Ben Hamdi

Shikano Yahia

Etienne Masahiro

Avdeev Gaudin

Chris D Maxim

Ling

Hamdi Ben Yahia
email: benyahia.hamdi@voila.fr

Masahiro Shikano
email: shikano.masahiro@aist.go.jp.

Etienne Gaudin

Maxim Avdeev

Chris D Ling

Single crystal and magnetic structures of maricite-type AgMnVO4

Keywords: Vanadate, Flux growth, Single crystal, Magnetic structure

come

Introduction

Phosphates with the general formula ABPO4 (A = K, Rb and Cs; B = transition metal) crystallize mainly in two different structural types, i.e., stuffed-tridymite and zeolite-ABW. Members of these families are characterized by a BPO4 -tetrahedral framework, which can show four-, six-, or eight membered rings. Sometimes, the transition metal is found at a trigonal bipyramidal site. In the more condensed phases ABPO4 (A = Li, Na; B = transition metal) with olivine and maricite-type structures, the transition metal is located at octahedral sites.

It is of interest to compare the structural evolution between homologous phosphates and vanadates, AMnPO4 and AMnVO4, respectively. In the AMnPO4 phosphates series, LiMnPO4 [1] NaMnPO4 [2] KMnPO4 [3] and CsMnPO4 [4] have been studied and their structures determined. Recently, we reported the structural and magnetic properties of RbMnPO4 [5,6] and AgMnPO4 [7]. In the homologous AMnVO4 vanadates, LiMnVO4 [8] is known to crystallize in the Na2CrO4-type structure.

Recently, the AMnVO4 vanadate series has been extended by the addition of AgMnVO4 [9], CuMnVO4 [10], NaMnVO4 [11], KMnVO4 [12], and RbMnVO4 [9]. AgMnVO4 and NaMnVO4 crystallize in the maricite-type structure and contains MnO4 chains made up of edge-sharing MnO6 octahedra. CuMnVO4 crystallizes in the Na2CrO4-type structure and also contains MnO4 chains made up of edge-sharing MnO6 octahedra. Surprisingly, KMnVO4 crystallizes in a new type of oxygen-deficient perovskite, and RbMnVO4 is the first vanadate crystallizing in the stuffed tridymite-type structure [9].

All the crystal structures of the AMnVO4 vanadate series have been solved from single crystal data, except AgMnVO4. Its structure had so far only been characterized on the basis of powder X-ray diffraction data, since this compound has an incongruent melting point and decomposes above 750°C [9]. Herein we report on crystal growth studies and a precise single crystal structure refinement for AgMnVO4. Furthermore, we have solved the magnetic structure from low temperature neutron powder diffraction (NPD) data. Results of our studies are described in the following.

Experimental

Synthesis

AgMnVO 4 was prepared by solid state reaction from a stoichiometric mixture of Ag 2 O, MnO and V 2 O 5 powders as previously reported [9]. AgMnVO 4 was then mixed with an excess of an equimolar amount of Ag 2 O and V 2 O 5 , pelletized, and fired at 700°C for 8 h under argon flow. During this step Ag 2 O reacts with V 2 O 5 forming AgVO 3 which melts at 476 °C [13] and therefore plays the role of flux for the crystal growth of AgMnVO 4 . By a relatively fast decrease of the temperature, at the rate of 15 °C h -1 to room temperature, red, black, and white single crystals were obtained. They have been identified, using EDX and single crystal diffraction analyses, as AgMnVO 4 , Mn 2 V 2 O 7 , and AgVO 3 , respectively.

Electron microprobe analysis

Semiquantitative EDX analyses of different single crystals including the ones investigated on the diffractometer were carried out with a JSM-500LV (JEOL) scanning electron microscope (SEM). The experimentally observed compositions were close to the ideal one AgMnVO 4 (Fig. 1). Some crystals showed compositions close to Mn 2 V 2 O 7 and AgVO 3 . AgMnVO4 single crystals suitable for single crystal X-ray diffraction were selected on the basis of the size and the sharpness of the diffraction spots. The data collections were carried out on a Smart Apex diffractometer using MoKa radiation.

Single crystal X-ray diffraction measurements

Data processing and all refinements were performed with the Jana2000 program package [14]. A Gaussian-type absorption correction was applied, and the shape was determined with the video microscope of the diffractometer. For the AgMnVO4 data collection details, see Table 1.

