Transient process spectroscopy has previously been thought to be applicable only to the analysis of intra-molecular processes. Two metal ion bridges used in the present work have allowed us to visualize real-time variations of the molecular vibration frequencies during photo-disproportionation inside bimolecule aggregates, which directly shows transient inter-molecular reactions. Rapid processes that cannot be tracked visually can often be observed by acquiring photographic images under rapid strobe lighting. The development of femtosecond strobe lights has enabled the study of the electronic and vibrational dynamics of transition states in photo-reactions. As a result, reaction pathways such as A → B → C in Fig. −1, corresponding to their molecular vibrational modes. As an example, the stretching modes of C=C and C-C bonds appear at 1600 and 1100 cm−1, in association with vibrational periods of 20 and 30 fs. These molecular vibrations can be temporally resolved by measurements employing sub-10 fs laser pulses that allow observations of vibrational motions in real-time. In addition, examination of changes in instantaneous molecular frequencies allows the visualization of transient processes such as chemical bond breaking and reformation. The observation of transition states has also been reported in the case of various intra-molecular reactions, based on the use of sub-10 fs laser pulses.The functional groups of organic compounds typically generate specific absorption bands in the range of 1000–2000 cmThe majority of organic photo-reactions proceed as inter-molecular reactions via inter-molecular collisions or inter-system crossing occurring in the picosecond to nanosecond time scale. However, the coherent molecular vibrations produced by impulsive photoexcitation dephase as fast as a few picoseconds,A Ti:sapphire regenerative amplifier  was used to generate near infrared (NIR) femtosecond pulses  so as to produce ultrashort visible pulses using a home-made non-collinear optical parametric amplifier (NOPA).A typical molecular vibration has a period of 20 fs, and so cannot be temporally resolved by time-resolved spectroscopy systems employing laser pulses with durations of 35 fs or longer. Therefore, ultrashort visible laser pulses with sub-10 fs durations have been developed, since these can temporally resolve real-time molecular vibrations. To allow the observation of both electronic and vibrational dynamics, a time-resolved absorption pump-probe spectroscopy system was designed in the present work, as follows.In this system, the intense broadband visible laser pulse generated by the NOPA was separated into two copies, at a power ratio of 10:1, and these were employed as the pump and probe pulses in pump-probe spectroscopy. The chirps of the pump and probe pulses were adjusted to have a pulse duration of less than 10 fs at the point of impingement on the solution sample held in a synthetic fused silica glass cell. The chirp adjustment was accomplished as follows.μm thickness to generate the sum frequency between the two pulses. The sum frequency pulse was clipped by an iris and coupled into optical spectral analyzer. The sum frequency spectrum was measured as a function of the delay between the pump and probe pulse in the second harmonic generation (SHG) frequency resolved optical gating (FROG) measurement. Therefore, the pulse duration estimated in the SHG FROG measurement reflects that of the pulse in the glass cell for the solution sample. We have adjusted the grating compressor to let the estimated pulse duration as short as possible in this situation. Thus, the visible pulse transmitted through one glass wall plate has a pulse duration of sub-10 fs. On the measurement of the solution sample, we have removed the glass wall plate and put the solution sample filled in the glass cell. Therefore, the solution sample was excited and probed by the sub-10-fs ultrashort visible pulse.The glass cell used in the present work  has 1-mm optical path length and its glass walls have a thickness of 1.1 mm. We have broken one of the glass cells into half to get one of the glass walls of the glass cell. The glass wall plate was inserted in front of the beam sampler in the pump-probe setup. Thus, both the pump pulse and the probe pulse transmit through the glass wall plate for once. By using a parabolic mirror with 101.6-mm reflected focal length, these pulses were focused in the beta barium borate (BBO) crystal with 10-T + ΔT) and the signal from the sample in the unexcited state (T) for every two probe pulses. Absorption changes were calculated as ΔA = −log10 (1 + ΔT/T). The absorption changes were determined by scanning the optical delay between the pump and probe pulses at 500 ms intervals with a step-size of 0.2 fs, from −30 to 1800 fs. In the subsequent data analysis, we averaged every five delay points to improve the signal-to-noise ratio.The probe pulse transmitted through the sample was coupled into a polychromator . The probe spectrum was subsequently dispersed by the polychromator and measured by a fast scan rate CCD line scan camera  with a line scan rate of 1 kHz. The probe spectrum was acquired for every pulse at a repetition rate of 1 kHz. The pump pulse was modulated by a mechanical chopper running at a frequency equal to half the laser repetition rate. Thus, the acquired probe spectrum contained the signal from the sample excited by the pump pulse (+2[TCNQ−•]2). A transition from the original yellow color of the initial TCNQ solution to green provided evidence for the charge transfer (CT) band of the [TCNQ−•]2 at 643 nm.+2[TCNQ−•]2 solution, with a concentration of 3.6 × 10−4 M, was transferred into a fused silica cell in preparation for the pump-probe measurements. The glass cell was put on a mechanical motorized stage to be continuously moved in a circle shape in a plane orthogonal to the light path. Thus, the system has been adjusted to implement that the sample probed by the last pulse will not be probed by the next pulse coming 1 ms later. The sample existing at the irradiated point goes away by convection in the glass cell after running the glass cell through the whole arc of the circle. Therefore, the degradation effect of the sample can be excluded during the measurement. It was also confirmed that the stationary absorption spectrum of the sample does not show any recognizable difference between before and after the measurement. All trials were performed at a room temperature of 22 ± 1 °C.A saturated methanol solution of 2,2′- dimalononitrile (TCNQ)+2[TCNQ−•]2 was performed replacing sodium with potassium. Geometric optimization was performed at the B3LYP/6–31G* level, and 5d functions were employed for the d orbitals. Raman active molecular vibration frequencies were calculated for each of the obtained structures at the same level.The Gaussian 09 program+2[TCNQ−•]2  and the gate function g(t − τ) shifting τ (the center position of the gate function). When τ = 0τ, the product ΔA(t)g(t − τ) contains oscillating components around t = 0τ because g(0t − τ) is non-zero only in −T/2 < t − 0τ < Τ/2. Therefore, its Fourier spectrum, i.e., spectrogram for τ = 0τ, S, represents the vibrational spectrum at around t = 0τ. Calculating the Fourier transform shifting τ, spectrogram is obtained reflecting the dynamics of the molecular vibrational spectrum. The spectrogram was calculated using the Blackman window function with a full width at half-maximum of 200 fs, as shown in the below equationThe transient absorption trace includes slow relaxation reflecting electronic dynamics and fast oscillation reflecting vibrational dynamics. We have an applied high pass filter to the transient absorption trace and got the fast oscillating components, which is plotted in Fig. The resulting spectrogram is provided in Fig. +2[TCNQ−•]2. The frequency region below 820 cm−1 shows inter-fragment vibrational modesδTCNQ-TCNQ), while the peaks at 1060, 1200, 1425, and 1560 cm−1 are assigned to the symmetric stretching mode of the benzene ring (νBn), the C-H bending mode (δCH), the C-C 1.5 bond stretching mode of the side chain group (ν1.5CCs), and the C-C double bond stretching mode of the benzene ring (νCCr), respectively.The vibrational modes immediately after photo-excitation reflect those of NaδTCNQ-TCNQ modes between the TCNQ anion radical dimer below 820 cm−1 were found to disappear approximately 300 fs after photo-excitation. This result implies that the dimer dissociates within this time span.The ν1.5CCs peak at 1425 cm−1 just after photo-excitation was separated into blue-shifted and red-shifted bands and transitioned to a new peak at approximately 400 fs. The mode separation between the blue-shifted mode and the red-shifted mode is comparable with the bandwidth of the modes appearing in the spectrogram. The result shown in ν1.5CCs mode has a bond order of 1.5. One of the disproportionation products, TCNQ, has a C-C double bond, which generates the blue-shifted band. The other product, TCNQ2−, has a C-C single bond that corresponds to the red-shifted band. The TCNQ is generated in the electronic ground state and generates a peak at 1470 cm−1, corresponding to the C-C double-bond stretching mode (ν2CCs). In contrast, the TCNQ2− was in the electronic singlet excited state and produced a band due to the C-C single bond stretching mode (ν1CCs) at 1340 cm−1. This frequency of νCCs1 higher than C-C single bond stretching mode agrees with the theoretical calculation. The blue shift of the frequency is thought to be because the methylene carbon in malononitrile unit has a negative charge of −0.5.The 52 (ϕ53), is the bonding (antibonding) orbital of the C-C bond of the side chain group  of TCNQ, ϕ−1 peak seen just after photo-excitation broadens 400 fs after photo-excitation. The DFT calculations  were studied. In this compound, two TCNQ–• molecules are bridged by two metal ions, and this structure serves to suppress inter-molecular collisions, which in turn tends to maintain the coherence of molecular vibrations. As such, changes in the molecular vibration frequency during the disproportionation of bimolecule aggregates could be visualized in real time. The data obtained in this manner were found to be in good agreement with the results of DFT calculations. Transient process spectroscopy was previously thought to be solely useful for the observation of intra-molecular reactions. However, the present work has proven that this technique is applicable to many chemical reactions, even those classified as inter-molecular.The ultrafast molecular vibrational dynamics of an inter-molecular reaction (the photo-disproportionation of Na


The molecular clock is a valuable and widely used tool for estimating evolutionary rates and timescales in biological research. There has been considerable progress in the theory and practice of molecular clocks over the past five decades. Although the idea of a molecular clock was originally put forward in the context of protein evolution and advanced using various biochemical techniques, it is now primarily applied to analyses of DNA sequences. An interesting but very underappreciated aspect of molecular clocks is that they can be based on genetic data other than DNA or protein sequences. For example, evolutionary timescales can be estimated using microsatellites, protein folds, and even the extent of recombination. These genome features hold great potential for molecular dating, particularly in cases where nucleotide sequences might be uninformative or unreliable. Here we present an outline of the different genetic data types that have been used for molecular dating, and we describe the features that good molecular clocks should possess. We hope that our article inspires further work on the genome as an evolutionary timepiece. Estimates of evolutionary rates and timescales form an important component of research in biology. These can be obtained from genomes using molecular clocks, which describe the relationship between genetic change and geological time. The idea of a molecular clock was first put forward >50 years ago. In their seminal study, With major advances in DNA-sequencing methods from the 1980s, the use of amino acid sequences in molecular dating has declined substantially. Nevertheless, they are still often employed in studies of deep timescales, particularly when nucleotide sequences exhibit high degrees of saturation or variability in nucleotide composition . NucleotMolecular clocks are based on the assumption that genetic change can be described as a simple function of time . An ideaAny particular molecular clock is unlikely to be reliable across a broad range of timeframes. For example, some genomic characters are so mutable that they cannot be compared between species. This might make them useful for studying the evolutionary process at the population scale, but it places strong limits on their taxonomic scope. The presence of intraspecific polymorphism, for example, can introduce considerable noise into the dating analysis, particularly when using methods based on linear regression . To provPrior to the wide availability of DNA and protein sequences, a range of biochemical techniques were used to estimate genetic distances between pairs of taxa . Early iDrosophila found that genetic distances based on RAPD gel bands were proportional to time since divergence, suggesting some degree of rate homogeneity across lineages  are coded from sections of DNA generated using restriction enzymes and amplified using PCR. AFLPs have been used to provide a shallow-time molecular clock in several species of alpine plants  and perclineages . These mlineages .Genome complexity, measured by the size of the nonredundant functional genome, has been proposed to exhibit clocklike evolution . This “cLooking towards the future, other features of the genome offer more promising sources of data for molecular clocks. There have been several dating studies that have focused on short tandem repeats of DNA sequences, or microsatellites. Various distance metrics have been proposed for microsatellite data, allowing them to be used in molecular dating based on linear regression . More reWhen looking at the timescales of ancient evolutionary events, such as the deep divergences in archaea, bacteria, and eukaryotes, nucleotide and amino acid sequences are often saturated with substitutions and cannot be aligned with confidence . HoweverRecombination events were recently used to estimate the ages of ancient human genomes . If two Various features of the genome can potentially be used for molecular dating, though they are likely to differ in their ability to keep evolutionary time. There have been few direct comparisons of the performance of different molecular clocks , but somWhen comparing different types of data for molecular clocks, an important consideration is whether an explicit model of evolution is available. For example, models have been developed for nucleotide sequences, amino acid sequences, binary characters, and microsatellites . ExpliciThe rapid growth in genomic data opens up unprecedented opportunities for evolutionary analyses. Although molecular dating is almost invariably performed using DNA sequences, genomes offer a rich variety of data that can be used to construct molecular clocks. These other sources of data provide an exciting and potentially valuable avenue of research, particularly in cases where molecular clocks based on DNA sequences might be expected to fail.


