The third compound is a molecular thiostannate (Sn2S6(AEPzH2)2) composed of dimeric Sn2S64− and AEPzH22+. In preparation of the layered compounds, the use of AEPz as the only solvent results in AEPz-SnS-1 with regular hexagonal pores and crystallographically disordered organic cations. In contrast, a mixture of AEPz and absolute ethanol gives AEPz:EtOH-SnS-1 with distorted hexagonal pores and ordered cations between the layers. The influence of cation order on the light absorption properties and the material thermal stability was investigated through thermal treatment of the layered compounds up to 200 °C. Both compounds show colour changes when heated, but cation order results in larger thermal stability. For AEPz-SnS-1, a decreased inter-layer distance and substantial loss of organic matter was observed when heated. However, pair distribution function analysis reveals that the local in-layer thiostannate structure of AEPz-SnS-1 remains unchanged. In contrast, AEPz:EtOH-SnS-1 does not undergo noticeable structural changes by the thermal treatment. All materials are optical semiconductors with band gaps of 3.0–3.1 eV.We present the synthesis, crystal structures and optical properties of three thiostannates prepared by using 1-(2-aminoethyl)piperazine (AEPz) as structure directing agent. Two of the thiostannates are layered materials (AEPz-SnS-1 and AEPz:EtOH-SnS-1) consisting of [Sn For example, tin sulfides and thiostannates comprise a family of structurally diverse compounds, and layered SnS2 has been studied thoroughly due to its photocatalytic properties with7 and without dopant modifications9. Multiple thiostannates have been reported, including molecular compounds containing the Sn2S64− ion20, polymeric layered materials containing [Sn3S72−]n or [Sn4S92−]n sheets25, and three-dimensional compounds containing [Sn2S52−]n26. The negative charge is balanced by molecular organic cations such as alkylammonium ions25, protonated amines23, or by metal ions or metal complexes16. The thiostannates are optical semiconductors; the molecular compounds with metal-free cations have band gaps in the range ~ 3.5–4.2 eV28, while band gaps of  ~ 2.5–3.2 eV have been reported for the layered materials24. Hence, the properties of thiostannates might be tailored due to their structural diversity and optical properties.A range of properties including photocatalysis has been reported for structurally complex metal sulfides2Sn3S7 family of layered materials (R is a cation), which are also denoted R-SnS-129. Some alkylammonium containing R-SnS-1 type compounds were reported to be photocatalytically active for dye degradation using visible light24. Other R-SnS-1 compounds have shown ion exchange properties, allowing for modification of their light absorption properties32. Recently, we observed that some R-SnS-1 compounds retain their optical properties, despite undergoing a crystalline to amorphous phase transition in water, while conserving their local structure33.A range of properties has been reported for members of the R3S72−]n layer in R-SnS-1 is the Sn3S44+ broken-cube cluster, which are connected by double sulfur bridges to create a 24-atom hexagonally shaped pore piperidine)23, or disordered as observed in e.g. trenH-SnS-1 (tren = tris(2-aminoethyl) amine)23. Depending on the structure directing agent used in the solvothermal synthesis (e.g. amines), the pores are either regular or distorted hexagons, and the inter-layer separation spans widely from e.g. 7.40 Å in FJSM-SnS-1 ([Me2NH2]4/3[Me3NH]2/3Sn3S7)31 to 9.52 Å in trenH-SnS-123.The crystal structure and compound stability are of key importance in relation to understanding the properties and potential of R-SnS-1 type materials. The fundamental building unit of the n. Lastly, these layers stack to give a layered compound with intercalated of cations R in-between35. While R-SnS-1 is considered the final product, the formation of the layered compound has previously been haltered and allowed isolation of crystals of the corresponding dimeric compound R2Sn2S6. As an example, polymeric trenH-SnS-123 and molecular Sn2S6(trenH2)217 have been synthesised selectively at different conditions by using the same amine.The formation mechanism of R-SnS-1 type compounds has been reported to occur via a polymerization process in which a cationic species R is used as structure directing agent under solvothermal conditions. Initially, a dimeric SnTo tailor materials towards specific applications, it is crucial to obtain synthesis control allowing preparation of products with desired properties. Even though several R-SnS-1 type compounds have been reported, many unanswered questions remain with respect to the role of the structure directing agent on the resulting structure in terms of e.g. cation order/disorder and regular versus distorted hexagonal pores. These structural properties might influence the material band gap and the compound stability.36, but without focusing on the crystal structures in detail. Instead, in the study by Walther et al.36 enzyme-like activity was demonstrated for the compounds, and only the fundamental structure was reported for layered AEPz:EtOH-SnS-1 and molecular Sn2S6(AEPzH2) without in-depth studies of e.g. cation–anion interactions and crystal packing. Finally, AEPz-SnS-1 was proposed to be isostructural to trenH-SnS-1 based on X-ray diffraction data36. In this paper, we present optimized synthesis protocols giving molecular Sn2S6(AEPzH2)2 and two R-SnS-1 type compounds (AEPz-SnS-1 and AEPz:EtOH-SnS-1) by simple variation of stoichiometry, solvent, and temperature. Moreover, the crystal structures of the three thiostannates are presented in detail. Finally, as the same structure directly amine (AEPz) uniquely gives two different layered R-SnS-1 compounds with ordered and disordered cations, respectively, the influence of cation order on some material properties were studied. This was done by investigating the light absorption properties and thermal stability of the compounds by diffuse reflectance spectroscopy (DRS), PXRD and X-ray total scattering and pair distribution function (PDF) analysis.In recent publications, we have presented the synthesis of thiostannates using 1-(2-aminoethyl)piperazine 2 was obtained as irregularly shaped transparent reddish crystals 2, ~ 8% in AEPz-SnS-1, and ~ 11% in AEPz:EtOH-SnS-1 . Based on the proposed chemical formulas (Table 2S6(AEPzH2)2 at 1.83 g·cm−1, while the R-SnS-1 compounds have increased densities of 1.98 and 2.21 g·cm−1, respectively, for AEPz-SnS-1 and AEPz:EtOH-SnS-1. The latter indicates a more compact crystal packing of AEPz:EtOH-SnS-1 in comparison with AEPz-SnS-1.The crystal structures of the three thiostannates were solved by single crystal X-ray diffraction. A summary of the crystallographic details is shown in Table 2S6(AEPzH2)2 2 Fig. , but alsP63/mmc (no. 194)  Å between opposite bridging sulfur atoms. The molecular cations were found to be crystallographically disordered, and the large interlayer spacing between the thiostannate layers suggests the cations to be located between these. We previously reported that the compound contains two protonated AEPz (AEPzH+) per [Sn3S72−] moiety according to CHNS analysis33. Herein, by using the SQUEEZE procedure39, 102 electrons were removed per [Sn3S72−], equivalent of approx. 