Magnetic susceptibility and specific heat capacity measurements

DC magnetic susceptibility data for AgMnVO4 were collected in the zero field cooled (ZFC) and field cooled (FC) modes on a Physical Property Measurement System (PPMS, Quantum Designs) in magnetic field of 5 kOe over the temperature range of 2-300 K. Isothermal magnetization was measured in the field of up to 10 kOe. Heat capacity measurements were performed on AgMnVO4 pellet using a PPMS Quantum Design in a temperature range from 4 to 30 K.

Neutron powder diffraction

Neutron powder diffraction (NPD) data were collected on the high-resolution diffractometer Echidna at the OPAL facility (Lucas Height, Australia) using neutrons of wavelength 2.4395 Å. For the measurements the sample in the form of ~1 g of powder was loaded in a 6 mm diameter cylindrical vanadium can and the data were collected at 3 K and 30 K using a closed-cycle refrigerator. The Rietveld analysis of the data was performed using the Fullprof Suite with the default neutron scattering lengths and Mn +2 magnetic form-factors.

Results and discussion

Structure refinement

The extinction conditions observed for single crystal AgMnVO and Table 3, respectively.

Crystal structure of AgMnVO4

AgMnVO4 is isostructural with maricite-type NaMnPO4. The structure consists of edge-sharing chains of MnO6 octahedra running along the b axis. The MnO6 octahedra are cross-connected by the VO4 and AgO4 tetrahedra (Fig. 2). No significant differences were observed compared to the structure solved from powder diffraction data [9]. The interatomic distances and the bond valence sums (BVS) [15,16] are given in Table 4.

Magnetic susceptibility and specific heat measurements of AgMnVO 4

Based on the previously reported magnetic property measurements, a long-range antiferromagnetic (AFM) ordering transition was expected at 12.3 K [9]. Our magnetic susceptibility data also revealed the presence of a ferromagnetic impurity with a transition around 45 K (Fig. S1). However, the field used for the measurements was apparently sufficiently high to suppress its contribution as analysis of the data in the range above 200 K with the Curie-Weiss law yielded the results in close agreement with those previously reported: µeff = 5.74µB and q = -55 K compared to 5.96 µB and q = -62 K [9]. In addition, our heat capacity data showed a clear lambda-type (Fig. S1, inset) anomaly at 12.1 K signalling a long-range magnetic ordering transition in excellent agreement with the previously reported measurements [9].

Crystal and magnetic structure from Neutron powder diffraction measurements

Neutron powder diffraction (NPD) data collected for AgMnVO4 at 30 K (i.e., above the transition expected based on our heat capacity measurements) were successfully analysed using the structure model determined from the X-ray single crystal diffraction data. Since NPD is unable to precisely determine vanadium atom positions due to its very low scattering length, the coordinates were fixed to those from Table 2. The final Rietveld fit and crystallographic information for AgMnVO4 at 30 K are presented in Fig. S2 and Table S1, respectively. Examination of the NPD data collected at 5 K revealed additional intensity due to long-range magnetic ordering (Fig. 3). All the diffraction peaks with magnetic contributions could be indexed to a unit cell with doubled a-and c-parameters, i.e., with the propagation vector k = (1/2, 0, 1/2). Results of representational analysis for the 4a (0,0,0) Wyckoff site of the Pnma space group, yielding such a propagation vector, were previously reported in Ref. 18 [18]. Although by symmetry the four manganese sites split into two pairs, which are allowed to carry different magnetic moments, we assumed equal moment values on all Mn atoms. The best agreement between experimental and calculated NPD patterns was obtained for the combination of G1 + G5

irreducible representations [18]. Examination of the data showed no evidence for scattering due to a magnetic moment component along the b-axis, therefore, only the parameters defining the moment in the ac-plane were refined. The final Rietveld plot and crystallographic information are presented in Fig. 3 and Table S2. The resulting magnetic structure, which can also be described by the P2a21/m group (Opechowski-Guccione #11.6.64) is illustrated in Fig. 4. The components of the magnetic moment along the a-and c-axes are 2.73(8) and 2.93(8) μB, respectively, yielding the total moment of 4.01(8) μB. The moment value reduction compared to the theoretical value of 5 μB for high-spin d 5 (S = 5/2) Mn +2 is likely due to the quasi-one-dimensional magnetic topology of AgMnVO4 (Fig. 4) in which exchange along the edge-sharing octahedral chains can be easily disrupted by local chemical disorder.