A numerical analysis was conducted to study a room temperature magnetocaloric refrigerator with a 16-layer parallel plates active magnetic regenerator (AMR). Sixteen layers of LaFeMnSiH having different Curie temperatures were employed as magnetocaloric material (MCM) in the regenerator. Measured properties data was used. A transient one dimensional (1D) model was employed, in which a unique numerical method was developed to significantly accelerate the simulation speed of the multi-layer AMR system. As a result, the computation speed of a multi-layer AMR case was very close to the single-layer configuration. The performance of the 16-layer AMR system in different frequencies and utilizations has been investigated using this model. To optimize the layer length distribution of the 16-layer MCMs in the regenerator, a set of 137 simulations with different MCM distributions based on the Design of Experiments (DoE) method was conducted and the results were analyzed. The results show that the 16-layer AMR system can operate up to 84% of Carnot cycle COP at a temperature span of 41 K, which cannot be obtained using an AMR with fewer layers. The DoE results indicate that for a 16-layer AMR system, the uniform distribution is very close to the optimized design. Barclay and Steyert2 introduced the AMR concept to address the problem, in which a regenerative cycle is applied to create temperature spans comparable to conventional cooling systems. Following their work, investigations in the magnetic refrigeration community focused on the AMR as it provided a methodology to realize an alternative refrigeration technology with significant potential energy savings compared to conventional vapor compression refrigeration technology at room temperature. A large number of experimental and numerical studies have been conducted on AMR research, which have been illustrated in the review papers10.The magnetocaloric effect (MCE) is a phenomenon where a temperature change of a magnetocaloric material is caused by exposing the material to a changing magnetic field. A cooling technology was developed based on MCE, however, studies of this technology were limited to the maximum adiabatic temperature change of 5 K due to the MCE limitations of most MCMs at moderate magnetic fields (up to 1.5 T)12. The major breakthrough occurred in 1997 when the giant MCE in a GdSiGe alloy was discovered by Pecharsky and Gschneidner13, which had twice the magnetic entropy change compared to that of Gd. The discovery of the giant MCE, which is due to the first-order phase transition (FOPT), stimulated the research on MCMs worldwide. This was followed by the discoveries of a number of other alloys with FOPT and pronounced MCE, based on MnAsSb14, MnFe15, MnFe15, LaH16 and LaFeSi18. Among the newly discovered materials, the LaFeSi based ones are very promising candidates as the working materials for magnetic refrigeration at room temperature. Even though their adiabatic temperature change is about the same as Gd, LaFeSi based MCMs have higher entropy change. They also have small hysteresis, and relatively low price 19.The research in magnetocaloric refrigeration in the past decades led to an increased interest in the search for new MCMs with high MCE. Initially, the materials used in the AMR studies focused on the ones with second-order phase transition, such as Gd and its alloys based on rare-earth materials . However, the commercial use of those materials is limited not only by their relative low MCE, but also by their relatively high costet al.20, ternary La13 alloys have a Curie temperature around 200 K. By adding a fourth element, such as Co or H, the Curie temperature is able to increase up to room temperature. Moreover, by controlling the quantity of additional elements, the Curie temperature can be tuned to cover a wide temperature range.However, disadvantages of FOPT exist for AMR implementation. One of its fatal disadvantages is the relatively narrow temperature range of the MCE which tremendously limits the temperature span of the AMR using FOPT materials as MCM. In order to provide the largest temperature span across the AMR, or the largest MCE in the entire AMR, several different MCMs with different Curie temperatures must be layered along its length. These materials can be synthetized by adding additional elements, such as Co or H, to the base materials (e.g. LaFeSi). The Curie temperature and the MCE of the synthesized material can be tuned by controlling the quantity of the added elements. As reported by Liu et al.21 reported the refrigeration performance improvement in their experiments by using a 3-layer AMR. Richard et al.22 conducted a series of experiments to compare the single-layer and multi-layer AMRs, in which the two-layer regenerator was found to produce a higher temperature span and more cooling power than the single material regenerator under certain operating conditions. Rowe and Tura23 extended the work from Richard et al.22 to a three-material layered AMR using Gd based materials. Recently, Teyber et al.24 reported on their two-layer AMR system using Gd based materials. They discovered that for the no-load conditions, the largest performance improvements were observed in the two-layer regenerators.Due to the necessity of layered materials with different Curie temperatures in AMR, it is desired to study the AMR with multi-layer MCMs. Currently, available literature on the multi-layer AMR is far less than that on the single layer AMR literatures. In 1997, Tsukagoshi 25 experimentally compared four AMRs using three different materials 13 and LCS) including a two-layer regenerator made of La13 compounds. He concluded that multi-layer regenerator presents a better efficiency than the single-layer. Similar experimental research was conducted by Legait et al.26 by comparing four AMRs using three different materials 13−xSix and Gd) including a four-layer regenerator. They also observed that the layered regenerator presents a better efficiency than the single layer. Tusek et al.27 experimentally compared the performance of the AMRs in room temperature magnetic refrigerators using multi-layer LaFeCoSi and single-layer Gd. They tested 2-, 4- and 7-layer LaFeCoSi AMRs and revealed that multi-layer LaFeCoSi AMRs provide higher cooling power than single layered Gd AMR. In their study, it can be concluded that the 7 and 4-layer AMRs exhibited very similar performance, which is mostly due to the relatively poor heat-transfer properties and the heat losses to the surroundings. Most recently, Govindappa et al.28 conducted an experimental investigation of FOPT material based multilayer AMR, in which 3-layer, 6-layer and 8-layer AMRs were studied. They discovered that it is necessary to have enough MCM length in every layer as the number of layers increases (e.g. 8-layer) to enable all layers to be active.With the discovery of the giant MCE, studies turned to FOPT material based multi-layer regenerator. Engelbrechtet al.29 conducted a numerical analysis of multi-layer (up to 6) AMR with GdxTb1-x and GdxDy1−x over the temperature range of 275–295 K and 260–280 K, respectively. However, the MCMs limited the working temperature range to non-room temperature. Monfared and Palm30 numerically studied a 6-layer packed bed regenerator using Gd based MCMs and optimized the design to obtain maximum performance. Hsieh et al.31 and Cararo et al.32 modeled 2-layer and 3-layer AMRs for a room temperature refrigeration also using Gd-based materials as MCMs. Kamran et al.33 conducted a performance optimization of a layered AMR with up to four different MCMs, suggesting using a smaller Curie temperature difference between the adjacent MCM layers in order to gain higher COP/capacity, which is a rare numerical study using FOPT materials as MCM (LaFe13−x−yCoxSiy).In addition to experimental studies, a few numerical studies are reported in the literature. Aprea et al.34 and Lei et al.35 reported their modeling of layered AMR with up to 60 layers, the properties of different MCM layers were approximated by shifting experimental data obtained for one layer according to Curie temperature. By doing so, there was not much increase in computational resources comparing to the single layer case. In present work, a 16-layer AMR was numerically studied. Numerical studies highly depend on experimental data, e.g. MCM properties, so 16 is the maximum number of layers in this paper due to the property data limitation. The MCMs are LaFeMnSiH alloys, and the property data was derived from the experimental measurements provided by VACUUMSCHMELZE GmbH & Co. KG. The model presented in this paper was tailored to accelerate the simulation speed, which significantly decreased the processing time for a multi-layer AMR  such that it approached the speed of a single-layer AMR simulation. Based on the model, a performance study was conducted to investigate the effects of frequency and utilization on the cooling power and COP of the multi-layer AMR. Then a DoE was employed to optimize the lengths of each of the 16 MCM layers.One of the most significant barriers to modeling layered AMR systems is lack of experimental data for the MCM properties. In addition, typical simulation codes suffer from significant increase in computational resources required (processor and memory) as the number of MCM layers increases. Therefore, previous modeling using properties directly from experiments only addresses AMRs with a few layers (up to 6). Although Lei L, 0.75 mm as height H, and a unit length width as shown in Fig. x and y are the direction of fluid flow and the direction perpendicular to the fluid flow, respectively. Figure The numerical simulations of the 16-layer magnetocaloric parallel plates regenerator are based on layers LaFeMnSiH with different Curie temperatures and a water/glycol mixture. The magnetic refrigerator is comprised of parallel plates with 200 mm as length ε in present work is 1/3. The cold end temperature TC and hot end temperature TH are fixed at 266 K (−7.15 °C) and 307 K (33.85 °C), based on the Curie temperature of MCM materials at the two ends, so the temperature span in present work is fixed at 41 K. The maximum applied magnetic field Bmax is 1.5 T. The density and thermal conductivity of MCM are 7000 kg/m3 and 10 W/(m⋅K), respectively, as reported by the MCM provider. Therefore, the total mass of MCM in one plate is 1.05 kg. The properties of the water/glycol mixture are obtained from36 as a function of temperature except the density, since the variance of fluid density has a trivial effect on the results. Similar to the single-layer AMR refrigeration system, the refrigeration cycle with 16-layer parallel plates regenerator also consists of four steps, which are magnetization, cold-to-hot blow, demagnetization, and hot-to-cold blow. The details of the cycle can be found in the literature37 and will not described here. Figure The effect of plate orientation with respect to external magnetic field on the magnetization is not considered for simplicity. The porosity 39.In order to analyze and optimize the magnetic refrigerator system with a multi-layer regenerator, a 1D numerical model was employed, which applies the first law of Thermodynamics to the MCM plates and the heat transfer fluid. The governing equations that describe the heat transfer in MCM plates and heat transfer fluid are shown in Equations , respectT, ke, ρ, and c denote temperature, effective thermal conductivity, density, and specific heat, respectively, while subscripts s and f indicate MCM and fluid respectively. a, ε, s, B, p and u are