1.5 AEPz (110 electrons). Due to the moderate crystal quality (see ESI), we suggest two AEPzH+ per [Sn3S72−], leading to a charge neutral compound. Thus, we can conclude that the thiostannate sheets of trenH-SnS-123 and AEPz-SnS-1 are isostructural as previously proposed36 despite of using structure directing organic amines with substantially different size and shape.The structure of the thiostannate layers in AEPz-SnS-1 was solved in the hexagonal space group 2:S precursor ratio of 1:10 and a temperature of 150 °C, formation of the polymeric sheets can be haltered to give crystals of dimeric Sn2S6(AEPzH2)2. The synthesis of Sn2S6(AEPzH2)2 mainly gives irregularly shaped crystals in various sizes, of which a representative example is shown in Fig. P2S64− ions (two edge sharing tetrahedra) and charge stabilising ammonium ions o and thereby deviate substantially from the ideal tetrahedron. Finally, the Sn-S-Sn’ angle across the sulfur bridge is found to be 88.15(1)o. Overall, the bond distances and angles are in good agreement with previously reported R2Sn2S6 compounds18. The lengths of the –N–H 2S6(trenH2)218. The protonated primary (N1) and secondary (N3) amino groups show strong interactions with neighbouring Sn2S64− clusters, by each interacting with two thiostannate dimers 2 was calculated. Overall, there is good agreement between the qualitative analysis of the hydrogen bonding interactions described above and the fingerprint plot ·0.5(Pz), but the positions of the hydrogen atoms, which is required to obtain charge neutrality of the compound, could not be identified from the electron density map.AEPz:EtOH-SnS-1 also consists of layers with the stoichiometry n layers (Table i) the secondary amino groups of AEPz (N5a/b and N8a/b), (ii) N2 on Pz, and (iii) one of the primary amino groups (N3a/b or N6a/b). This gives four ammonium groups per [Sn3S72−]2, hence a net charge of zero.In the structure of AEPz:EtOH-SnS-1, the ammonium functionalities of the AEPzHrs Table . The str) Å Fig. c. The coInterestingly, despite of adding ethanol as a co-solvent, the alcohol was not incorporated into the structure according to CHNS analysis see  section.33, the band gap of AEPz-SnS-1 was determined to 3.0 eV piperidine23. Interestingly, despite the structural differences, AEPz:EtOH-SnS-1 also has a band gap of 3.0 eV 2 was determined to be 3.1 eV . Kubelka–Munk transformed spectra are shown in Fig.  eV Fig. b, but th33. AEPz:EtOH-SnS-1 with ordered cations does not lose its crystallinity in water suspension according to PXRD  undergo a phase transition from crystalline to amorphous when dispersed in water. Based on these observations we suggested that the cation disorder in R-SnS-1 compounds might play a role on their stability in waterXRD Fig. . This obThe fact that the same structure directing amine uniquely resulted in the synthesis of R-SnS-1 type materials with cation disorder (AEPz-SnS-1) and order (AEPz:EtOH-SnS-1), respectively, motivated us to investigate the influence of cation order/disorder on the thermal stability and light absorption properties of the two layered compounds. The two R-SnS-1 compounds were heated to 100, 150 and 200 °C in air, after which they were cooled to room temperature and analysed. Interestingly, after heating to 100 °C, both compounds show a slight colour change, before they started to turn red-brown at 150 °C and dark red-brown at 200 °C ⋅r, which highlights subtle features at high r  and Fig. G(r)⋅r in an extended range of r). Inspection of the PDF data below 8.5 Å confirms the in-layer local structure to be maintained for all samples, as no new peaks appear or disappear, and no significant shifts in peak positions are observed. Peaks at distances larger than the inter-layer distance show minor changes including some slightly decreasing correlation lengths, which is in agreement with the inter-layer distance contraction according to PXRD  analysis based on X-ray total scattering data. Figure XRD Fig. g. For inXRD Fig. i. Howeve33, while heat treatment led to a substantial loss of organic matter. While the compounds were seen to either remain crystalline (AEPz:EtOH-SnS-1) or have an unchanged in-layer local structure (AEPz-SnS-1), CHNS reveals that the incorporated AEPz molecules are more easily evaporated from the disordered AEPz-SnS-1 relative to ordered AEPz:EtOH-SnS-1. We suggest evaporation of AEPz to be the main mechanism behind the decrease in inter-layer distance observed for AEPz-SnS-1, and the colour change may be due to surface oxidation or charring of residual organics on the surface from the evaporation process.To summarise, we propose that cation order has a significant impact on the material stability. This is observed by the increased water and heat stability of the ordered AEPz:EtOH-SnS-1. For comparison, suspension of AEPz-SnS-1 in water led to amorphizationOverall, for the R-SnS-1 compounds we suggest that the light absorption of the pristine materials appears to be largely independent of cation order and symmetry of the hexagonal pores. However, cation order, which is indicative of strong guest molecule-thiostannate framework interactions, indeed affects the stability of the materials.2S6(AEPzH2)2. The compound Sn2S6(AEPzH2)2 was prepared at 150 °C and contains AEPzH22+ and Sn2S64− motifs. AEPz-SnS-1 was obtained when using AEPz as the only solvent at 190 °C. In this compound, protonated cations of AEPz are intercalated between [Sn3S72−]n layers with regular hexagonal pores, and the cations are crystallographically disordered. When mixing AEPz with ethanol, AEPz:EtOH-SnS-1 was obtained at 190 °C with ordered cations between layers of [Sn3S72−]n with distorted hexagonal pores. Actually, in AEPz:EtOH-SnS-1 two different organic species are intercalated, namely protonated AEPz and piperazine formed in situ by cleavage of the aminoethyl group from AEPz. The ordering of the organic species reduced the inter-layer spacing by approx. 20% resulting in an increased density in comparison with AEPz-SnS-1. The three materials have band gaps in the range of 3.0–3.1 eV.In this study, we present synthesis protocols for three thiostannates by variation of temperature, stoichiometry and solvent. All procedures use AEPz as the structure directing agent and solvent. We have solved the crystal structures of two layered R-SnS-1 compounds (AEPz-SnS-1 and AEPz:EtOH-SnS-1) and molecular SnFor the R-SnS-1 compounds, the influence of cation order on the thermal and water stability was investigated. The stronger cation–anion interactions in AEPz:EtOH-SnS-1 causes the material to be substantially more stable in air up to 200 °C and in water at room temperature in comparison with AEPz-SnS-1. Upon heating, the inter-layer distance of AEPz-SnS-1 was found to decrease by approx. 0.2 Å, and strain was induced in the crystalline powder, but the local structure was preserved. The decrease in inter-layer distance is associated with loss of AEPz, as observed by elemental analysis. We also conclude that the optical properties of the pristine materials have low dependence of the inter-layer distance and cation order, but the latter substantially modifies the material stability.2 (≥ 99.5%), 1-(2-aminoethyl)piperazine  and absolute ethanol (C2H5OH) were purchased from Sigma-Aldrich and used without further purification.Elemental sulfur (≥ 99.9%), SnO2S6(AEPzH2)2 and AEPz:EtOH-SnS-1 were synthesised similarly to our previously reported description using stainless steel autoclaves with a 23 mL Teflon liner36. For all syntheses, 2 mL AEPz and 0.2 g sulfur were used, while the amount of SnO2 was adjusted according to the stoichiometry of the desired products. The precursor mixtures were stirred in the Teflon liner for ~ 5 min, before the autoclave was sealed and placed in a convection oven (Memmert UF-30plus) for 6 days at the target temperature. Subsequently the oven was turned off, and the autoclaves were cooled to room temperature over approx. 4 h, unless otherwise specified. Finally, the powders were isolated by suction filtration, washed with absolute ethanol, and dried in air.AEPz-SnS-1, Sn2  and sulfur , i.e., in a molar ratio of 3:8, at 190 °C. Approx. 0.6 g was obtained (yield 90% based on Sn). To improve the growth of single crystals for structure determination, a 30-hour linear cooling ramp from 190 °C to room temperature was applied, following the 6 days at 190 °C.AEPz-SnS-1 was synthesised by mixing SnO2S6(AEPzH2)2 was synthesised at 150 °C using SnO2  and sulfur  corresponding to a molar ratio of ~ 1:10. Approx. 