Examination of the magnetic structure reveals that the orientation of Mn +2 magnetic moments is almost identical to that of Fe +2 in isostructural maricite-type NaFePO4 [18]. However, while in NaFePO4 the edge-sharing octahedral chains are ferromagnetic, in AgMnVO4 they are antiferromagnetic. This suggests that direct Mn +2 -Mn +2 exchange plays a more significant role compared to the weak ferromagnetic coupling promoted by superexchange via shared oxygen atoms with Mn-O-Mn angles of ~93 -103 º [19].

Concluding remarks

The crystal structure of AgMnVO4 was determined using single crystal X-ray diffraction data for the first time. In agreement with previous results based on powder diffraction data, the structure was found to crystallize with the maricite type. Lowtemperature physical property measurements and neutron powder diffraction experiments showed that the compound undergoes long-range antiferromagnetic ordering transition at 12.1 K. Although AgMnVO4 and NaFePO4 crystallize in the maricite-type structure, the magnetic structures of the two compounds are significantly different in that the spins are antiferromagnetically-and ferromagnetically-coupled within the chains of AgMnVO4 and NaFePO4, respectively. This difference is explained by the influence of direct Mn +2 -Mn +2 exchange in the former.

Fig. 1 .

 1 Fig. 1. Image and EDX analyses of the AgMnVO4 single crystal used for the data

4

 4 agree with the Pnma and Pn21a space groups. The refinement was performed taking into account the centrosymmetric space group Pnma. Most of the atomic positions were found by superflip program implemented in Jana2000 program package. With anisotropic atomic displacement parameters, the final residual factors converged to the value R(F) = 0.0307 and wR(F 2 ) = 0.0790 for 41 refined parameters, 387 independent reflections and difference-Fourier residues in the range between -0.82 and +0.61 eÅ -3 . The refined atomic positions and anisotropic displacement parameters (ADPs) are given in Table2

Fig. 2 .

 2 Fig. 2. Projection view of the structure of AgMnVO4.

a

  bond valence, BV = e (r0-r)/b with the following parameters: b = 0.37, r0 (Ag I -O) = 1.843, r0 (Mn II -O) = 1.790 and r0 (V V -O) = 1.803 Å[15,16].

Fig. 3 .

 3 Fig. 3. The Rietveld plot for AgMnVO4 refined against NPD data collected at 5 K. The red crosses and black and green solid lines indicate the observed and calculated patterns and their difference, respectively. The tick marks from top to bottom indicate the position of the diffraction peaks of the nuclear and magnetic structure, respectively. Rp = 4.23%, Rwp = 5.58%, χ 2 = 2.34, Rmag = 9.24%. The blue curve in the inset shows the magnetic contribution only.

Fig. 4 .

 4 Fig. 4. Left: general view of the crystal and magnetic structure of AgMnVO4. Right:

Table 1 .

 1 Crystallographic data and structure refinement for AgMnVO4.

	Chemical formula	AgMnVO4
	Crystal color	red
	Crystal size mm	0.100×0.090×0.045
	M, g mol -1	277.7
	Crystal system	orthorhombic
	Space group	Pnma
	a, Å	9.5393(12)
	b, Å	6.8132(9)
	c , Å	5.3315(7)
	V, Å 3	346.51(8)
	Z	4
	Density calcd., g cm -3	5.32
	Temperature, K	293(1)
	F(000), e	508
	Diffractometer	SMART APEX
	Monochromator	graphite
	Radiation Å	MoKα , 0.71069
	Scan mode	multi-scan
	h k l range	±12, -8≤ k ≤7, -7≤ l ≤6
	θmin, θmax, °	4.27, 28
	Linear absorption coeff., mm -1	11.67
	Absorption correction	Gaussian
	Tmin/Tmax	0.3573/0.6248
	No. of reflections	2304
	No. of independent reflections	387
	Reflections used [I ≥ 0 s(I) ]	387
	Rint	0.024
	Refinement	F 2
	No. of refined parameters	41
	R factors R(F) / wR(F 2 )	0.0307/0.0790
	g. o. f.	1.33
	Weighting scheme

w = 1/(s 2 (I) + 0.0016I 2 ) Diff. Fourier residues, e -Å -3 -0.82/ +0.61

Table 2 .