volume-specific surface area, porosity, MCM entropy, applied magnetic field, fluid pressure, and fluid velocity, respectively. The ke,s and ke,f are defined as Engelbrecht suggested37, based on the axial dispersion for a fluid flowing between two infinite plates from Beard40,In these two equations, ks and kf are the thermal conductivities of MCM and fluid, respectively. Pr and Re are the Prandtl Number and Reynolds Number, respectively. ke,s relates the actual rate of axial conduction through the composite MCM/fluid matrix in the absence of fluid flow to the rate of conduction heat transfer that would be experienced by a comparable solid piece of material. ke,f considers an augmentation of the thermal conductivity in the fluid due to the eddy mixing of the fluid that occurs when fluid flows37.where cs = Ts(∂s/∂Ts). Therefore, for the 16 MCMs with different Curies temperatures, 16 different sets of s property data have to be utilized, which are from the experimental measurements provided by the material provider.In Equations  and 2),,2), the hsf, is calculated from correlations for the flow between parallel plates41,dh and Nuf are the hydraulic diameter and Nusselt number. The Nusselt number is calculated asGz is the Graetz number in the regenerator, which is defined asxf, is measured from the fluid inlet.The convection heat transfer coefficient, 42,The pressure drop is calculated from Equation 42,9\docuThe friction factor, which includes the viscous developing region of the flow, is shown in Equation .10\documThe boundary conditions are shown in Table The cooling capacity, rejected heat, COP, and utilization, are calculated using the following equations,τ is the cycle time, which is the multiplicative inverse of frequency f. τf is the time for cold flow and hot flow, since they are identical in present work. 43. The cooling power density  of the regenerator from cycle to cycle is less than a specified tolerance. To verify the developed model, simulation of a single-layer AMR made of Gd using water as the heat transfer fluid with published results from Petersen et al.44  were compared, since Petersen et al.44 provide the comprehensive parameters in their paper to benefit the comparison. The comparisons in Fig. et al.45. As shown in Fig. Qc from our numerical model due to the lack of external losses.The single-layer code needs to be extended for multi-layer AMRs. Extension of the original single-layer model to multi-layer suffered from excessively long computation time. The speed issue may be insignificant if only four or six layers are considered. However, for a 16-layer AMR, the increase in computational time makes the model undesirable, e.g. the time required for a 16-layer case using this model running 10 cycles was 10809 seconds, while a one-layer case under the same conditions needed only 1804 seconds. In the simulation, the MCM properties (entropy and specific heat) are preprocessed as matrices in MATLAB that are used as lookup tables, so for multi-layer AMR, every MCM layer has two property matrices. The property matrices need to be interpolated to obtain the entropy and specific heat for a specific temperature and magnetic field. It was found that the most time consuming process in the simulation is the interpolation in properties matrices. The time consumption increases significantly with the number of layers as 2*Nlayer property matrices have to be interpolated every time step in the simulation. It is also found that the calculation speed of the native MATLAB interpolation function is almost independent of the size of matrix, i.e. the time consumption to conduct an interpolation in a large matrix is almost as same as in a small one. This finding was exploited to reduce the simulation computation time. Instead of using multiple matrices for every layer separately, the multi-layer properties were integrated into two large matrices for entropy and specific heat, individually. A mathematical transformation was employed to change the temperature index of different layers, i.e. the temperature index for layer N will add N*100 K, so the properties can be identified for each layer in one matrix. As a result, the speed of 16-layer cases has been significantly increases by a factor of 6. As a result, the 16-layer case solved by the new speed improvement model only needs 1867 seconds to run 10 cycles, which is very close to the time needed for single-layer model.The 1D model was implemented in MATLABQc from Nx = 256 to 512. According to the mesh and time step study, Nt = 40000 (Δt = 1/4000 s) and Nx = 256 (Δx = 0.78 mm) are chosen as the parameters for an optimal balance between accuracy and simulation speed.Figure Qc and COP for different utilization and frequencies for single layer AMR have been well investigated by the previous work experimentally, e.g. Trevizoli et al. and Trevizoli et al.47, so the discussions in this section will focus on the performance of the 16-layer AMR.U on the performance of the refrigerator. Figure Qc change with utilization at different operating frequencies. For a fixed frequency, the cooling power increases with U initially and then decreases after reaching a maximum value. When the utilization is low, the heat transfer fluid moves slowly and small quantity of low temperature fluid reaches the cold side. Therefore, only little cooling power is produced. On the other hand, when the fluid moves too quickly, the temperature profile of the AMR is disturbed, resulting in a loss of cooling power as well48. Note that the cooling power is negative, and not shown in this plot, when U > 0.255 and f = 0.5 Hz, indicating that the disturbed temperature profile leads to Tf (x = L) > Tc in the cooling processing and the refrigerator does not produce cooling effect under this condition. Consequently, for a 16-layer AMR running under a certain frequency, there exists a U, as shown in Fig. U of the maximum cooling power increases as frequency decreases. It is because a higher U (e.g. fluid speed) is required to disturb the temperature profile in a lower frequency AMR. Figure With the configuration shown in Fig. U at different operating frequencies. The COP at f = 0.5 and U > 0.255 is not shown here because it is less than zero due to the negative cooling power discussed above. Results indicate that when f = 0.5, the COP always decreases with U from 0.05 to 0.25. The trends of the COP of other frequencies are similar to the cooling power, i.e. there exists a utilization that results in a maximum COP. Figure f = 0.167, 0.1, and 0.05 Hz are 4.7 and 5.5 and 5.0 occurring at U = 0.1, 0.15, and 0.2 respectively, so the COP of the 16-layer AMR system reaches its maximum at frequency around 0.1 Hz. This is attributed to the competition between Qc and Qc, leading to reduction of COPmax with increasing frequency. When frequency is less than 0.1 Hz, the increase of Qc overcomes max increases with increasing frequency. The corresponding U of the COPmax increases as the frequency decreases, although it is smaller than that of maximum cooling power at the same frequency. This is due to the mismatch between the maximum values of Qc and Qc and COP in one configuration simultaneously. Therefore, after considering the priority of COP and comparing the 3 optimized point candidates, which are f = 0.167, f = 0.1 and f = 0.05 Hz at U = 0.1, U = 0.15, and U = 0.2, the point of f = 0.1 Hz and U = 0.15 was selected as the optimized working condition of the present 16-layer AMR since it provides the highest COP and an acceptable Qc. This frequency, f = 0.1 Hz, will be used in the discussions below.Figure Nlayer) were investigated, including 4-layer, 6-layer, 8-layer, 10-layer and 16-layer AMRs at a frequency of 0.1 Hz determined from the performance analysis. Since the Curie temperatures of the MCM layers are different, various combinations of MCM layers were chosen to comprise the AMRs. The configurations of AMRs with different Nlayer are depicted in Fig. Nlayer were conducted under the same conditions to compare their performances.Different regenerators with different number of layers  at about U = 0.4. As expected, the 4-layer AMR demonstrates the lowest cooling power, i.e the highest cooling power is less than 6 W from the 4-layer AMR. With the Nlayer increasing from 6 to 10, the cooling power is improved significantly, which reaches as high as 14.3 W for the 10-layer AMR. To clearly illustrate the relationship between Qc,max and number of layers in AMR, Fig. Qc,max as a function of the numbers of layers. Figure Qc,max of the 6-layer AMR is less than the 8-layer one, while the 10-layer AMR provides the greatest Qc,max. Since the Curie temperatures of the MCM layers of AMRs fit the temperature distribution along the length of the regenerator, the shorter layer length leads to a narrower temperature span in one single layer of 10-layers AMR than AMR with less layers. This allows AMRs to take full advantage of the cooling ability of the individual MCM layers, leading to a higher cooling power. However, when Nlayer = 16, the maximum cooling power drops. As shown in Fig. Qc,max of the 16-layer AMR is less than the 6-layer one. This is because going from 10-layer to 16-layer AMR, the arrangement of the MCM layers becomes denser. This means that the adjacent layers have only 2.5 or 2.25 K temperature difference for the 16-layer AMR, which may lead to a mismatch in the regenerator operating temperature. This mismatch in the regenerator operating temperature range in 16-layer AMR may not provide the optimal total entropy change comparing to the AMR with fewer layers (e.g. 10-layer), which has been reported by Govindappa et al.28. This phenomenon was also observed by Lei et al.34. Their results indicate that the cooling power does not monotonically increase with Nlayer. Since the properties of different layers in their simulation is identical (only shifting by Curies temperatures), it only occurs when the temperature span is low. However, due to the difference of entropy curve for every layer, the phenomenon observed in present work happens in a greater temperature span. Moreover, it implies that an optimal Nlayer exists (here is Nlayer = 10) for the AMRs in present work, that could provide the highest cooling power among the candidate options.Figure U for AMRs with different Nlayer, which illustrates that the COP from the 4-layer regenerator is always less than 1.7 for any utilization. When Nlayer is increased from 4 to 6, 8 and 10, improvement in COP in these regenerators is observed (COPmax from 1.6 for 4-layer to 3 for 10-layer) due to the cooling power enhancement of the AMR with more layers. However, the COPmax in these three regenerators never exceeds 3.1 as shown in Fig. Qc,max and COPmax as a function of U, i.e. a certain value of utilization cannot provide both the highest Qc and the highest COP simultaneously. This trade-off between COPmax and Qc,max also can be observed for the performance variance as a function of Nlayer. Figure Nlayer = 10 is the optimal number of layers for cooling power, it is not the optimum choice for COP; the16-layer AMR offers the highest COP (about 5.5) at U = 0.15. As discussed above, due to the competition between Qc and U for the COPmax is always less than the U for the Qc,max, so COPmax occurs at U = 0.15 or 0.2 for all the AMRs instead of U = 0.4, corresponding to Qc,max. It can be observed that in the range of U = 0.15 to 0.2, except the 4-layer AMR, the cooling powers of the different AMRs are very close. Therefore, the reason for the high COPmax of the 16-layer AMR can be attributed to the low Qc and U and the low