private sector. While opacity on price is well noted in developed settings , we are As the sole regulator of clinical laboratory services in Kenya, KMLTTB is mandated with five main responsibilities: indexing or examination of college students taking up careers in medical laboratory sciences; registration and licensing of all laboratory staff in Kenya; developing policy guidelines for the implementation of the Continuing Practice Development (CDP); and regulation of all In vitro Diagnostics (IVDs) intended for laboratory testing. Most importantly, KMLTTB regulates the conduct, practices and licensing of laboratory businesses in Kenya . HoweverThe average test price among private-in-facility laboratories was 468% of the average test price in public laboratories. Similar findings on test price variations have also been reported among private laboratories in Kampala, Zimbabwe and the USA . The higSlightly more than half of the participants interviewed 59.6%) said that they value quality when seeking for laboratory tests. Unfortunately, many of them find it difficult to differentiate between good and poor-quality laboratories. At least 13% of the interviewed patients said that they relied on government laboratories for quality tests while 10% mentioned that they trusted laboratories to which their clinicians or doctors referred them. Some clinicians have relationships with laboratories and are incentivized to send their patients to those laboratories while in other cases, they simply want a reliable result for their patient and for themselves. The fact, however, remains that government laboratories have limited test menus 47%-65%) mainly due to lack of proper equipment, reagent stock outs and inadequate personnel 9.6% said. A simil%-65% maiDiagnostic testing is comprised of many processes that happen behind the closed doors of clinical laboratories. Evaluating quality and issuing certification requires both access and expertise. It would be desirable to educate patients and clinicians to seek services from laboratories that have certificates of participation in PT or verified quality accreditations. Laboratories taking part in such programs usually display these certificates publicly at the reception areas of their laboratories and on their websites. The main challenge, however, is that such certifications are not mandatory, and indeed, they come with costs that mean that most clinical laboratories do not have them. Although all the private laboratories that we surveyed were accredited, this is because we only included large private laboratories with bigger market share in this study. Unlike these few bigger laboratories, many of the small and medium private laboratories in Kenya are not accredited. There is an opportunity to make this information available to potential laboratory test seeking clients. Given the high literacy and smart phone ownership level in Nairobi city , one posRegulation of laboratory test prices, especially in the private sector, is a complex issue and seemingly beyond the powers of most laboratory regulatory agencies. However, one possible way of ensuring accessibility to diagnostic testing is for the Kenyan government to introduce a policy ensuring that all public laboratories are well equipped to conduct all the essential diagnostic tests as set out by the WHO . Such a Although the mystery shopper approach is an accepted way of collecting withheld information, especially in the business sector, we did not obtain consent from the non-public laboratories from which we collected the pricing data and it would have been preferable to have had their full cooperation. However, this was the only viable approach of collecting the exact test prices charged to patients. The pricing data obtained in this survey were completely anonymized such that they cannot be linked back to the respective laboratories. Separately, we did not physically confirm from the laboratories whether they had the capacity to conduct the tests as informed during the mystery shopper call. This could have led to overreporting of test availability although it is rare for a laboratory to give false information regarding test availability to a prospective client whom they are expecting to visit their premises to have the test conducted. Most importantly, the findings reported in this study were summarized as proportions, percentages and means. The authors acknowledge that further statistical approaches would have been useful in drawing conclusive inferences from this study. For instance, inferential statistics comparing the average test prices across laboratory categories would confirm if the deviations in test prices were statistically significant. Nonetheless, it is clear from the calculated average test prices that different laboratory categories have different price ranges. It is also possible that our finding on low knowledge of test use among the participants could be attributed to literacy levels, but we are certain that cities such as Nairobi have the highest number of literates compared to non-urban settlements. Finally, this was a small study that aimed to collect preliminary data on test availability, test costs and patients’ access to such information within purposively selected facilities. The patients interviewed in the patient exit interviews within the two public hospitals were also selected based on convenience sampling to avoid delayed care consequently resulting to a small sample size. Therefore, the results obtained here may not be a representative of all the medical laboratories in Nairobi and patient responses were only gained from those using the public sector.Under the current system, patients have limited access to information regarding the key criteria required to make a rational decision. This has significant impact on the quality, price, and TAT offered by the medical laboratories that operate in this dysfunctional market. There is a clear need for all clinical laboratories to provide this vital information to their clients. Patients should also be given access to simpler metrics that they could use to distinguish poor and good quality laboratories. The opaque nature of testing information in the current clinical laboratory market in Nairobi limits access to high quality diagnostics and in turn, this limits the potential for higher quality medical care. There is an opportunity to enable this market to work better. In addition to more traditional means of working with the regulator and the ministry to encourage more open information in the market, we are excited by the prospect of digital health interventions. One possibility is to use a digital marketplace app, in the same way that has been done in other sectors where customers choose taxi drivers, second-hand sellers or holiday lettings.