 2 Fractional atomic coordinates and isotopic atomic displacement parameters (Å 2 ) for AgMnVO4.

	Atom	Wyck.	Symm.	x	y	z	Ueq (Å 2 )
	Ag	4c	.m.	0.35033(7)	1/4	0.00305(7)	0.0208(2)
	Mn	4a	-1	1/2	1/2	1/2	0.0106(3)
	V	4c	.m.	0.67960(11)	1/4	0.02069(14)	0.0050(3)
	O1	4c	.m.	0.8636(4)	1/4	0.0587(8)	0.0096(10)
	O2	4c	.m.	0.6198(4)	1/4	-0.2881(6)	0.0106(10)
	O3	8d	1	0.6159(3)	0.4465(4)	0.1725(5)	0.0121(7)

Table 3 .

 3 Anisotropic atomic displacement parameters (Å 2 ) for AgMnVO4.

	The

Table 4 .

 4

		distance	B.V.
	Ag-O3 (×2)	2.293(3)	0.296
	Ag-O1	2.340(4)	0.260
	Ag-O2	2.479(4)	0.179
		<2.351>	1.031 a
	Mn-O3 (×2)	2.099(2)	0.434
	Mn-O1 (×2)	2.166(2)	0.362
	Mn-O2 (×2)	2.342(2)	0.225
		<2.202>	2.042 a
	V-O1	1.767(4)	1.102
	V-O2	1.742(4)	1.179
	V-O3 (×2)	1.678(3)	1.402
		<1.716>

Interatomic distances (in Å) and corresponding bond valences (BV) for AgMnVO4. Mean distances are given in brackets.

  The Mn-O distances range from 2.099 Å to 2.342 Å with a mean distance of 2.202 Å. This is very similar to the results found for NaMnPO4 and NaMnVO4, in which the Mn-O distances range from 2.077 Å to 2.397 Å and from 2.099 Å to 2.368 Å with mean distances of 2.229 Å and 2.216 Å, respectively. The BVS of 2.042 is in good agreement with the expected value of +2 for high-spin Mn +2 (d 5 )[17].

	The vanadium tetrahedra are quite regular with distances ranging from 1.678 Å
	to 1.767 Å and a mean value of 1.716 Å. The O-V-O angles range from 105.82(13) ° to
	115.68(19) ° with a mean value of 109.39 °. The BVS of 5.085 is in agreement with the
	expected value of +5 for V +5 .

The Ag + ion is bonded to four O atoms belonging to four different MnO6 groups to form an irregular tetrahedron. The Ag-O distances range from 2.293 Å to 2.479 Å with a mean value of 2.351 Å. The O-Ag-O angles range from 86.32(7) ° to 128.84(9) ° with a mean value of 108.21 °. The BVS is calculated to be 1.031 for the fourcoordinate silver atom.

Acknowledgments

Part of this work was supported by Grant-in-Aid for the Japan Society for the Promotion of Science (JSPS) Fellows Grant Number 24•02506.

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jssc.2014.06.011.


Benjamin D Hobson
email: benjamin.hobson@cuanschutz.edu

Etienne Herzog
email: etienne.herzog@u-bordeaux.fr

Methodological concerns and lack of evidence for single synapse RNA-seq

 develop a new droplet-based RNA sequencing (RNA-seq) method and apply it to particles derived from subcellular fractionation of brain tissue 1 . The authors first establish that their new method, Multiple-Annealing-and-Tailingbased Quantitative scRNA-seq in Droplets (MATQ-Drop), is compatible with aldehyde-fixed human and mouse nuclei. The authors apply MATQ-Drop to study the transcriptome of subcellular particles from the mouse hippocampus, identifying numerous transcriptomic clusters. They further employ MATQ to study subcellular particles from frozen human brain samples of healthy controls and patients with Alzheimer's disease. The goals of this paper are laudable, and the MATQ-Drop method as applied to fixed nuclei may be widely useful to researchers. However, we have major technical concerns about the methods employed to generate and isolate synaptosomes. Due to a lack of critical control and validation experiments, the authors present little or no evidence that they are analyzing the transcriptomes of single synapses.