max from 16-layer AMR. Also, it is not feasible to obtain a highest Qc and COP from an AMR simultaneously.Figure max of different investigated AMRs with varying number of layers. It can be concluded that significant COP enhancement was achieved by utilizing the 16-layer AMR. As mentioned above, the COP of the 16-layer AMR can reach as high as 5.5, which amounts to 84.6% of the Carnot cycle COP.Figure 32. It is essential to investigate the optimal distribution of the 16-layer parallel plates regenerator since it could provide guidance to achieve higher cooling power and COP. To accomplish this task, a DoE was employed to guide the parameters in numerical experiments and condense the essential number of numerical simulations. After the simulations were conducted following the DoE, a plot was generated to pick the cooling power and COP. Since the total length of the MCM layers is L, which is a constraint on the component proportions, a constrained mixture design was used to assess the influence of the length of every MCM layer on the overall regenerator performance49. Three levels of individual layers’ lengths were considered in present work, which are L, L, and L, while the MCM with uniform length is L. The details of the constrained design are shown in Table L1, L2, …, L16 denote the length of layer # 1, 2, …, 16 in Fig. L in turn with other layers length L, while the runs 17 to 136 set the lengths of two layers as L each in turn with other layers length L. The last run is the reference run in which all layers have the same length, L.Concerns over optimal distribution of MCM layers in multi-layer AMRs have stimulated studies to optimize the MCM layersf = 0.1 Hz and U = 0.15 obtained in Section 1, since the two parameters are the optimized working conditions for COP of all of the 137 runs. The cooling power and COP results from these simulations are compiled in Fig. Ri − Rref)/Rref, where subscript i and ref indicate the ith run from DoE and the reference case, respectively, and R represents either Qc or COP.Following the guidance of the DoE, a set of 137 simulations were conducted for Qc from the DoE cases are close to the reference case as shown by the negative relative difference with small absolute values. Only a few runs return a higher cooling power, which are more than a 10% increase from the reference case. By examining the DoE parameters, it was discovered that every high cooling power result corresponds to the cases with a longer layer# 16, e.g. case 16, 31, 45, 135 and 136 in Table Figure Qc that is as great as the decrease in COP (about 10% for both). This is due to the competition between Qc: the longer layer# 16 not only enhances Qc, but also requires more power to overcome the increasing Qc enhancement, resulting in a lower COP output. Therefore, although the longer layer# 16 provides a higher cooling power, the dramatic COP drop eliminates them from the list of optimization.Moreover, Fig. Qc and a much lower COP compared to the reference case; (2) Although some cooling power benefits could be obtained from some cases with a longer layer# 16, all these cases result in a significant COP drop; (3) only a few combinations of non-uniform layer length are capable of providing COP benefits, which are no more than 1.2% compared to the reference case. Therefore, some optimization work may be required for the previous work with fewer layers MCM; however, this work suggests limited benefit of optimizing the length of every MCM layer for present 16-layer AMR case. This is because the Curie temperatures of the adjacent MCM layers of the 16-layer regenerator are very close to each other. As a result, there is no space to improve the COP.The DoE results can be summarized as: (1) Most of non-uniform layer cases lead to a lower A room temperature magnetic refrigerator with a 16-layer parallel plates regenerator has been numerically investigated in present work. The property data of these 16 MCMs with different Curie temperatures was derived from experimental measurements provided by the material manufacturer. The Curie temperatures were from 268.4 K to 305.4 K with temperature steps 2.25 or 2.5 K. A 1D model was employed based on the previous single layer AMR model, in which a new method was developed to accelerate the model to make it executable for the multi-layer AMR model. Results indicate that the computational time of a 16-layer case is very close to the single-layer case using the new method, while, without the new method, the time required is almost 6 times that of the single-layer case.Qc, max decreases as frequency increases. The same maximum COP with frequency variance is also observed except for the highest frequency case. The results reveal that around f = 0.1 Hz, the 16-layer AMR reaches its highest COP.Using the new model, performance analysis of the 16-layer AMR was conducted between 41 K temperature span and a maximum applied magnetic field of 1.5 T. It was found that for every frequency, there exists a maximum cooling power for the 16-layer AMR. The max, which could reach 84% COP of the Carnot cycle. These results guided an optimization investigation of layer length of the 16-layer AMR. A constrained mixture design, as a kind of Design of Experiments, was employed to generate a series of numerical experiments for the optimization. The design includes 137 simulations. According to the results, it is not essential to use a 16-layer AMR with non-uniform layer lengths, since it cannot find a significant COP enhancement by adjusting the layer lengths in DoE. This is explained because the Curie temperature between the adjacent layers is very close (2.25 or 2.5 K), and there is no room to improve COP by adjusting the space length of the layers.Additionally, a performance comparison between AMRs with different layers is provided in the present work. The results suggest that the AMR with a 10-layer regenerator offers the greatest cooling power. However, the AMR with a 16-layer regenerator can significantly enhance the system COPAll data generated or analysed during this study are included in this published article.


Alpha-1 antitrypsin deficiency (AATD) is an inherited disorder characterized by early-onset emphysema and liver disease. The most common disease-causing mutation is a single amino acid substitution (Glu/Lys) at amino acid 342 of the mature protein, resulting in disruption of the 290–342 salt bridge , protein misfolding, polymerization, and accumulation in the endoplasmic reticulum of hepatocytes and monocytes. The Z allele causes a toxic gain of function, and the E3 ubiquitin ligase gp78 promotes degradation and increased solubility of endogenous ZAAT. We hypothesized that the accumulation of ZAAT is influenced by modulation of gp78 E3 ligase and SVIP  interaction with p97/VCP in ZAAT-expressing hepatocytes. We showed that the SVIP inhibitory effect on ERAD due to overexpression causes the accumulation of ZAAT in a human Z hepatocyte–like cell line (AT01). Overexpression of gp78, as well as SVIP suppression, induces gp78-VCP/p97 interaction in AT01 cells. This interaction leads to retro-translocation of ZAAT and reduction of the SVIP inhibitory role in ERAD. In this context, overexpression of gp78 or SVIP suppression may eliminate the toxic gain of function associated with polymerization of ZAAT, thus providing a potential new therapeutic approach to the treatment of AATD. Alpha-1 antitrypsin (AAT), is a 52-KD globular protein mostly produced in hepatocytes. AAT is the most abundant serum serine protease inhibitor exerting its neutrophil elastase-neutralizing action throughout the body and, in particular, in the lung . SeveralThe ability of a cell to maintain quality control of misfolded proteins is critical for cellular vitality . The accp97/VCP, a member of the AAA (ATPase associated with various cellular activities) ATPase family, participates in protein degradation through interaction with a large number of partners and protein cofactors, such as gp78. The interaction between p97/VCP and gp78 enhances the binding of p97/VCP to polyubiquitinated proteins, such as ZAAT , 24. RecThe highly conserved VIM is important for interaction with p97/VCP. gp78 has a VIM, which allows the two partners to complete the cycle of retro-translocation and ubiquitination. In 2002, Nagahama et al. identified a small p97/VCP-interacting protein (SVIP), which contains the same VIM domain. SVIP has 76 amino acids with two putative coiled-coil regions . SVIP shThis study has been designed to investigate whether SVIP has a regulatory role in gp78-mediated ERAD in hepatocytes with ZAAT aggregation. The data indicate that SVIP regulates the formation of a gp78-p97/VCP complex, thereby changing ZAAT ERAD efficacy. Furthermore, SVIP is upregulated in the presence of ZAAT and inhibits ERAD through gp78-p97/VCP interaction, leading to increased ZAAT accumulation and AATD-associated liver disease.Primers, Dynabeads protein A, Superscript VILO cDNA synthesis kit, VCP siRNA, and SVIP siRNA were purchased from Life Technologies , and X-tremeGENE siRNA and, HP DNA transfection reagent were purchased from Roche Applied Science . The rabbit polyclonal antibodies against AMFR and ubiquitin and anti-rabbit IgG (conformation specific) were purchased from Cell Signaling . The rabbit polyclonal antibody against N-terminus AMFR was purchased from LifeSpan BioScience . Polyclonal anti-rabbit antibody against SVIP and mouse monoclonal antibody against actin were purchased from Sigma-Aldrich  Polyclonal anti-rabbit antibodies against p97/VCP and IP lysis buffer were purchased from Thermo Fisher scientific . Polyclonal anti-rabbit antibody against AAT was purchased from Dako .Anonymized human tissue for this study was obtained through Alpha-1 Foundation DNA and Tissue Bank, University of Florida IRB number 201500842, ClinicalTrials.gov identifier: NCT00884455. Samples where anonymized by Bank personal and then disturbed to the researchers. The reported study was specifically approved by University of Florida IRB#201700065 and entitled Molecular Mechanisms of Alpha-1-Antitrypsin Accumulation in the Liver. Written consent was obtained from all study subjects enrolled the Alpha-1 Foundation DNA and Tissue Bank. The Bank and UF IRB assure that all samples were obtained according to the principles expressed in the Declaration of Helsinki. Three cell lines were initially included in the present study. Our group developed two of the cell lines: AT01 and Hu339. The AT01 (PiZZ) and Hu339 (PiMM) cell lines was derived from human liver tissue. The cells were immortalized with infection of adenovirus-expressing human telomerase reverse transcriptase (hTERT) gene , 35. HowHepatocyte-like MM cell line (Hu339) and hepatocyte-like ZZ cell line (AT01) were cultured in DMEM/F12 with 10% bovine serum and 5% penicillin/streptomycin and were checked for liver cell gene markers, such as alpha fetal protein (AFP), albumin, and AAT. Cells were transiently transfected with the indicated plasmids by the X-TremeGENE transfection reagent, according to the manufacturer’s instructions. Stably-transfected Huh 7.5 cells were generated by selecting for successful integration of the RFP-ZAAT gene using the antibiotic Geneticin. Selection occurs in a 10-cm tissue culture plate, where it takes approximately 2–3 weeks for foci of Geneticin-resistant colonies to appear, after which the resistant foci are clonally expanded over a period of approximately 2–3 weeks.Anonymized human tissue for this study was obtained through Alpha-1 Foundation DNA and Tissue Bank, University of Florida IRB number 