0.15 g was obtained (yield 64% based on Sn).Compound Sn2  and sulfur  were heated to 190 °C in a molar ratio of 3:7, and a solvent mixture of 2 mL AEPz and 2 mL absolute ethanol was used. 0.6 g product was obtained (90% based on Sn). CHNS found/calculated % for AEPz:EtOH-SnS-1: C, 12.5(1)/12.7%; H, 3.04(1)/2.94%; N, 7.10(5)/7.42%; S, 25.2(1)/29.7%. Calculated values are based on the sample stoichiometry according to a composition of Sn6S14·C16N8H42  in 1 h. Following, the sample was heated at the target temperature for 1 h, and the oven was subsequently ramped down to room temperature over 1 h.Kα1 radiation (λ = 1.54056 Å) and a D/TEX Ultra 250 multi-channel detector was used for collection of PXRD data. Samples were packed in glass capillaries (inner diameter 0.5 mm) and convergence beam geometry was applied. The FullProf suite was used for Le Bail and Rietveld refinements40.A Rigaku SmartLab diffractometer using Cu 2S6(AEPzH2)2 were acquired on a Bruker Apex-II CCD diffractometer using monochromatic Ag Kα radiation (λ = 0.56086 Å). The Apex-III software was used for data integration and correction, specifically data were corrected for absorption in SADABS-2016/2 by applying a multi-scan method41. Data on crystals of AEPz-SnS-1 and AEPz:EtOH-SnS-1 were measured on an Oxford Diffraction Supernova diffractometer with a molybdenum microfocus sealed X-ray tube , and an Atlas CCD detector. The CrysAlisPro software (version 1.171.40.67a) was used for integration, and a spherical absorption correction was performed in ABSPACK (included in the CrysAlisPro software package). Crystals of Sn2S6(AEPzH2)2 were cooled to 100 K during data collection using a nitrogen cooler from Oxford Cryosystems, whereas data on AEPz-SnS-1 and AEPz:EtOH-SnS-1 were measured at room temperature due to cracking of the crystals upon cooling. The structures were solved using SHELXT42 or SHELXS and refined against F2 in SHELXL44 by using the Olex2 interface45. The SQUEEZE procedure was used in the analysis of AEPz-SnS-1 data to deal with disordered organic substances39.Single crystal X-ray diffraction data on Sn46. Hirshfeld surface analysis was used to analyse molecular interactions between AEPzH22+ and Sn2S64− in Sn2S6(AEPzH2)2. Close contacts were visualised through a fingerprint plot, which represents molecular interactions in a two-dimensional map. Along the abscissa is plotted the distance from the Hirshfeld surface to the nearest internal atom (di), while the distance from the surface to the nearest external atom (de) is plotted along the ordinate. For molecules with hydrogen bonding, sharp needle-like features are expected47.Hirshfeld surface analysis was carried out using the CrystalExplorer softwareλ = 0.2072 Å, beam size 1 × 1 mm). All samples were packed in Kapton capillaries (inner diameter 1.0 mm) and placed in a home-built sample changer. Scattered intensities were collected on a 2D detector (Perkin Elmer XRD1621) at a sample-to-detector distance of ~ 20 cm . For each sample, data were measured for 5 × 1 min in 1 s exposures, and the 1 min frames were integrated and subsequently summed using Dioptas V0.448. Thereby, data in the Q-range of 0.5–20 Å−1 were obtained. The total scattering data were background corrected by subtraction of the scattered intensities from an empty Kapton capillary. The corrected data were transformed to Pair Distribution Functions (PDF), G(r), using xPDFsuite49 based on the PDFGetX3 engine50. Finally, pattern calculations and refinements were performed using PDFgui51.X-ray Total scattering data were collected at beamline P02.1 at PETRAIII at the Deutsches Elektronen-Synchotron (DESY), Germany, using an energy of 60 keV (Scanning electron microscopy (SEM) images were captured using a FEI-Nova Nano SEM 600 in high vacuum. Samples were immobilized on double-sided carbon tape and coated with approx. 10 nm platinum to prevent charging. The platinum layers were deposited using a LEICA EM SCD 500 vacuum film deposit system equipped with a LEICA EM QSG100 Quartz Crystal Film Thickness Monitor to track the film thickness.4 as a reference. Band gaps of the materials were determined by transforming the reflectance data using the Kubelka–Munk function, where R is the reflectance, and F is the transformed data.Diffuse reflectance spectroscopy (DRS) data were measured on the samples in the range 200–800 nm in 1 nm steps on the Shimadzu UV-3600 spectrophotometer against BaSO3 as catalyst.Elemental analyses of AEPz:EtOH-SnS-1 and AEPz-SnS-1 and their heat treated samples were performed on a Elementar Vario MACRO cube in CHNS mode. Sulfanilamide was used as a reference (approx. 25 mg for each measurement). Triplicates were analysed with approx. 20–25 mg sample and approx. 10 mg WOSupplementary Information.


In this report, we have developed a simple approach using single-detector fluorescence autocorrelation spectroscopy (FCS) to investigate the Förster resonance energy transfer (FRET) of genetically encoded, freely diffusing crTC2.1 (mTurquoise2.1–linker–mCitrine) at the single molecule level. We hypothesize that the molecular brightness of the freely diffusing donor (mTurquoise2.1) in the presence of the acceptor (mCitrine) is lower than that of the donor alone due to FRET. To test this hypothesis, the fluorescence fluctuation signal and number of molecules of freely diffusing construct were measured using FCS to calculate the molecular brightness of the donor, excited at 405 nm and detected at 475/50 nm, in the presence and absence of the acceptor. Our results indicate that the molecular brightness of cleaved crTC2.1 in a buffer is larger than that of the intact counterpart under 405-nm excitation. The energy transfer efficiency at the single molecule level is larger and more spread in values as compared with the ensemble-averaging time-resolved fluorescence measurements. In contrast, the molecular brightness of the intact crTC2.1, under 488 nm excitation of the acceptor (531/40 nm detection), is the same or slightly larger than that of the cleaved counterpart. These FCS-FRET measurements on freely diffusing donor-acceptor pairs are independent of the precise time constants associated with autocorrelation curves due to the presence of potential photophysical processes. Ultimately, when used in living cells, the proposed approach would only require a low expression level of these genetically encoded constructs, helping to limit potential interference with the cell machinery. FRET is in vivo . FRET ap in vivo , conform in vivo , and env in vivo .Steady-state spectroscopy can be used to measure the energy transfer efficiency of FRET pairs based on the comparison of the time-averaged fluorescence intensity of the donor in the presence and absence of an acceptor . This mein vivo studies of macromolecular crowding or ionic strength  studies have been reported on a wide range of immobilized biomolecules on surfaces . In smFRDifferent modalities of fluorescence correlation spectroscopy (FCS), a single-molecule approach, have been used for non-invasive applications to study translational diffusion, chemical kinetics, and photophysical processes such as intersystem crossing to triplet electronic states and fluorescence blinking in fluorescent proteins . FluoresIn this report, we highlight a simple concept of using the fluorescence fluctuations and molecular brightness (number of emitted photon per molecule) for FRET analysis of a donor-linker-acceptor model construct at the single-molecule level using a traditional, single-detector FCS setup. As a proof of concept, we used crTC2.1 construct (mTurquoise2.1–linker–mCitrine)  as a modThe molecular brightness (the average number of fluorescence photons per molecule during their residence time in the observation volume) of the donor in the presence and absence of an acceptor can be used for FRET analysis of D-L-A constructs at the single-molecule level. We hypothesize that the molecular brightness of the donor alone is larger than that in the presence of an acceptor at close proximity. In this concept , the fluUsing steady-state spectroscopy, the energy transfer efficiency in donor-acceptor FRET pairs is determined using the time-averaged fluorescence signal of the donor in the presence (such thatE(%)=(1−FF) of an ensemble of fluorophores, excited by light intensity  of the fluorophore can be related to the number of molecules (N) in a given volume (V) using Avogadro’s number  under 488 nm illumination can be determined using a photostable fluorphore (rhodamine-110) with