Main

The principles, advantages and limits of subcellular fractionation were established by decades of work in the laboratory of Nobel laureate Christian de Duve [START_REF] Duve | Exploring cells with a centrifuge[END_REF] . Based on this work, Whittaker and Gray established a sucrose gradient fractionation of neural tissue leading to several discrete fractions, including one enriched in synapses in suspension that they termed "synaptosomes" 3 . Synaptosomes are described as resealed presynaptic varicosities bound to a rarely resealed post-synaptic membrane that displays the post-synaptic density. Classical synaptosome preparations are known to contain around 60% of genuine synaptosome particles and around 40% of non-synaptic contaminants of all kinds that display a similar size and density. Recent work suggests that local translation within the nerve terminals and at postsynapses may contribute to the synaptic proteome [START_REF] Hafner | Local protein synthesis is a ubiquitous feature of neuronal pre-and postsynaptic compartments[END_REF] . High-throughput analysis of the synaptic transcriptome at single particle resolution might shed light on the heterogeneity in this fascinating neuronal process. Unfortunately, Niu et al. [START_REF] Niu | Droplet-based transcriptome profiling of individual synapses[END_REF] present no experimental evidence that their droplet-based sequencing data are derived from uncontaminated single synaptosomes. To describe their approach, the authors use the term "synaptome" as the contraction of synapse and transcriptome. However, the term "synaptome" is widely used in the field to name the ensemble of features of all synapses of a given neural system [START_REF] Zhu | Architecture of the Mouse Brain Synaptome[END_REF] . Here we raise the following major concerns:

Quality control of the subcellular fractionation protocol

The classic protocol for preparing synaptosomes employs liquid shearing homogenization of fresh brain tissue in isosmotic sucrose buffer [START_REF] Gray | The isolation of nerve endings from brain[END_REF] . Several aspects of the protocol employed by the authors are unconventional. First, the authors use liquid nitrogen flash-frozen brain tissue, which is likely to damage synaptic membranes and compromise mechanical integrity of nerve terminals. In fact, synaptosome preparation from frozen samples has been optimized for functional assays, and requires a slow freezing of the tissue in isosmotic sucrose buffer [START_REF] Dodd | Optimization of freezing, storage, and thawing conditions for the preparation of metabolically active synaptosomes from frozen rat and human brain[END_REF] . Yet this protocol on frozen tissue lacks several controls of molecular morphology to be fully validated for single synaptosome studies 7 . Second, Niu et al 1 conduct their studies on preparations derived from the first pellet (P1) obtained from the centrifugation of the brain homogenate at 1,500xg for 10 minutes. Although Niu et al 1 provide no reference for this procedure, it resembles a protocol proposed in 2006 8 , one that has not been properly validated nor accepted by core laboratories in the field. Indeed, the archetypal elements of P1 are cell soma and nuclei 3 , while the vast majority of synaptosomes remain in the supernatant (S1) (Figure 1a). S1 centrifugation at around 10,000 -12,000xg yields a crude synaptosomal pellet (P2) containing synaptosomes, and various light membrane contaminants. P2 is further split by a discontinuous 3 layer sucrose density gradient centrifugation to enrich synaptosomes at the intermediate interface 3 (Figure 1a). As shown in the original paper, P1 studied by electron microscopy includes "large numbers of neuronal and glial nuclei, together with tissue fragments, portions of blood vessels, myelin fragments, mitochondria and vesicular masses that are unlike synaptic vesicles or other known structures" 3 . While some genuine synaptosomes are probably present in P1 derived fractions, they are likely a small minority of the particles of the preparation. Conversely, they will represent a small minority of all synaptosomes, with, potentially biased features that make them prone to fractionate in P1. Given the unconventional nature of their fractionation methods and the lack of a robust qualitative and quantitative validation dataset, it is unlikely that the particles studied by the authors reflect the true nature of synaptosomal transcriptomes.