201500842, ClinicalTrials.gov identifier: NCT00884455. Samples where anonymized by Bank personal and then disturbed to the researchers. The reported study was specifically approved by University of Florida IRB#201700065 and entitled Molecular Mechanisms of Alpha-1-Antitrypsin Accumulation in the Liver. Written consent was obtained from all study subjects enrolled the Alpha-1 Foundation DNA and Tissue Bank. The Bank and UF IRB assure that all samples were obtained according to the principles expressed in the Declaration of Helsinki.CRISPR  knockouts were created as described previously . BrieflyA total of six explanted liver tissue samples  were used for Western blotting (see IRB protocols listed in Human Tissue and Cell Lines section). Pi stands for protease inhibitor. PiMM is the term for being homozygous for the normal AAT variant, and PiZZ stands for being homozygous for the ZAAT variant. Each sample was lysed using RIPA buffer containing 1:1000 phosphatase inhibitor cocktail and PI EDTA-free from Calbiochem . The concentration of protein lysates was determined by the Thermo Fisher scientific  protein assay kit, according to the manufacturer's instructions, using bovine albumin as a protein standard.5´AAGCTTATGCCGGTGCTCTTCCTCGA3´ and 5´ACGCGTCTAATCTGACCGCTGTGTAGGAA3´ to introduce stop codon TAG before C terminal 135 amino acid. The fragment was cloned into pCR3.1-GP78-3 Flag plasmid to swap GP78 fragment from HindIII and MluI sites to make pCR3.1-gp 78 Δ135. pCR3.1-EGFP-gp 78 was a gnerous gift from Dr. Allan M Weissman from the NIH .The full-length cDNA of gp78 fragment was cut from pCMV6-Entry-AMFR plasmid  by KpnI and NotI sites, which was sub-cloned into the same sites of pTR2-CB-MAAT vector (a kind gift from Dr. Shihong Song’s Lab at the University of Florida) to swap the MAAT gene. The plasmid was digested with Hind III and NotI and cloned into the same sites in pCR3.1 plasmid  to form pCR3.1-gp78 plasmid. pCR3.1-T plasmid (Invitrogen) was self-ligated to form pCR3.1-empty plasmid. The gp 78 Δ135 amino acid fragment was obtained from PCR with primer pair: pCMV6-Entry-SVIP plasmid was purchased from OriGene Technologies, HA-Ubiquitin plasmid and pSpCas9(BB)-2A-GFP encoding Cas9 and sgRNA plasmid were purchased from AddGene , and pCDNA3.1-SVIP mutant with five amino acid mutations  and Myc tag and Flag tag on 3´ end was synthesized and cloned into pCDNA3.1 vector by Life Technologies. The ZAAT with mRFP cloned into BamH1 site was sub-cloned into Hind III and Not I sites of PCR3.1 plasmid (Invitrogen) to make the Z-RFP plasmid. The DNA sequence was confirmed by Sanger sequencing.−ΔΔCt equation.The total RNA was extracted from liver tissue samples and selected cell lines, and the first complementary strand was generated. Expression level of SVIP and p97/VCP gene products were analyzed by quantitative real-time PCR using an Applied Biosystems 7500 fast Real-Time PCR system from Life Technologies and TaqMan Universal PCR Master Mix from Roche Applied Science. Pairs of genes were analyzed simultaneously, and h18S ribosomal RNA was used as an endogenous control. The specificity of the priming and amplification was verified with a melt curve for each amplicon. The quantitative real-time PCR was performed in duplicates, and results were averaged. Results are presented as the relative quantification (RQ) as determined by the 25/well in 6-well plates 24 hours prior to the day of transfection with X-TremeGENE HP DNA transfection reagent  or electroporation  (for plasmids) and X-TremeGENE siRNA transfection reagent (Roche) (for siRNAs). Transfected cells were collected 48 hours post transfection and lysed in RIPA buffer containing 50 mM Tris, 150 mM NaCl, 0.5% SDS, 0.5% sodium deoxycholate, and 1% NP-40. For immunoprecipitation (IP), lysates were incubated with 1 μg of antibody and 50 μL of Dynabeads protein A beads (Novex) for 2 hours at 4°C. Beads were washed three times in buffer containing 1× PBS and 0.1% Triton X-100 before processing for immunoblotting (IB). Total protein was resolved on Tris glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels . Proteins were transferred to nitrocellulose membranes. The blots were blocked and incubated with antibodies overnight at 4°C. The immunocomplex was washed three times and then incubated with either goat anti-mouse or goat anti-rabbit IgG IP-specific secondary antibodies (Abcam) at 1:1000 dilution for 1 hour at room temperature. Proteins were detected by SuperSignal West Dura Extended Duration Substrate Kit (Thermo Scientific).AT01 and Hu339 cells were seeded at 3 × 10AT01 cells were transfected with or without empty plasmid, WT gp78, gp78 Δ135, WT SVIP plasmid. After 48 hours, cells were incubated with 20 μM proteasome inhibitor MG132 for 6 hours and lysed in IP lysis buffer containing 10 mM iodoacetamide. Total ubiquitination was examined by Western blotting for polyubiquitin and actin as a loading control, and ZAAT ubiquitination was assessed by co-IP for ZAAT followed by SDS-PAGE and IB for ubiquitin.PIZZ and PIMM cells were transfected with or without 22 μg WT SVIP plasmid or siSVIP and were preserved in 1% glutaraldehyde in Tyrode’s buffer, rinsed with Tyrode’s buffer, scraped from the culture dishes, and pelleted in centrifuge tubes. Pelleted cells were rinsed with 2-mercaptoethanol in 0.1M Na cacodylate buffer (2-ME buffer), post-fixed in 1% osmium tetroxide in 2-ME buffer, and washed in 2-ME buffer. The samples were dehydrated in a graded series of ethanols and propylene oxide and embedded in epoxy resin . Ultrathin (60–70 nm) sections were counterstained with uranyl acetate and lead citrate and observed using a Hitachi 7600 transmission electron microscope  equipped with a Macrofire monochrome progressive scan CCD camera  and AMTV image capture software .35S] methionine  [Nearly confluent monolayers of AT01 cells were co-transfected with eukaryotic expression vectors containing Z-RFP and SVIP or empty plasmid in 100-mm diameter culture dishes. The cells were incubated for 30 minutes with [ham, MA)  and thenAAT knock out cells were co-transfected with 1 μg Z-RFP and 2 μg empty vector, gp78-EGFP plasmid , SVIP, op < 0.05 was considered statistically significant.All results are expressed as mean ± S.E. Statistical analysis was performed using Prism 6 software program. Values of gp78 enhances the interaction of misfolded proteins with p97/VCP and facilitates their degradation . To asseTo examine the AAT degradation facilitation by the overexpression of gp78, the level of ubiquitinated AAT was detected in AT01 cells co-transfected with 2 μg ZAAT plasmid and WT gp78. At 48 hours post transfection, cells were incubated with 20 μM proteasome inhibitor MG132 for 6 hours and subjected to co-IP for AAT, followed by WB using ubiquitin antibody. MG132 caused a significant accumulation of polyubiquitinated AAT in WT gp78-transfected cells as compared with control cells . To inveProteins that fail to meet conformational standards, such as misfolded proteins, are selected and ultimately degraded by the ubiquitin-proteasome system of ERAD . gp78 reTo assess whether gp78 had a role in the degradation of ZAAT, we transfected WT gp78 into Huh 7.5 knockout cells stably expressing RFP-ZAAT. At 48 hours post transfection, the cells were processed to determine the levels of ZAAT by pulse chase experiments. As compared with the control cells, gp78 overexpression significantly decreased the level of accumulated ZAAT and caused faster ZAAT clearance from the ER without significant changes in the level of extracellular ZAAT . Then, wp97/VCP is a known chaperone that inhibits protein aggregation. To assess the association between gp78 and p97/VCP in control and deficient liver tissue samples, 10 μg of total lysate was subjected to IB to determine and confirm the high level of antitrypsin and ER stress in deficient samples . AAT aggAT01 (PiZZ genotype) and Hu339 (PiMM genotype) are immortalized cell lines from human liver. Although these cell lines sufficiently proliferate, they display a mesenchymal phenotype, express low levels of AAT, and do not aggregate AAT compared with Huh 7.5 cells; Huh 7.5 cells are a hepatocyte-derived cellular carcinoma cell line that the good levels of hepatocyte-specific functions . We usedTo determine the role of SVIP in AATD, first we examined the expression level of SVIP in normal and AATD human liver tissues and Hu339 and AT01 cell lines by qPCR and IB. The accumulation of misfolded ZAAT using pTR2-CB-ZAAT in the ER of the Hu339 cell line significantly upregulated SVIP; this was comparable to AT01 cells, which show an elevated level of SVIP protein. This result is consistent with a previous report that ER stress upregulates SVIP, which is expected to inhibit ERAD [In our real time qPCR analysis, SVIP mRNA expression was relatively high in the AT01 cell line and Hu339 transfected with ZAAT, compared with control Hu339 samples . SVIP mROverexpression of SVIP induces cellular vacuolation, which represents enlarged ER . There a35S pulse chase analysis in AT01 cells transfected with the Z-RFP plasmid with overexpression of SVIP, as well as stable RFP-ZAAT–expressing Huh 7.5 cells. Overexpression of SVIP significantly inhibited ZAAT degradation in both cell lines with no significant changes in extracellular level of ZAAT  compared with WT gp78. The gp78 mutation in the VIM domain could not enhance clearance of ZAAT aggregates. Moreover, p97/VCP co-immunopreciptates with ZAAT in the presence of WT gp78 but not gp78 Δ135. This result sugests that gp78 and p97/VCP are involved in ZAAT degradation and the VIM motif of gp78 is necessary to form the complex.In this study, we also demonstrated that SVIP, a previously identified p97/VCP-interacting protein, negatively regulates gp78-mediated ZAAT degradation. This report may reveal a few clues with respect to ZAAT degradation. Our current study using liver tissue samples shows higher levels of SVIP in liver samples from AATD individuals, as well as in a PiZZ liver cell line. This phenotype is seen in the cells under prolonged ER stress. SVIP is an endogenous ERAD inhibitor  expresseIn addition, overexpressing SVIP reduces clearance of ZAAT, and knocking down SVIP increases ZAAT clearance in presence of p97/VCP. Inhibition of p97/VCP by siRNA also stabilizes ZAAT. Therefore, we demonstrate that clearance of ZAAT can be accomplished by overexpressing gp78 or by knocking down SVIP in the presence of functional p97/VCP.SVIP causing vacuolation is a very interesting cytopathic effect . In a prIn the present study, we demonstrate that the high level of SVIP competes with gp78 to interact with p97/VCP, attenuates the interaction of gp78 and p97/VCP, and inhibits ZAAT retro-translocation and ERAD. In summary, our results demonstrate that SVIP may be a regulator of the gp78-mediated ubiquitin-dependent degradation of ZAAT in hepatocytes. We propose that p97/VCP can form a complex with either gp78 or SVIP. SVIP is a negative regulator of gp78-p97/VCP–mediated ERAD. We believe that the misfolded and aggregated ZAAT affects the stoichiometry between these complexes and establishes a positive feedback cycle that reduces degradation of ZAAT. As intracellular ZAAT inclusions are believed to be hepatotoxic and the main cause of early liver disease in PiZZ individuals, clarifying the molecular mechanisms associated with gp78-mediated ERAD may lead to the identification of novel therapeutic targets.Nazli Khodayari, Rejean liqun Wang, George Marek, Karina Krotova, Mariana Kirst, Chen Liu, Farshid Rouhani, and Mark Brantly declare that they have no conflict of interest.