known diffusion coefficient as a reference 6A (GSG)6A (EAAAK)6A (GSG)6–   as a mod (GSG)6– . This mo (GSG)6–  to test  (GSG)6–  6– . The abs (GSG)6– , where t2 and 103.9 kW/cm2, respectively. Under 488 nm excitation of the acceptor, however, the average power of the laser was about 2 μW with laser intensity at the sample of 1.6 kW/cm2. A dichroic mirror (395DM for 405 nm excitation or 490DM for 488 nm excitation) was used to separate the fluorescence emission from the excitation laser. The collimated epifluorescence signal was first filtered  and then focused on a confocal pinhole . The fluorescence signal was then detected by an avalanche photodiode , amplified, and correlated using an external multiple-tau-digital correlator .A home-built fluorescence correlation spectroscopy (FCS) setup has been described in detail elsewhere . BrieflyIn a single-detector FCS, the time-dependent fluorescence fluctuations, uch that δF(t)=F of molecules in the observation volume due to translational diffusion, chemical reactions, and fast photophysical processes such as fluorescence blinking in fluorescent proteins, conformational changes of biomolecules, or intersystem crossing . The coruch that G(τ)=〈δF Gaussian profile of the observation volume, the fluorescence fluctuation autocorrelation curve, ollowing G(τ)=1N is the ratio of the axial (z) to the lateral (r) extension of that volume such that Here, D) of a given molecule are related, where:The measured diffusion time  with a known diffusion coefficient is used to determine the radius of the observation volume .ith species  equals the inverse of the average number of molecules (According to N) in the observation volume, the molecular brightness  buffer (pH 7.4) at room temperature.In these experiments, we measured time-dependent fluorescence fluctuations of the donor (mTurquoise2.1) in crTC2.1 construct both in the presence  and absence  of the acceptor (mCitrine). The measured time-dependent fluorescence fluctuation traces of the donor were then used to calculate the corresponding autocorrelation curve for each trace, where the corresponding number of molecules was determined and used for molecular brightness per trace prior to statistical analysis. This experimental design helps to eliminate the possible effects of fluorescence blinking on the estimated FRET analysis under 405 nm excitation.2versus 103.9 kW/cm2), within the linear regime of laser intensity dependence of the fluorescence signal. These box and whisker plots display the median as well as the upper and lower quartiles, which best reflect the range of the experimental data at the single-molecule level. For example, the molecular brightness of the intact construct (1.5 ± 0.1 kHz/molecule) is lower than that of the cleaved counterpart (1.8 ± 0.1 kHz/molecule) under the 405-nm excitation laser intensity (55.4 kW/cm2) as shown in 2), the molecular brightness of the intact construct (1.9 ± 0.1 kHz/molecule) is lower than that of the cleaved counterpart (2.3 ± 0.1 kHz/molecule) as shown in 2 excitation laser intensity. A statistically insignificant difference in the calculated FRET efficiency was observed (17.4 ± 5.6)% under 103.9 kW/cm2 excitation laser  was measured on the same construct . The staTaken together, our experimental data support our hypothesis as well as the proposed concept for FRET analysis at the single molecule level using the simple, conventional FCS setup, where the fluorescence fluctuation of the donor in both the intact and cleaved donor–linker–acceptor construct can be measured directly.2) of the acceptor (mCitrine), detected at 531/50 nm. Representative fluorescence fluctuations  construct under 488 nm excitation, which reveal similar molecular brightness for both cleaved (2.7 ± 0.2 kHz/molecule) and intact (2.7 ± 0.2 kHz/molecule) construct (data not shown). The results on GE (mCerulean3–linker–mCitrine) construct support the general applicability of the proposed FCS-FRET approach.It is worth mentioning that the measured translational diffusion times of cleaved and intact crTC2.1  are consAs a proof of concept, we have demonstrated a simple approach for FRET analysis of freely diffusing crTC2.1 construct, at the single molecule level, using a traditional, single detector FCS setup. In this approach, the donor in mTurquoise2.1–linker–mCitrine construct was excited at 405 nm and the fluorescence fluctuation of the donor (475/50 nm) in the presence and absence of the acceptor (mCitrine) are directly measured under the same experimental conditions of laser intensity and detection efficiency. The initial amplitude of the corresponding autocorrelation curve was used to calculate the average number of molecules residing in the observation volume. Then, we calculated the corresponding molecular brightness of both cleaved and intact crTC2.1 construct under the same experimental conditions to rule out any minor changes in the concentration of the prepared samples. These molecular brightness values were then used to determine the energy transfer efficiency in crTC2.1 construct under 405-nm excitation of the donor (mTurquoise2.1).Our results support our stated hypothesis that the molecular brightness of the donor alone is larger than that of the intact mTurquoise2.1–linker–mCitrine construct, which is attributed to FRET. These single-molecule studies also yield a relatively larger FRET efficiency as compared with ensemble studies using time-resolved fluorescence on the same construct . The staControl experiments on the intact and cleaved crTC2.1, under 488 nm excitation of the acceptor, yield a slightly larger molecular brightness of the intact crTC2.1 construct than the cleaved counterpart, which is in contrast to our 405-nm excitation results, due to the absence of FRET. Additional control studies were carried out on another GE (mCerulean3-linker-mCitrine) construct under 488-nm excitation of the acceptor, where the observed molecular brightness of both cleaved and intact construct were the same.It is worth noting that intrinsically fluorescent proteins and their mutations are known to undergo both pH- and light-induced fluorescence blinking . Light-iThe proposed FCS approach takes advantage of the traditional, single-detector FCS setup for selective excitation of the donor in the presence and absence of the acceptor for FRET analysis at the single-molecule level. Another key advantage here is that the proposed approach minimizes the ensemble averaging by at least three orders of magnitude and therefore interesting single molecule events or conformations will not be washed out. Finally, this FCS-FRET approach does not rely on the exact nature of the autocorrelation decay that could be complicated in the presence of a number of photophysical processes in any given donor–linker–acceptor construct.The proposed concept would eliminate the need for high expression levels of these sensors when used in living cells while also eliminating the ensemble averaging that is inherent in other techniques. This FCS concept is generally applicable for FRET analysis in a wide range of donor–linker–acceptor constructs, where the donor and acceptor are fluorescent proteins linked together with amino acid sequence that can be enzymatically digested for control studies. This could be considered as a limitation of the proposed approach here, but that is also the same for any reliable time-resolved fluorescence measurements where control studies on the donor alone must be carried out under the same experimental conditions. To overcome this limitation in future studies in living cells, for example, it is possible to repeat the same site-specific FCS measurements on two cell cultures, one expressing donor–linker–acceptor construct only and another expressing the donor alone. Laser-induced quenching of the acceptor in living cells expressing donor–linker–acceptor construct may also work. For solution studies on untethered FRET pairs, it is also conceivable to vary the relative concentrations of the acceptor with respect to the donor while monitoring the molecular brightness of freely diffusing molecules using a traditional FCS setup.