Fluorescence-activated microparticle sorting

Niu et al 1 applied fluorescence activated sorting to the P1 samples using forward scattering of light (FSC) triggering, a strategy of exclusion of DNA positive particles based on Hoechst staining, and a selection of particles with FSC lower than beads of 5µm diameter. Niu et al 1 Supplementary Figure 3 showing this strategy does not include a reanalysis of the sorted sample that would demonstrate the homogeneity of the sorted population of particles. Based on our experience, it is unlikely that such a sort would provide a homogeneous population of microparticles. Indeed, flow cytometers and sorting instruments traditionally rely on an FSC threshold to trigger particle detection and sorting, but we have shown that single synaptosomes bearing endogenous fluorescence (VGLUT1 VENUS ) cannot be reliably detected and sorted using FSC triggering [START_REF] Biesemann | Development of Fluorescence Activated Synaptosome Sorting (FASS) and analysis of VGLUT1 synapses from mouse brain[END_REF][START_REF] Biesemann | Proteomic screening of glutamatergic mouse brain synaptosomes isolated by fluorescence activated sorting[END_REF] . We established a fluorescent triggering method using the amphiphilic styryl dye FM 4-64 that enables more sensitive particle detection and mitigates the limitations of FSC resolution and noise. We found that around 75% of the particles in synaptosomal preparations are undetectable using FSC triggering [START_REF] Biesemann | Development of Fluorescence Activated Synaptosome Sorting (FASS) and analysis of VGLUT1 synapses from mouse brain[END_REF][START_REF] Hobson | Critical Analysis of Particle Detection Artifacts in Synaptosome Flow Cytometry[END_REF] . Of note, individual synaptosomes and other microparticles are among these populations with low FSC that cannot be reliably distinguished from noise. We have also confirmed that polystyrene bead standards are not appropriate for scatter-based size estimation of biological microparticles: the higher refractive index of polystyrene means that biological particles are much larger than the polystyrene beads with similar FSC [START_REF] Biesemann | Development of Fluorescence Activated Synaptosome Sorting (FASS) and analysis of VGLUT1 synapses from mouse brain[END_REF][START_REF] Hobson | Critical Analysis of Particle Detection Artifacts in Synaptosome Flow Cytometry[END_REF] . Indeed, despite their size of 0.5 -1 µm in diameter, single VGLUT1 synaptosomes produce FSC signals that are unresolved by a conventional flow cytometer and lower than 0.5 µm diameter polystyrene beads [START_REF] Biesemann | Development of Fluorescence Activated Synaptosome Sorting (FASS) and analysis of VGLUT1 synapses from mouse brain[END_REF] . Additionally, subcellular fractionation samples tend to contain debris of an infinite range of sizes and prone to aggregation. This produces growing diagonals in the dot-plots with FSC (as seen in Niu et al 1 Supplementary Figure 3). Hence, the sorting procedures employed by Niu et al 1 are likely to target a random collection of cellular debris and aggregated particles an order of magnitude larger than the genuine synaptosomes (Figure 1b). Meanwhile, the small biological microparticles that fall below the FSC threshold are ignored by the cell sorter and randomly distributed in the sorted droplets, and shearing forces during sorting tends to disrupt aggregates and large debris. Thus, the occurrence of small microparticles under transmitted light microscopy shown in Niu et al 1 Supplementary Figure 3 can be explained by both the disruption of aggregates and the random inclusion of under-threshold microparticles. Especially given that P1 samples are poor in genuine synaptosomes, the sorting strategy of Niu et al 1 is likely to target a random collection of cellular debris and microparticles.

To establish the synaptic nature of the subcellular particles, Niu et al 1 performed a immunoblot analysis of a few markers and a flow cytometry-based immunostaining analysis of the presynaptic marker synaptophysin and the post-synaptic marker PSD95. We notice that on the immunoblot analysis, a significant level of the astrocyte marker GFAP is still detected after sorting. GFAP is known to be a good marker of astrocyte cell soma and primary processes, but mostly absent from the fine processes engulfing a fraction of synapses. This observation is in line with the previous points and suggests contamination of the sorted samples with cell debris. Of note, the normalization of immunoblots with "house-keeping proteins" such as actin is not possible with subcellular fractionation because all proteins will have differential abundance in each fraction. Regarding the flow cytometry of immunostained fractions, we have shown that synaptosomal aggregation promoted by the immunostaining procedure leads to erroneous colabeling of markers that should be mutually exclusive in single synaptosomes [START_REF] Hobson | Critical Analysis of Particle Detection Artifacts in Synaptosome Flow Cytometry[END_REF] . The presence of aggregates and false co-localization is enhanced in the FSC-triggered detection range studied by the authors [START_REF] Hobson | Critical Analysis of Particle Detection Artifacts in Synaptosome Flow Cytometry[END_REF] . Also, synaptophysin and PSD95 co-labeling is expected at most excitatory synapses and for aggregates. Meanwhile, a significant extra-synaptic fraction of these proteins exists in soma and dendrites that will probably be stained within cellular debris. In addition to isotype controls for specificity of the antibody staining, a formal demonstration of the presence and purity of single synaptosomes in P1 samples requires a lack of co-labeling for markers known to be largely mutually exclusive (e.g., VGLUT1 and VGAT).