Mechanistically, we found that TIMP2/3, which are critical metastasis inhibitor of prostate cancer, were down-regulated by DANCR synergistically with EZH2 through epigenetically silencing their promoter by chromatin immunoprecipitation assay. In addition, we further investigated whether DANCR is regulated by the differentiation-promoting androgen-androgen receptor (AR) pathway and found that DANCR expression is repressed by androgen-AR; furthermore, DANCR impedes the upregulation of TIMP2/3 and the suppression of invasion and migration by androgen-AR. On the other hand, interestingly, we found that in prostate cancer cells DANCR knockdown decreased the promotion of invasion and migration by the treatment of enzalutamide, which is an AR inhibitor. In summary, our results indicate that DANCR promotes prostate cancer invasion and metastasis through repressing the expression of TIMP2/3, and suggest that DANCR could be a potential target for preventing prostate cancer metastasis, and knockdown DANCR may lessen the potential side effect of AR inhibitor.LncRNA DANCR suppresses differentiation of epithelial cells, however, its function in prostate cancer development is still unknown. In the present study, we found the expression of DANCR increases in prostate cancer tissues and cells compared to normal prostate tissues and cells, moreover, DANCR promotes invasion and migration of prostate cancer cells  Prostate cancer is one of the most prevalent solid tumor and a leading cause of cancer-related deaths among males in the U.S., with an estimated 29,720 deaths in 2013 –3. In prLong Noncoding RNAs (lncRNAs) are a class of noncoding RNAs which are longer than 200 nucleotides without evident protein coding function , and thein vitro and in vivo. Moreover, we determined whether DNACR regulated cell invasion synergistically with EZH2 and whether DANCR affected the effect of androgen-AR signaling and enzalutamide, an inhibitor of AR, on migration and invasion of prostate cancer cells. Finally we found that DANCR could be a potential target for preventing the metastasis of prostate cancer.Given the importance of lncRNAs in prostate cancer, in the present study, we investigated the lncRNA DANCR (Differentiation Antagonizing Non-Protein Coding RNA), which suppresses the differentiation of epidermal cells  and promhttp://www.ncbi.nlm.nih.gov/geo/). Furthermore, elevated expression of DANCR was associated with high-grade tumors (Gleason score 5 samples versus Gleason score 3 samples) . To confirm the result from the GEO database analysis, we detected the expression of DANCR by RT-qPCR in clinically collected human prostate adenocarcinoma specimens and their paired adjacent normal tissues using laser capture micro-detection, and we found that DANCR level in prostate cancer specimens are much higher than that in adjacent normal tissues  than that in normal prostate samples (n = 29) [From the Oncomine database, we found that lncRNA DANCR is up-regulated in prostate cancer. To determine the expression of DANCR in prostate cancer, we first searched multiple microarray data in the GEO database. As shown in Figure s Figure . These r(n = 29) . In addiTo further determine the function of DANCR in prostate cancer, lentivirus containing short hairpin RNA (shRNA) against DANCR was infected into androgen- independent C4-2B and CW22Rv1 prostate cancer cells and empty vector-containing lentivirus was used as control. As shown in Figure Since DANCR promoted cell invasion of C4- 2B and CW22RV1 cells, we further investigated the mechanism of how it regulates target genes. We examined the expression of 37 invasion related genes in DANCR knockdown C4-2B cells by RT-qPCR assay and found that DANCR knockdown significantly changed the mRNA level of MMP2, MMP9, TIMP2, TIMP3, SERPINB and TM4SF1 Figure . ConsideTo further confirm that EZH2 is necessary for DANCR to inhibit TIMP2/3, we used 3-Deaza-naplanocin A (DZNep), an inhibitor of EZH2, in C4-2B cells and CW22Rv1 cells and we detected the expression of TIMP2/3 and examined the invasive and migratory activity in DANCR knocked down cells and control cells. As shown in Figure in vitro analysis, further confirming that DANCR represses the expression of TIMP2/3 and promotes the metastasis of prostate cancer cells.To further determine whether DANCR promotes metastasis of prostate cancer, CW22Rv1-shDANCR or CW22Rv1-shNC cells were injected into nude mice by tail vein. More lung metastatic foci were detected in shNC group compared with the shDANCR group when mouse died or 8 weeks after injection by histological analysis Figure  and 5B. Androgen receptor (AR) plays a critical role in the development and treatment of prostate cancer. It was previously documented that AR is an inhibitor of prostate cancer metastasis –21. To iWe further investigated whether DANCR affects AR function on the invasion of prostate cancer cells. Through western blot analysis and RT-qPCR assay, we detected TIMP2/3 expression at both protein level and mRNA level in C4-2B and CW22Rv1 cells with DANCR knockdown and DHT treatment, and found that TIMP2/3 were upregulated by DHT treatment and a higher basal mRNA level of TIMP2/3 in DANCR knockdown groups Figure . MoreoveAndrogen deprivation therapy, which disrupts AR signaling through androgen ablation or AR antagonists, is the first-line treatment for disseminated prostate cancer. Enzalutamide (MDV 3100), an AR inhibitor, is widely used with prostate patients. However, it has been reported that enzalutamide promotes prostate cancer metastasis in different models , 23, morin vitro and enhanced metastasis of xenograft prostate tumor in mouse model. Mechanically, we found that TIMP2/3 are target genes of DANCR and knockdown of DANCR leads to up-regulation of TIMP2/3, decreased binding of EZH2 and H3K27me3 on the promoter of TIMP2/3. Furthermore, we confirmed that DANCR repressed expression of TIMP2/3 synergistically with EZH2. In summary, our results indicate that DANCR promotes invasion and metastasis of prostate cancer, and DANCR could be a potential target for the treatment of prostate cancer.In the present study, we found that lncRNA DANCR expression increased in prostate cancer, moreover, DANCR promoted invasion and migration of prostate cancer cells TIMP2/3 promoter, indicating that DANCR may mediate the binding of EZH2 on them. On the other hand, we also found that DANCR and EZH2 synergistically repressed the expression of TIMP2/3, suggesting that the epigenetically silencing by EZH2 could play an essential role in the repression of TIMP2/3 expression by DANCR. Notably, it is possible that DANCR may activate EZH2 beyond its enzymatic activity since lncRNA can function as a scaffold to bind and bring transcription regulators to the promoter region of the target genes [Expression of EZH2, which functions as an epigenetic gene silencer, increases in prostate cancer and it plays an oncogenic role in prostate cancer that is typically associated with increased risk of metastasis and recurrence –27. In tet genes , and EZHet genes –29. ThusAndrogen-AR signaling pathway is crucial in driving the terminal differentiation of prostate epithelial cells –31. InteRecent studies, utilizing specific cell surface markers and genetic lineage tracing approaches, have produced direct evidence that prostate stem cells are most likely AR-negative and it seems that androgen-AR pathway suppresses the stem cell phenotype and promotes cell differentiation –34. ConsAlthough DANCR expression elevates in prostate cancer, the clinical relevance between DANCR and prostate cancer is still not clear. More human specimens are needed to clarify whether DANCR can be used as diagnostic and prognostic marker of prostate cancer. Moreover, several lncRNAs, such as PCA3 and MALAT-1, can be used as diagnostic urinary biomarkers for prostate cancer –36, thusin vitro and metastasis of tumor xenografts in nude mice, and decreases expression of TIMP2/3 synergistically with EZH2 through epigenetically silencing their promoter; moreover, DANCR expression is repressed by androgen-AR signaling pathway and DANCR knockdown facilitates the upregulation of TIMP2/3 and the suppression of invasion and migration by androgen-AR, while DANCR knockdown decreased the promotion of invasion and migration in prostate cancer cells by enzalutamide treatment. Our results suggest that DANCR could be a potential target for preventing prostate cancer metastasis, and knockdown DANCR may lessen the potential side effect of AR inhibitor.In conclusion, our results indicate that lncRNA DANCR promotes invasion and migration of prostate cancer cells 2. DNZep and enzalutamide (MDV3100) were purchased from Selleck Chemicals . DHT (dihydrotestosterone) were purchased from Sigma-Aldrich . All the reagents were reconstituted and stored following the protocol.Human prostate cancer cell lines CWR22Rv1 and PC-3 were purchased from American Type Culture Collection . C4-2B cell lines were a gift from Dr. Jer-Tsong Hsieh of University of Southwestern Medical Center. The three cell lines are all cultured in RPMI1640 medium supplemented with 10% fetal bovine serum  at 37°C, in humidified air containing 5% of COHuman prostate cancer specimens were obtained from the Health Sciences Tissue Bank at the University of Pittsburgh Medical Center under approval by the UPMC Institutional Review Board following a standard protocol. All the specimens were from patients with prostate cancer with Gleason score 7 or above. The tissue process for laser-capture microdissection and mRNA level detection procedure were described previously , 38. A dCells were washed once with cold PBS and lysed in RIPA buffer  containing protease inhibitors. Approximate 30 μg of protein was separated with 8–12% SDS-PAGE gel and blotted onto nitrocellulose membranes. Then membranes were blocked with 5% skim milk at room temperature for 1 hour and then incubated with primary antibodies against GAPDH , TIMP2, TIMP3 and androgen receptor (AR)  at 4°C overnight, followed by TBST wash and 1 hour incubation with horseradish peroxidase-conjugated secondary antibodies at room temperature. Protein bands were visualized by a Molecular Imager ChemiDoc XRS System .