Blood-brain barrier (BBB) injury is prevalent in patients with HIV-associated dementia (HAD) and is a frequent feature of HIV encephalitis. Signs of BBB damage are also sometimes found in neuroasymptomatic HIV-infected individuals without antiretroviral therapy (ART). The aim of this study was to investigate the integrity of the BBB before and after initiation of ART in both neuroasymptomatic HIV infection and in patients with HAD.We determined BBB integrity by measuring cerebrospinal fluid (CSF)/plasma albumin ratios in archived CSF samples prior to and after initiation of ART in longitudinally-followed neuroasymptomatic HIV-1-infected individuals and patients with HAD. We also analyzed HIV RNA in blood and CSF, IgG Index, CSF WBC counts, and CSF concentrations of β2-micoglobulin, neopterin, and neurofilament light chain protein (NfL).We included 159 HIV-infected participants; 82 neuroasymptomatic individuals and 77 with HAD. All neuroasymptomatic individuals (82/82), and 10/77 individuals with HAD, were longitudinally followed with a median  follow-up of 758 (230–1752) days for the neuroasymptomatic individuals, and a median (IQR) follow-up of 241 (50–994) days for the individuals with HAD. Twelve percent (10/82) of the neuroasymptomatic individuals and 80% (8/10) of the longitudinally-followed individuals with HAD had elevated albumin ratios at baseline. At the last follow-up, 9% (7/82) of the neuroasymptomatic individuals and 20% (2/10) of the individuals with HAD had elevated albumin ratios. ART significantly decreased albumin ratios in both neuroasymptomatic individuals and in patients with HAD.These findings indicate that ART improves and possibly normalizes BBB integrity in both neuroasymptomatic HIV-infected individuals and in patients with HAD. Human immunodeficiency virus type 1 (HIV-1) invades the central nervous system (CNS) early in primary infection . IntrathPrior to the introduction of modern antiretroviral therapy (ART), approximately 30 to 40% of patients with AIDS developed HIV-associated dementia (HAD) , 11. BBBART drastically reduces CSF HIV RNA levels  and CSF The effect of ART on the BBB has only been studied in a few case reports, where patients with HAD showed normalized BBB function after initiation of ART , 22. TheIn this retrospective longitudinal study, we analyzed archived CSF samples from HIV-infected adult (≥ 18 years) individuals with no neurological symptoms, and from individuals with HAD, both before and after initiation of ART, which included three active agents: two nucleoside reverse transcriptase inhibitors plus one non-nucleoside reverse transcriptase inhibitors, protease inhibitor, or integrase inhibitor. The samples were collected from 23 September 1986 until 1 June 2017 at three locations: Sahlgrenska University Hospital, Gothenburg, Sweden; San Francisco General Hospital, San Francisco, CA, USA, and St. Vincent’s Hospital, Sydney, Australia. To meet the inclusion criteria, participants showing no neurological symptoms had to be treatment-naïve, or off treatment for a minimum of 6 months prior to initiation of ART. To study the effect of efficient ART, they had to reach viral suppression within 7 months and remain virally suppressed for more than 6 months. Viral suppression within 7 months was chosen to allow individuals who had a reduction in plasma HIV RNA levels as expected on treatment, but where the lumbar puncture was not performed exactly at 6 months. Blips with a maximum of 500 HIV RNA copies/mL at the time of lumbar puncture during follow-up were considered acceptable. For individuals who had > 1 valid sample date according to the inclusion criteria, the most recent sample before initiation of ART was included in the study. Neuroasymptomatic participants were clinically assessed for neurocognitive and neurologic symptoms. Those who had neurologic symptoms or complaints at baseline, or developed during follow up, were excluded in order to more clearly evaluate the BBB and the associations with other biomarkers. No routine neurocognitive testing was performed on neuroasymptomatic patients.Participants without neurologic signs or complaints were designated neuroasymptomatic. The diagnosis of HAD was based on the Centers for Disease Control and Prevention and American Academy of Neurology Task Force criteria, using standard laboratory, neuropsychological, and clinical evaluations , 24. TreInclusion in the HAD cohort required a lumbar puncture sample both prior to and while on ART. Participants had to be treatment-naïve or off treatment for 6 months or more, and must have reached viral suppression. In accordance with the inclusion criteria participants with HAD who recovered on treatment were not excluded. Since HAD is a rare condition and few individuals had been followed longitudinally prior to and during ART, we also included individuals with HAD who had a lumbar puncture either prior to initiation of ART or while on ART, and who had been virally suppressed for 6 months or more.Lumbar punctures during follow up in all participants were performed according to different schedules depending on the research site. In Gothenburg, lumbar punctures after initiation of ART were made after 3 months, 12 months, and yearly thereafter. In San Francisco lumbar punctures were made frequently during the first 3 months and thereafter with a few months to 1 year’s interval. From Sydney, only cross-sectional data on patients with or without ART was provided.CSF samples were obtained in accordance with research protocols approved by the institutional review boards of each of the study sites, and in compliance with the Helsinki Declaration, either within the context of studies of the natural history of HIV infection , or during diagnostic evaluations (for patients with HAD). All participants gave their informed consent or, if their capacity to provide consent was questioned, consent was also obtained from those with power of attorney.Quantitative determinations of CSF and plasma albumin was performed with nephelometry . Albumin ratio was calculated as CSF albumin (mg/L)/plasma albumin (g/L) and used to evaluate BBB function. Reference values were < 6.8 for individuals younger than 45 years and <  10.2 for individuals ≥45 years of age .+ T-cell counts, CSF leukocytes (WBC), IgG index, and CSF/blood albumin measurements were performed at the local clinical laboratories. IgG index as a marker of intrathecal IgG synthesis was defined as [CSF IgG (mg/L)/serum IgG (g/L)]/[CSF albumin (mg/L)/serum albumin (g/L)] [CSF neopterin was analyzed using a commercially available immunoassay  according to the manufacturer’s instructions. The normal upper reference value for CSF neopterin is 5.8 nmol/L.  CSF NfL n (g/L)] . The refn (g/L)] ; and for10 transformed to reduce skewness.Descriptive statistics were performed using Prism, version 9.0  or SPSS, version 27  or R, version 4.0, software. All continuous variables except age and leukocyte count were logAssociations were analyzed with Pearson correlation coefficients. Independent Student’s t-test was used for comparison between two groups, and Wilcoxon signed rank test was used for evaluation over time at 6 months and 3 years with interpolated values using LOCF (last observation carried forward) if all values were prior to these time points.n = 47) or on ART (n = 20); for these individuals only cross-sectional data were available (Table p < 0.0001), albumin ratio (p < 0.0001), CSF neopterin (p < 0.0001), and CSF NfL (p < 0.0001), and significantly lower CD4+ T-cell count (p < 0.0001) and CD4 nadir (p < 0.0001), compared to neuroasymptomatic participants. There was no significant difference in age between the groups.In total, we included 159 HIV-infected participants; 82 neuroasymptomatic individuals, and 77 with HAD. All of the neuroasymptomatic individuals and 10 of those with HAD were followed longitudinally with lumbar punctures both pretreatment and during ART. Sixty-seven of the participants with HAD had lumbar punctures either only pre-ART  had an elevated albumin ratio at baseline. Baseline characteristics for these ten did not differ compared to the others in the neuroasymptomatic cohort. At the last