Validation of omics clusters by independent methods

The real added value of single particle RNA-seq should be to learn more about the heterogeneity of the given population of the targeted particles. In Niu et al 1 , one can expect that the transcriptomic clusters derived from the RNA-seq data reflect a heterogeneous distribution of mRNA at synapses in brain tissue. Do these transcriptomically distinct synapse clusters "Syn1-12" actually exist in the hippocampus? How are these synapse types organized in the hippocampal neuropil? Unfortunately, Niu et al 1 do not attempt to make this connection using established techniques such as in situ hybridization. This lack of validation further limits the degree of confidence one can have in the relevance of their RNA-seq data.

RNA quantification, contamination, and background

The quantity and relative proportion of mRNA, rRNA, and other RNA species contained within synaptosomes has not been well studied, even in bulk preparations. The proportions of mRNA and rRNA seem roughly similar to RNA from tissue homogenates, based on bioanalyzer traces showing the two characteristic rRNA peaks. Our current estimates based on total RNA quantification (Qubit) from 100 million sorted synaptosomes [START_REF] Hafner | Local protein synthesis is a ubiquitous feature of neuronal pre-and postsynaptic compartments[END_REF] or UMI-based RNA-seq from 1 -5 million sorted synaptosomes are in the range of 0.1 -10 mRNA molecules per particle [START_REF] Hobson | Subcellular and regional localization of mRNA translation in midbrain dopamine neurons[END_REF] . Thus, whether all single genuine synaptosomes contain mRNA remains an open question. Niu et al 1 report a median detection rate of 306 genes per particle 1 . If the MATQ-Drop protocol has an efficiency of 10 -50%, this would suggest the presence of 612 -3,060 mRNA molecules per particle (an underestimate based on genes per particle, since UMI counts per particle were not provided). These estimates are several orders of magnitude higher than previous studies, and should be supported by quantitative analysis of the RNA content of bulk synaptosomes. In the absence of such evidence, these data further suggest to us that the droplet-based RNA-seq data are derived from biological particles much larger than single synaptosomes.

Also, given the low mRNA content of synaptosomes, careful attention must be paid to the mRNA contamination of the particles and the droplets. All droplet-based RNA-seq experiments are susceptible to background contamination by ambient mRNA molecules in the cell suspension. Furthermore, snap-freezing leads to mechanical damage of synaptic membranes, which can result in molecules from the homogenate entering the cytoplasmic interior of synaptosomes. This phenomenon has been exploited to load membrane-impermeable molecules into synaptosomes via inclusion in the initial buffer during tissue homogenization [START_REF] Raiteri | Entrapping of impermeant probes of different size into nonpermeabilized synaptosomes as a method to study presynaptic mechanisms[END_REF] . Niu et al 1 make no attempt to control for either of these possible sources of mRNA contamination. A mixedspecies MATQ-Drop experiment is critically absent for the subcellular particles. Human and mouse brain tissue should be mixed during homogenization, ideally in the presence of soluble spike-in RNA standards, in order to control for general cytoplasmic RNA cross-contamination.

Although such an experiment would help control for aggregation and contamination, it would not substitute for biochemical and microscopic evidence that the particles analyzed are single synaptosomes per droplet. In addition, an RNAse protection assay on unfixed samples may provide further evidence for the specific mRNA content within single particles versus mRNA contamination outside the particles in the droplets.

Concluding perspective

A single hippocampal CA3 axon is estimated to harbor ~30,000 -60,000 presynapses [START_REF] Li | The hippocampal CA3 network: an in vivo intracellular labeling study[END_REF] . Given that the data presented by Niu et al 1 suggest ~600 -3,000 mRNA molecules per synaptosome, the axon of a single hippocampal CA3 pyramidal neuron would be expected to harbor 18 -180x 10 6 mRNA molecules. Yet, a typical mammalian cell is thought to contain at most 1x10 6 mRNA molecules [START_REF] Shapiro | Single-cell sequencing-based technologies will revolutionize whole-organism science[END_REF] . There is a clear gap between these estimates that is reconciled by the possibility that the experiments from Niu et al 1 are performed on large brain tissue debris and aggregates rather than on genuine single synaptosomes. Hence, we believe that the claims of Niu et al 1 are not supported by the data and evidence provided.