−ΔΔCt method. The primer sequences were given as followings: 18S, ; DANCR, ; PSA, , TIMP2, , TIMP3, , GAPDH, .Cells were harvested with Trizol reagent  to extract total RNA, which was reverse-transcribed to cDNA using PrimeScript™ RT reagent kit . Then the cDNA was studied using CFX96 real-time PCR system  using SYBR Green PCR Master Mix  to determine the transcriptional expression of specific genes. GAPDH was used for normalization. Relative gene expression was calculated by the 2LV5 lentiviral vectors encoding short hairpin RNA (shRNA) targeting non-specific control (NC) or human Lnc RNA DANCR (5′- GGAGCTAGAGCAGTGACAATG -3′) were constructed by GenPharma . The siRNAs targeting Lnc RNA DANCR and non-specific control (NC) were purchased from RiboBio . Cells achieved 70–80% confluence for lentiviral transfection or 30–50% confluence for siRNA transfection, and were transfected with X-tremeGENE siRNA Transfection Reagent  for 2–3 days, and harvested for the next experiments.in vitro invasion assays, the upper chambers of transwell were pre-coated with diluted matrigel , while for in vitro migration assays, no matrigel was used. 105 × cells were seeded onto upper chamber in serum free medium and medium containing 10% serum were added to the lower chamber as a chemoattractant. After incubation for 24 h, the upper surface of the insert was wiped with a cotton swab and cells that migrated to the lower surface were fixed by 4% paraformaldehyde and stained with crystal violet. Cell numbers were counted in 6 random fields (200 ×) per well. Quantitation indicates mean ± SEM of triplicate repeats.Transwell migration and invasion assays were performed in 8-μm-pore transwell inserts . For Prostate cancer C4-2B or CW22RV1 cells with stably knocked down DANCR were seeded onto six-well plates. When the cell density reached above 90%, scratch wounds were made by scraping the cell layer in each culture plate using the tip of a 200-μl pipette. Then the cells were washed with PBS and cultured in serum-free medium with different treatment times, and then five fields (×100) were randomly chosen from each scratch wound and visualized by microscopy to evaluate the ability of cell migration. The experiments were performed in triplicates.® Enzymatic Chromatin IP Kit (Magnetic Beads) from Cell Signaling Technology  following the manufacturer's protocol. Antibodies against EZH2 (CST), H3K27me3  or normal rabbit IgG were used to precipitate protein/DNA complex. Precipitated DNA was analyzed by PCR with region-specific primers [ChIP assays were performed using SimpleChIP primers : TIMP2-sOligonucleotides of the specific-region sets of TIMP2 and TIMP3, with biotin labeled on the 5′-end, were synthesized by GENEWIZ . The oligonucleotide sequences were as followings: TIMP2-set2 , TIMP2-set3 , TIMP3-set1 , TIMP3-set2 , TIMP3-set3 . Each DNA fragment was amplified by PCR and purified with standard protocol. DANCR was knocked down in prostate cancer cells before cells were lysed. Procedures for pulling down DNA-bound proteins were described previously –40. FinaProstate cancer cells were lysed with RIPA buffer containing RNase inhibitor  and cell lysates was incubated with biotin-labeling oligonucleotides for the specific-region sets of TIMP2 and TIMP3 overnight at 4°C. Then the biotin-oligonucleotides-protein-RNA complexes were pulled down with ImmunoPure streptavidin-agarose beads  and the complexes were freed from streptavidin-agarose beads by using 0.1 M biotin. The solution containing the complexed was treated with DNase I (CWBio) and Proteinase K (CWBio) before the RNA extracted with Trizol reagent, and then the RNA was reverse-transcribed to cDNA and PCR was performed to detect DANCR, and the primer sequences are as followings: F:5′-GCCACTATGTAGAGGGTTTC-3′, R:5′-AC-CTGCGCTAAGAACTGAGG-3′.6 CWR22Rv1-shDANCR or CWR22Rv1-shNC cells were injected into seven male, 5 weeks old nude mice through tail vein, and 8-weeks later, mice were sacrificed and the metastases were examined by H&E staining. All animal studies were performed under the supervision and guidelines of the Institutional Animal Care and Use Committee of the Medical School, Xi'an Jiaotong University .CWR22Rv1 cells were stably knocked down DANCR and 4 × 10The Immunohistochemistry staining were performed with EnVisionTM System , and the slides were de-paraffinized, rehydrated, followed by 5 min antigen retrieval, 10 minutes of endogenous enzyme block, and incubated with primary antibody  overnight at 4°C. Then the slides were incubated with EnVision-HRP secondary antibody for 1 hour and the signal were detected by diaminobenzidine (DAB), followed by hematoxylin counterstaining. Staining signals were photographed using an Olympus BX51 Microscope .t test was used when two groups comparison. P < 0.05 was considered statistically significant.All the statistical analyses were performed by GraphPad Prism (vesion5.0) software, and Student's


Shigella species, a priority AMR organism, in men who have sex with men to gain insight into AMR emergence and spread. Using genomic epidemiology, we show that repeated horizontal transfer of a single AMR plasmid among Shigella enhanced existing and facilitated new epidemics. These epidemic patterns contrasted with slighter, slower increases in disease caused by organisms with vertically inherited  AMR. This demonstrates that horizontal transfer of AMR directly affects epidemiological outcomes of globally important AMR pathogens and highlights the need for integration of genomic analyses into all areas of AMR research, surveillance and management.Horizontal gene transfer has played a role in developing the global public health crisis of antimicrobial resistance (AMR). However, the dynamics of AMR transfer through bacterial populations and its direct impact on human disease is poorly elucidated. Here, we study parallel epidemic emergences of multiple  Shigella species is a public health concern. Here, Baker et al. show how horizontal transfer of an AMR plasmid among Shigella species contributes to epidemics in men who have sex with men in England.Antimicrobial resistance (AMR) in  Despite this inferred importance of HGT in global AMR emergence, the real-time epidemiological impact of HGT on AMR emergence has never been observed so is poorly understood. Observations have been made that are suggestive of HGT of AMR 5, and such evidence would be enhanced by strong epidemiological data to support the plausibility of HGT occurring, and information on the epidemiological fate  of the newly formed pathogen-AMR combination. Thus, to understand the impact of HGT in the emergence of new antimicrobial resistant pathogens, it is essential that we study HGT alongside associated epidemiological information, and at the population level, where epidemiological outcomes are less stochastic.Bacterial genomics has revealed that antimicrobial resistance (AMR) phenotypes are conferred largely by either de novo mutation in the chromosome or the acquisition of new AMR genes through horizontal gene transfer (HGT), both of which can then be vertically propagated. The importance of HGT in the current global AMR crisis is evidenced by repeated observations of a new AMR gene in one organism in one location being followed by global reports of the gene in other organisms and geographical areas. This has happened for the carbapenemase-encoding Shigella in MSM is a relevant model for studying AMR emergence owing to features of both the pathogen and its AMR, as well as features characteristic of the epidemiological community of MSM. Shigellae cause a large burden of disease and are globally distributed. They are one of the most common causes of childhood diarrhoeal disease in low-to-middle income nations and also cause disease in high income nations often, but not always, linked with travel7. In addition to its concerning disease burden, shigellae are increasingly resistant to antimicrobials 9 and ciprofloxacin-resistant Shigella, a phenotype conferred by mutations in the Quinolone Resistance Determining Regions (QRDR) of the chromosome, is recognised by the World Health Organisation as a priority AMR pathogen10. Within the epidemiological community of MSM in England there are dense transmission networks of individuals with similar risk profiles  currently permissive of pathogen epidemics, including some that are highly resistant to antimicrobials. This permissiveness is evidenced by recent, simultaneous epidemics of multiple sexually transmissible illnesses (STIs) in this community, including Lymphogranuloma venereum, syphilis, and gonorrhoea, the latter of which has become highly multi-drug resistant16.Shigella has emerged as a sexually transmissible enteric infection in MSM that is capable of global dispersal and is associated with AMR18. An MSM-associated (MSMA) S. flexneri 3a lineage that emerged epidemically in England in 2009 has since disseminated across four continents through international transmission among MSM20, and successful sublineages of this MSMA lineage carried azithromycin resistance on the conjugative plasmid pKSR10019. Since 2012, England has experienced two further, parallel epidemics of domestically acquired shigellosis thought to be associated with MSM .In addition to these more traditional STIs, Shigella epidemics in a densely connected epidemiological community to elucidate the role of HGT on the epidemiology of AMR emergence and spread. We show that HGT of a single azithromycin resistance plasmid enhanced and facilitated epidemic emergence of multiple Shigella species. This demonstrates the importance of tracking the genetic context of AMR within pathogen populations.Here, we study these parallel S. flexneri 2a and S. sonnei isolates and associated epidemiological metadata were selected from the national archive of isolates held by the national reference laboratory at Public Health England collected between 2008 and 2014. The national archive contains all S. flexneri and ~ 70% of S. sonnei isolated in hospital, private and regional laboratories , and basic epidemiological data were extracted from sample submission forms, i.e., region, gender, patient age and travel history where noted (as in22). The time-period selected encapsulated three parallel epidemic emergences of Shigella sp. in England, and isolates were subsampled proportionately to the number of cases by year with a greater weighting of domestically acquired infections (non-travel-associated isolates) and patient age stratification (16–60 year olds only) for S. sonnei owing to the large number of isolates of this species (Table n = 366) were whole-genome sequenced (as in ref. 17) and whole-genome phylogenies were constructed  of isolate designation to an MSMA sublineage with recent (within 1 month of diagnosis) patient MSM activity (Methods). The MSMA sublineages were designated major and minor in S. flexneri 2a and 1–4 for S. sonnei . For S. sonnei, it was also possible to explore persistence of MSMA sublineages in England over time. This persistence was evaluated by combining the 2008–2014 representative subsample with data from isolates of MSMA shigellosis clusters detected England in 2015/201622  was assessed. The French and English MSM epidemiological communities are known to be connected as they were previously shown to have phylogenetically admixed isolates of the intercontinentally distributed sublineage of MSMA S. flexneri 2a and sublineage 3 of S. sonnei performed poorly in their natural transmission setting relative to the other MSMA sublineages. In addition to causing a comparatively low number of cases and not persisting through to 2015–16 in UK MSM, they were not found in France, and were also the only two MSMA sublineages not observed in the final years of the sampling window, despite having been the earliest two sublineages in existence . For both species, vertically inherited triple QRDR mutations responsible for resistance to ciprofloxacin were exclusively associated with