follow-up 9 % (7/82) had elevated albumin ratios.p < 0.05) and 3 years (p < 0.01) , CSF neopterin , CSF β2-microglobulin , WBC , and CSF NfL  also decreased significantly. There was a trend towards a significant decrease in the IgG index at 6 months (p = 0.06), and a significant decrease after 3 years of treatment (p < 0.001) p < 0.05)p < 0.001) and a significant correlation with age . No correlations were found between albumin ratio and CSF NfL, inflammatory markers such as CSF neopterin, β2-microglobulin, or WBC; nor with plasma or CSF HIV RNA, CD4+ T-cell count, or CD4 nadir.At baseline, there was an inverse correlation between albumin ratio and IgG Index . At the same defined time points, there was a significant reduction of CSF HIV RNA  and CSF neopterin (p < 0.01), p < 0.01),  showed elevated albumin ratios. In the cross-sectional HAD cohort of those with only one lumbar puncture who were on ART, 25% (5/20) had elevated albumin ratios. In the longitudinal HAD cohort, 20% (2/10) had elevated albumin ratios at their last follow-up. For these ten individuals with HAD who were longitudinally followed, the albumin ratio declined, although not significantly, at 6 months; and significantly after 3 years .In the 57 untreated individuals with HAD , no correlation was found between albumin ratio and plasma HIV RNA, CSF HIV RNA, CD4In all longitudinally followed individuals (82 neuroasymptomatic and 10 with HAD), the decrease in CSF viral load and neopterin occurred early after initiation of ART, whereas the decrease in CSF β2-microglobulin and IgG index occurred later. Despite the normalization of CSF HIV RNA, levels of inflammatory markers in CSF remained slightly elevated. In individuals with HAD, the plateau of elevated CSF neopterin was higher than for neuroasymptomatic individuals Fig. .Fig. 3Tin = 631), where 16% of untreated neuroasymptomatic individuals showed elevated albumin ratios. It also is in accordance with other studies [In our study we found that BBB integrity improved after initiation of ART in both neuroasymptomatic individuals and in those with HAD. A small proportion 12%) of neuroasymptomatic individuals had elevated albumin ratios before initiation of ART, as opposed to 80% of the individuals with HAD. This is in the same range as in one of our previous cross-sectional studies n = 61, where % of neur studies , 28. How studies , 20. The studies –31. In o studies . The remSince the majority of the neuroasymptomatic individuals we examined had albumin ratios within the normal range, it is not surprising that the ratios remained fairly stable over time after initiation of ART. The neuroasymptomatic individuals whose BBB was impaired at baseline were similar in immune status and other baseline data. There was a slight, although not significant, difference in age: a median age of 40 years in those with elevated albumin ratios compared to 38 years for those with a normal albumin ratio, which would not explain the discrepancy between baseline levels of albumin ratios. The duration of HIV disease could not be assessed, this may be a cause for the discrepancy. Another possible explanation may be that the BBB impairment is not related to the HIV-infection. We have previously found that neuroasymptomatic HIV-infected individuals have albumin ratios in the same range in as those of HIV negative controls , 30, andIn agreement with the findings of earlier research, the neuroasymptomatic individuals in our study had a significant reduction of CSF viral load , 36 and The albumin ratio in individuals with HAD also declined, but was not statistically significant until the second time point, at 3 years, indicating that improvement of the BBB takes longer in individuals with HAD. Viral replication in individuals with HAD primarily takes place in cells in brain tissue or in tissue macrophages rather than in CSF, as for neuroasymptomatic individuals. This may be reflected in the slower reduction of HIV RNA after ART initiation. Nevertheless, a majority (75%) of those with elevated albumin ratios at baseline normalized this following the initiation of ART.n = 415) [An intact BBB is vital to maintain homeostasis in the CNS and protect the brain from peripheral, potentially neurotoxic, substances. It is composed of a semipermeable layer of a highly specialized capillary bed that surrounds the CNS. The endothelial cells are fused together with tight junctions . Viral pn = 415) , 3, 6. ACSF/plasma albumin ratio is a reliable marker of BBB impairment , 27, andOur study has several limitations. First, the sample size was fairly small, especially for the HAD cohort. As HAD is rare nowadays it is difficult to include many such individuals, although, to the best of our knowledge, ours is at present the largest study. Second, the number of samples during follow-up differs considerably between individuals, some having been followed for many years, and others only a few months. Also, some individuals had a very short interval before their first sample was taken, whereas others had been on ART for several years before their first follow-up sample. Due to a lack of some data in individuals with HAD, important biomarkers, such as IgG index, CSF β2-microglubulin, and CSF NfL, could not be analyzed during follow-up. An additional consequence of the currently rare HAD diagnosis is that data had to be collected over a very long period of time, during which ART has continued to be developed, something which also might have influenced results.In conclusion, albumin ratios declined significantly after initiation of ART in neuroasymptomatic HIV-infected individuals, as well as in patients with HAD, although the decline took longer for the latter group, indicating that healing may be slower in individuals with a more pronounced BBB injury. This would confirm the central role of BBB in the process of neuroinflammation and neuronal injury.


The data suggest that defective DC differentiation in malaria is caused by GM-CSF R dysregulation and GM-CSF R modification by lipoperoxidation product 4-HNE via direct interaction with its CD116 subunit.Malarial pigment hemozoin (HZ) generates the lipoperoxidation product 4-hydroxynonenal (4-HNE), which is known to cause dysregulation of the immune response in malaria. The inhibition of granulocyte macrophage colony-stimulating factor (GM-CSF)-dependent differentiation of dendritic cells (DC) by HZ and 4-HNE was previously described in vitro, and the GM-CSF receptor (GM-CSF R) was hypothesised to be a primary target of 4-HNE in monocytes. In this study, we show the functional impact of HZ on GM-CSF R in monocytes and monocyte-derived DC by (i) impairing GM-CSF binding by 50 ± 9% and 65 ± 14%, respectively ( Malaria is still the most important life-threatening parasitic disease according to the World Health Organisation report, with an estimated 229 million cases and 409,000 deaths occurring in 2019. The immune response during malaria is complex, and immune depression has been described as an important malaria complication ,4,5. MecStudies in malaria models and patients revealed a role of malaria pigment hemozoin (HZ) as pathomechanistic factor in malaria. The nanocrystal (core) of HZ is formed from hemoglobin-heme by the malaria parasite during its proliferation in the infected host red blood cell (RBC). The appearance of HZ in circulating monocytes ,7 is assOxidative stress and lipoperoxidation play important roles in malaria pathogenesis ,23,24,25Here, we show changes in GM-CSF R expression and posttranslational modifications of the receptor by 4-HNE, which both may explain the impaired DC differentiation in HZ-laden or 4-HNE-treated precursor cells. The observed modifications of GM-CSF R may contribute to gain insight into the inadequate immune response described in malaria.Unless otherwise stated, reagents were purchased from Merck Sigma Aldrich . Cell culture supplements were obtained from Invitrogen Thermo Fisher Scientific .Pf parasites  were kept in culture with donor RBC as described [g on a discontinuous Percoll-mannitol density gradient, HZ was collected from the 20/40% and trophozoites and schizonts from the 40/80% interphase. HZ was washed five times