More broadly, we foresee that single biological particle -omics may open exciting perspectives for a better understanding of cellular mechanisms. In this context, we encourage the community to take into account previous work in the field for rigorous experimental design, and to establish

Figures

  Figures

Figure 1 :

 1 Figure 1: Conventional synaptosome fractionation and sorting compared to Niu et al (a) Standard synaptosome fractionation protocol 3 . Note that pellet P1 is rich in nuclei, cells, and heavy debris, while most synaptosomes remain in supernatant S1. Even highly enriched synaptosomal fractions from sucrose density gradient centrifugation remain contaminated with glial membranes and non-synaptic fragments. (b) Sorting strategy employed by Niu et al compared to fluorescence-activated synaptosome sorting (FASS). Note that most particles detected using FSC-triggering are aggregates, while single synaptosomes produce FSC signals below the noise threshold and require fluorescencetriggering for detection 9-11 .

  1. Inadequate subcellular fractionation protocol and lack of quality control and validation 2. Inappropriate fluorescence-activated sorting methods and absence of proper validation of the chosen protocol 3. No validation of key marker mRNAs using in situ hybridization 4. Mismatch between the estimates of mRNA content per synaptosome and current established knowledge of the neuronal transcriptome


est destinée au dépôt

Deformation and hydrothermal alteration insights of a high strain sheared zone in a gneissic basement (Black Forest, Germany). An analogue for fractured geothermal crystalline reservoirs.

Benjamin Avakian * 1,2,3 , Béatrice Ledésert 1 , Ronan Hebert 1 , Philippe Claeys 2 , Steven Goderis 2 , Ghislain Trullenque 3 , and Sébastien Potel 3

1 CY Cergy Paris Université -Geosciences Environnement Cergy -France 2 Vrije Universiteit [Brussel] -Belgique 3 UniLaSalle -B2R -France
Enhanced geothermal systems in basement rocks consist in the exploitation of the hot brines circulating within the fracture network as the main fluid pathway within the reservoir. This is the case at Soultz-sous-Forêts and Rittershoffen (France). The fracture network must be clearly documented before developing a geothermal installation to reduce the risks of targeting low permeability fractures or induced seismicity related to reservoir stimulation. A potential sub-surface analogue to Soultz-sous-Forêts' reservoir is found in a highly strained zone rich in clay minerals in Shauinsland's mine (Black Forest, Germany). The analogue is crosscut by an ore vein that disappears close to the core of the fault zone and reapers on its other side. Petrographical and petrophysical characterizations were done to identify deformation and hydrothermal alteration markers such as grain size reduction, fracturation, illitization process and precipitation of carbonate veins. This was coupled with a geochemical study focusing on the effect of hydrothermal alteration on the chemical composition of the deformed host rock. Micro X-ray fluorescence element maps were gained at thin section scale (3x4 centimeters) to identify fluid circulation features within the host rock. Major element concentrations were quantified using powdered samples taken along the fault zone to identify hydrothermal alteration stages. Porosity, carbonate content and magnetic susceptibility increase towards the fault core and tend to be correlated with clay content. Fe precipitated massively in the core zone and Ca in veins of the damage zone. High fluid flow was channelled in the core zone which is no longer permeable as indicated by the high clay content. Combined with mechanical data and numerical modelling, this may help reduce the risks of "dry well" or generate seismic events during geothermal stimulation.

Mots-Clés: Enhanced geothermal systems, analogue, fault zone, fluid/rock interaction, permeability * Intervenant sciencesconf.org:rst2023-rennes:486777

  Deformation and hydrothermal alteration insights of a high strain sheared zone in a gneissic basement (Black Forest, Germany). An analogue for fractured geothermal crystalline reservoirs Benjamin Avakian, Béatrice Ledésert, Ronan Hebert, Philippe Claeys, Steven Goderis, Ghislain Trullenque, Sébastien Potel To cite this version: Benjamin Avakian, Béatrice Ledésert, Ronan Hebert, Philippe Claeys, Steven Goderis, et al.. Deformation and hydrothermal alteration insights of a high strain sheared zone in a gneissic basement (Black Forest, Germany). An analogue for fractured geothermal crystalline reservoirs. 28e Réunion des Sciences de la Terre, SGF; Géosciences Rennes, Oct 2023, Rennes (FR), France. hal-04311377