monophyletic sublineages related to travel from Asia  is thought to have arisen consequent to high treatment rates for other STIs commonly found in the shigellosis-affected MSM subpopulation25, rather than from empirical treatment for shigellosis. This ‘collateral damage’ hypothesis is supported by recent treatment reviews (both from the United Kingdom and the United States), which suggest that upwards of 80% of MSM with shigellosis receiving antimicrobial therapy are treated with ciprofloxacin26.To investigate the association of population-level success with AMR, genetic determinants of AMR were characterised and correlated with phylogeny. Specifically, this included the acquired resistance gene content and the presence of QRDR mutations  had minimum inhibitory concentrations (MICs) of ≥64 mg/l, whereas isolates containing only the mphA gene (n = 13) had MICs ≤32 mg/l and isolates with neither gene (n = 43) had MICs of ≤2 mg/l . Similarly, S. sonnei MSMA sublineages that were still present in the MSM transmission network in 2015/2016 and/or were present in France  also contained pKSR100. Contrastingly, the two MSMA sublineages that did not contain pKSR100 (the minor sublineage of S. flexneri 2a and sublineage 3 of S. sonnei) did not achieve such markers of population-level success , is the possibility that the increased success of pKSR100-containing sublineages reflect that different genetic backgrounds of important resistance genes also have a role in determining the population-level success of a pathogen. Differential fitness of mobile genetic elements in bacterial hosts is well established in vitro28, but efforts to link in vitro fitness with epidemiological, population-level outcomes at a hospital-level have been unsuccessful29. Although recapitulation of population-level success in vitro is unrealistic, these results frame an exciting line of future in vitrowork with the collection of clinically relevant organisms in our study (and others relevant to HGT of AMR as they arise), that may be useful in interpreting the apparent impact of mobile genetic element carriage in determining pathogen population-level outcomes.The exact reason behind the differential population-level success in pKSR100-containing sublineages is unknown. However, the two most likely explanations both have important consequences for public health. First, it is possible that the increased success of pKSR100-containing sublineages is attributable to the increased azithromycin resistance conferred. In which case, these results point to an epidemiologically relevant division of azithromycin resistance for Shigella MSMA sublineages. The exact route of transfer is unknown, but three non-mutually exclusive scenarios exist: 1 the HGT occurred directly between Shigella sublineages in patients simultaneously infected with multiple types; 2 during Shigella infection, pKSR100 entered another bacterial host  permitting subsequent transfer to an alternate Shigella sublineage on repeat infection of that individual; 3 an alternate bacterial host that contains pKSR100 (possibly acquired during a prior Shigella infection) was transmitted onward to another human host who was infected (or subsequently infected) with an alternate Shigella sublineage . In the first two scenarios, infections of patients with multiple types of Shigella is necessary. Among our samples, we found ten patients with repeat Shigella infections, including nine with repeat infections of MSMA sublineages, and two patients infected with different species of Shigella  were associated with three pKSR100 variants , and our subsample is only a representative quantitation of gene flow, our proposed combined genomic epidemiological model indicated that at least eight horizontal transmission events of pKSR100 occurred among genetically-distinct MSMA sublineages. This hypothetical model is summarised both statically  the three pKSR100-containing S. sonnei MSMA sublineages  In capturing the population-level epidemiological consequences of horizontal AMR transmission we have revealed heretofore unseen insights into AMR emergence and spread in important pathogens. Here, horizontal transmission of AMR among pathogenic bacterial species caused fundamental epidemiological shifts, including enhancing emerging epidemics and facilitating epidemics of previously circulating pathogens that were waiting for the ‘right’ AMR determinant to come along. These dramatic shifts contrasted with the incremental increases in pathogens carrying chromosomally encoded  AMR, and demonstrated how these two types of resistances  can combine to form new highly resistant sublineages. It is important to consider that these overarching observations about AMR emergence were made within a specific patient subpopulation known to have increased connectivity and antimicrobial usage . However, the genomic processes in play here and their impact on pathogen epidemiology are likely translatable to the broader trends responsible for elaborating the global AMR crisis.31. However, there is a clear and valuable lesson here for future AMR work. It is no longer enough to understand and trace AMR pathogen combinations as simple taxonomic compartments , as the genomic diversity contained within such compartments, and genetic exchange among them, also contributes to epidemiological outcomes . Clearly, we must continue to weave the sub-genomic study of pathogens into all facets of AMR research, surveillance and management, and work to determine the compartments, barriers and quantity of gene flow among pathogenic and non-pathogenic, as well as antimicrobial resistant and antimicrobial susceptible bacterial species.Of course, factors other than AMR, including changes in sexual behaviour and population immunity, will have also shaped these epidemics, and in truth the English MSM shigellosis epidemics are all but gone for nowTo infer association of sublineages with MSM, the number of patients who were male, who had low-risk travel histories, and who were between 16 and 60 years old were calculated (for each category) and compared with those numbers among the entire (within species) data set among the UK representative subsample. These proportions were then compared using a two-tailed Fisher’s exact test .To confirm our classification of phylogenetic sublineages as being MSMA, epidemiological interview data from patients whose isolates were submitted through Institut Pasteur were used to explore the association between recent MSM activity and the phylogenetic classification of isolates. These data are being collected as part of a public health enquiry into an ongoing outbreak of azithromycin-resistant shigellosis in France. Contact details for male patients without a recent history of high-risk travel , aged 16–60 years old were retrieved from clinical laboratories and interviewed by telephone by a Santé Publique France epidemiologist (who was blinded to the phylogenetic classification of the patient’s isolate). Of 57 patients that fit these criteria, attempts were made to contact 34, and 13 interviews were successfully completed. Interviews comprised a standard questionnaire to retrospectively identify travel history and sexual exposure ; sex with female partner(s); sex with both male and females partner(s); no sex partners; unknown; refused to respond) in the month prior to diagnosis. In total, 9 of 10 (90%) patients reporting recent MSM activity within the month prior to diagnosis had isolates belonging to MSMA sublineages and 3 of 3 (100%) patients not reporting such activity had isolates outside of these lineages. This resulted in a significant association of recent patient MSM activity with isolates belonging to MSMA lineages (32. Azithromycin-resistance gene-containing contiguous sequences were identified using Resfinder33. The length (in bp) of these contiguous sequences was extracted and correlated with phylogenetic position to identify clusters sharing the same azithromycin resistance determinant. Additional AMR genes co-inherited with the azithromycin-resistance genes . SMRT-sequenced AZM resistance determinants were unavailable for two sublineage representatives as the S. sonnei sublineage 1 representative failed to regrow for extraction, and on comparative genetic analysis, the plasmid from S. sonnei sublineage 3 representative had been lost. In the comparative genetic results for these sublineages  of all UK representative subsample isolates and the MSMA S. flexneri 3a sublineage was used to identify the presence and count of 86 pKSR100 orthologues among isolates. Those isolates containing ≥60 orthologues were mapped to a concatenated Shigella genome and pKSR100 pseudomolecule (as in ref. 19) and those with ≥90% coverage over the pKSR100 region were deemed to contain pKSR100  was performed with fsm-lite39. Population structure estimation was made using mash40 and converted into a three-dimensional distance matrix using scripts inbuilt to SEER39. Associations of kmers were then run with SEER and filtered for positive effect size; a minor allele frequency of >5%; an unadjusted pvalue of <10e-5; and an adjusted pvalue of 10e-8. Filtered kmers were then converted to fastq format, mapped to a draft assembly of an S. flexneri 2a major sublineage isolate, converted to a Manhattan plot (again using SEER inbuilt scripts) and visualised with Phandango41. This revealed a significant association of MSMA sublineage designation with pKSR100  t-test  and/or in-agar dilution using a frozen azithromycin reference panel with concentrations ranging from 1 to 256 μg/ml. In-agar antimicrobial susceptibility testing was performed according to Clinical Laboratory Standards Institute methods, with Staphylococcus aureus ATCC 29213/ NCTC 12973 used as a quality control strainE. coli O117:H7 isolated in 2014 from the outbreak was sequenced using SMRT sequencing as above, and de novo assembly was compared with azithromycin resistance encoding plasmids from MSMA Shigella sublineages in this study. This showed that the S. sonnei sublineage 3 plasmid was present in the E. coli outbreak isolate : a Bayesian-inferred phylogenetic tree of S. flexneri 3a based on 1243 variable sites (from ref. 19); the tree presented in Fig. S. flexneri 2a and the S. sonnei whole-genome phylogeny incorporating the MSMA clusters from the UK in 2015/16  as in ref. 19  and median . The project was reviewed and approved by the PHE Research Support and Governance Office and Caldicott Panel deemed to comply with public health surveillance standards. For data relating to isolates from Institut Pasteur: Data collected were part of an ongoing public health investigation into an ongoing outbreak of azithromycin-resistant shigellosis and as such were not subject to ethical approval (see Methods).All sequencing and genome assembly data for isolates in this study are available under project number PRJEB12097 in the European Nucleotide Archive. Further detail linking isolates and sample accession numbers within the project are available in Supplementary Data Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Data 1