with 10 mM HEPES (pH 8.0) containing 10 mM mannitol at 4 °C and once with phosphate-buffered saline (PBS), and stored at 20% (v/v) in PBS at −20 °C.This study was carried out in accordance with the Declaration of Helsinki and authorised by the local Ethical Committee. Blood from healthy adult donors of both sexes was obtained from the local blood bank , treated with heparin, and promptly used for RBC isolation. escribed . HZ was v/v), and latex beads (0.114 µm diameter) suspended at 5% (v/v) in RPMI 1640 were added separately to the same volume of fresh human AB serum and incubated for 30 min at 37 °C for opsonisation.HZ was thawed, washed once, and finely dispersed in PBS at 30%  suspended at 50% hematocrit in PBS, supplemented with 10 mM glucose (PBS-G), were incubated with half the volume of human anti-D IgG  for 30 min at 37 °C for opsonisation, and washed three times with PBS-G.The timeline for cell treatment and analysis is summarised in 6 cells per millilitre of RPMI 1640 cell culture medium supplemented with l-glutamine, sodium pyruvate, penicillin/streptomycin, nonessential amino acids (RS), and 1% (v/v) FCS (RS-FCS medium), were plated in each 35 mm diameter culture dish. After a 30 min incubation at 37 °C, dishes were washed three times with RPMI 1640 to remove non-adherent cells. Two millilitres of RS supplemented with 5% (v/v) human AB-serum  was added to each dish. Phagocytosis and cell treatment were started at time 0 by adding either opsonised HZ , RBC (50 cells per monocyte), latex beads (10 µL of a 100-fold dilution of the opsonised latex bead suspension per 106 monocytes), or 4-HNE at 1 µM final concentration to adherent monocytes. The addition of 4-HNE at 1 µM final concentration was repeated each second day after cell culture medium replacement before cytokine supplementation, unless otherwise noted. In some experiments, 10 µM 4-HNE was added once as bolus at time 0. 4-HNE was freshly activated from (E)-4-hydroxynonenal-dimethylacetal, according to manufacturer protocol, immediately before being added to cells. Untreated monocytes were kept in parallel as control under the same conditions. Dishes with control monocytes, HZ-, RBC-, latex-fed, and 4-HNE-treated monocytes were prepared in triplicate.Monocytes were isolated from freshly drawn peripheral blood of healthy human donors  by Ficoll centrifugation and lymphocyte depletion with PanT/PanB-Dynal beads , as described previously . Two milAfter a 3 h incubation, phagocytosis was stopped by three washings with RPMI 1640, and phagocytosis was assessed in one of the replicate dishes with RBC- and HZ-fed cells and compared to controls. Two replicate dishes with monocytes of any treatment were further incubated for differentiation to DC (see below) and analysis after 24 h and 7 days.HZ and RBC phagocytosis by monocytes was quantified by luminescence, as described previously , and visAdherent washed control monocytes, HZ-, RBC-, latex bead-fed, or 4-HNE-treated monocytes were supplemented with 80 ng/mL GM-CSF and 40 ng/mL IL-4 (R&D) in RS-HS medium for differentiation as shown in the timeline . At the Monocyte and DC phenotypes were characterised in terms of CD1a, CD14, CD40, CD83, CD87, MHC class II , or MHC class I  antigen expression by flow cytometry  before monocyte treatment at time 0, then at 24 h after starting phagocytosis and at day 7 of differentiation to DC . The surCD116 expression was assessed by FITC-conjugated mouse anti-human CD116 antibody   in humanw/v), and incubated with saturating concentrations of anti-4-HNE monoclonal mouse primary antibody (1:50 dilution) at room temperature for 1 h. After three washes, bound antibodies were revealed by FITC-conjugated anti-mouse IgG secondary antibody (1:300 dilution). MFI was measured and data were analysed using a FACS Calibur cytofluorograph  and CellQuest software (Becton–Dickinson).Conjugates of 4-HNE with cell surface antigens were quantified by anti-4-HNE-conjugate antibody  ,46 in stFor functional characterisation of GM-CSF R, FITC-conjugated GM-CSF was supplemented instead of unconjugated reagent, on the basis of the manufacturer’s indications . Ligand binding was assessed as MFI, and data were analysed using a FACS Calibur cytofluorograph .Adherent cells were washed with ice-cold PBS-G, containing 5 mM mannitol  and Complete protease inhibitor cocktail , and lysed with Laemmli sample buffer in the tissue culture dish to avoid artefacts by cell harvesting. The quantity of lysate proteins was determined by the Bradford protein assay . Equal amounts of proteins of different lysate samples were separated by SDS-PAGE and transferred to nitrocellulose membranes by Western blotting using standard protocols (Bio-Rad). Transfer efficiency and protein amount in each lane were checked by Ponceau red staining of the nitrocellulose membrane. CD116 presence in the lysate of different samples was detected by anti-CD 116  and visualised by horseradish peroxidase-conjugated secondary anti-mouse antibody and enhanced chemiluminescence (ECL) assay (Bio-Rad). To guaranty the comparison of same cell numbers, we detected actin as housekeeping protein by anti-actin C-2 monoclonal mouse antibody , and visualised it by secondary anti-mouse antibody and ECL as specified for CD116. The arbitrary optical density of labelled bands was acquired and compared using ImageLab software version 4.1 (Bio-Rad).Protein bands localised by anti-CD116 antibodies after Western blotting were excised from a duplicate gel and subjected to in-gel tryptic digestion and mass spectrometry analysis, as described previously . ProteinApoptosis was tested by FACS after annexin V–FITC staining, following the manufacturer’s specifications .p-value) was calculated by non-parametric Mann–Whitney test. For the significance test: * p < 0.01, ** p < 0.05.The values from at least three independent biological replicates are presented as the mean ± SE. Statistical significance . While RBC were digested and not seen after 24 h, HZ and latex beads remained visible inside differentiating monocytes in initial quantity for the whole period of observation (up to 7 days) confirming the persistent presence of ingested HZ (approximately 20 fmole heme for monocyte).Monocyte phenotype analysis at start of experiment (immediately before treatments/stimulations) and at 24 h after phagocytosis showed the cell positivity for CD14, MHC class II, MHC class I, CD40, and CD87, as well as negativity to apoptosis, as annexin-V-FITC low binding was observed in less than 2% of cells (data not shown). Cell phenotype analysis at day 7 of monocyte differentiation to DC revealed the inhibition of MHC class II and CD 1a (antigen presenting molecules), CD83 (accessory molecule), and CD40 (costimulatory molecule) expression on HZ-fed and 4-HNE-treated DC as compared to unfed control DC , mainly The modified cell phenotype at day 7 was associated with increased levels of 4-HNE-protein conjugates measured on the surface of GM-CSF/IL-4-stimulated HZ-fed or 4-HNE-treated monocytes 24 h after HZ phagocytosis and GM-CSF/IL-4 supplementation and on DC at day 7 of differentiation . CompareDue to the impaired GM-CSF/IL-4-dependent differentiation, we hypothesised that the binding capacity of GM-CSF R was affected after HZ phagocytosis. As shown in . CD116 cell surface expression was decreased by 36 ± 11% and 39 ± 13% after HZ phagocytosis or exogenous 4-HNE treatment, respectively (To explain the decrease in GM-CSF binding, we evaluated the surface expression of GM-CSF R on HZ-fed and 4-HNE-treated monocytes 24 h after the respective treatment and measured it as MFI A or sharectively A, and thectively B. We notectively B. The obAfter the observation of surface CD116 reduction, whole cell lysate proteins were checked for GM-CSF R α-chain expression. Decrease of CD116 after HZ by 58 ± 16% and 4-HNE treatment was observed , suggestGiven the impaired GM-CSF R functionality  and quanIn HZ-treated monocytes, we observed the binding of 4-HNE to distinct amino acid residues in CD116 . PosttraDC differentiation from human primary monocytes was formerly shown to be strongly impaired by malarial pigment HZ, which is avidly phagocytosed but not digested by the natural precursor cell of DC, in vitro and in vivo . ConsequThe present study confirms the formerly described inhibition of GM-CSF/IL4-dependent ex vivo differentiation of HZ-fed monocytes to DC, as concluded from the inadequate change in expression of functionally relevant surface antigens . The effLipoperoxidation as cause for downregulation of CD116, as observed during initial pro-inflammatory immune cell response to malaria product HZ, may not be restricted to malaria but may also underlay other inflammatory pathologies, accompanied by a decreased receptor expression such as chronic alcohol intoxication  or inflaFinally, 4-HNE affected not just the expression/degradation but also modified the protein structure of GM-CSF R in HZ-fed or 4-HNE-treated monocytes. The highly reactive aldehyde formed adducts with four cysteine residues of the CD116 alpha chain of the receptor, two of them, the cysteins in position 228 and 233, in close proximity to amino acids threonine 231 and histidine232, which are crucially engaged in alpha-beta-chain interface formation . Figure The introduction of 4-HNE adducts in the nascent protein chain during translation and export may also impact on CD 116 export to the membrane, thus resulting in decreased expression and enhanced degradation, a commonly observed event for 4-HNE tagged proteins . GM-CSF Pf malaria [In summary, HZ and HZ-derived lipoperoxidation product 4-HNE profoundly interfere with the expression, structure, and function of GM-CSF R in human DC precursor cells, resulting in the impairment of GM-CSF/IL4-dependent DC differentiation in vitro. Interference of 4-HNE with immune cells may also explain the inhibitory effects on peripheral blood DC observed in children with acute  malaria , and thuMalaria pigment HZ is known to strongly impair several immune responses such as DC differentiation. Oxidative stress and lipoperoxidation are hallmarks of malaria and other infectious and inflammatory diseases. HZ and lipoperoxidation-derived 4-HNE are shown here to modify the cellular GM-CSF R expression and provoke posttranslational modifications of the receptor, which both may explain the impaired DC differentiation in HZ-laden or 4-HNE-treated precursor monocytes. The observed modifications of GM-CSF R by lipoperoxidation products may contribute to gain insight into the inadequate immune response described in malaria but may also play a role in other inflammatory diseases.


Cerebrospinal fluid (CSF) circulating in the human central nervous system has long been considered aseptic in healthy individuals, because normally, the blood-brain barrier can protect against microbial invasions. However, this dogma has been called into question by several reports that microbes were identified in human brains, raising the question of whether there is a microbial community in the CSF of healthy individuals without neurological diseases. Here, we collected CSF samples and other samples, including one-to-one matched oral and skin swab samples (positive controls), from 23 pregnant women aged between 23 and 40 years. Normal saline samples (negative controls), sterile swabs, and extraction buffer samples (contamination controls) were also collected. Twelve of the CSF specimens were also used to evaluate the physiological activities of detected microbes. Metagenomic and metatranscriptomic sequencing was performed in these 116 specimens. A total of 620 nonredundant microbes were detected, which were dominated by bacteria (74.6%) and viruses (24.2%), while in CSF samples, metagenomic sequencing found only 26 nonredundant microbes, including one eukaryote, four bacteria, and 21 viruses (mostly bacteriophages). The beta diversity of microbes compared between CSF metagenomic samples and other types of samples (except negative controls) was significantly different from that of the CSF self-comparison. In addition, there was no active or viable microbe in the matched metagenomic and metatranscriptomic sequencing of CSF specimens after subtracting those also found in normal saline, DNA extraction buffer, and skin swab specimens. In conclusion, our results showed no strong evidence of a colonized microbial community present in the CSF of healthy individuals.IMPORTANCE The microbiome is prevalent throughout human bodies, with profound health implications. However, it remains unclear whether it is present and active in human CSF, which has been long considered aseptic due to the blood-brain barrier. Here, we applied unbiased metagenomic and metatranscriptomic sequencing to detect the presence of a microbiome in CSF collected from 23 pregnant women with matched controls. Analysis of 116 specimens found no strong evidence to support the presence of a colonized microbiome in CSF. Our findings will strengthen our understanding of the internal environment of the CSF in healthy people, which has strong implications for human health, especially for neurological infections and disorders, and will help further disease diagnostics, prevention, and therapeutics in clinical settings. First defined by Joshua Lederberg in 2001 28. It has9–13–Porphyromonas gingivalis was identified in brain regions, including the cerebral cortex and hippocampus, in patients with Alzheimer’s disease , circulating in the human central nervous system (CNS), has long been considered sterile, given that the blood-brain barrier can effectively protect against microbial invasions. However, the dogma has been challenged in recent years by several reports of microbes being detected in human brains and CSF. For example, the bacterial pathogen  disease . In addi disease . AlthougGiven the debate over the existence of any microbial community in CSF and the importance of understanding microbial infection in the human CNS, we have performed microbiome analysis to characterize bacteria, archaea, eukaryotes, and viruses in CSF samples from 23 donors without neurological disorders, as well as one-to-one matched positive controls  and negative controls . DNA/RNA extraction buffers and sterile swabs were collected as contamination controls. In total, 116 specimens were used in this study. Considering the limitations of the 16S rRNA gene-based approach in identifying microbes to the species or strain level –23, unbi–26–n = 26), negative controls (n = 49), and extraction buffers (n = 27) were relatively fewer  and 12 CSF specimens for metatranscriptomic sequencing (CSF RNA), 23 normal saline specimens as negative controls for CSF sampling, 23 skin and 23 oral swab specimens as positive controls, and 6 sterile swabs and 6 DNA/RNA extraction buffer specimens as contamination controls . These results were consistent with previous studies   were compared in this study. Nonmetric multidimensional scaling (NMDS) analysis , princip = 0.59) , but theEscherichia virus V5, Klebsiella virus KP27, Macaca mulatta polyomavirus 1, and Trichoplusia ni single nucleopolyhedrovirus), two bacteria , and one eukaryote (Aspergillus turcosus).We questioned whether these detected microbes were truly CSF inhabitants or simply brought in from external sources, such as skin, sampling equipment, and DNA extraction buffer. To reduce the exogenous DNA noise of CSF samples, we subtracted the microbes collectively detected in negative controls  and DNA extraction buffer specimens from the microbes of each CSF DNA specimen. After subtraction, 12 CSF DNA specimens had no remaining microbes, whereas the other 11 CSF DNA specimens contained a total of 14 microbes, including 11 viruses, 2 bacteria, and 1 eukaryote . Since aEscherichia coli in 11 specimens. Interestingly, when only considering the specimens with suspected colonized microbes by metagenomic sequencing after subtraction of microbes found in negative controls and DNA extraction buffers, bovine alphaherpesvirus 1 and human alphaherpesvirus 1 were found in CSF by both metagenomics and metatranscriptomics. However, human alphaherpesvirus 1 was also detected in skin swab specimens and bovine alphaherpesvirus 1 appeared in unmatched CSF DNA and CSF RNA specimens. It is worth noting that we detected bovine alphaherpesvirus 5 in 13 negative controls and 1 skin swab specimen. At the genus level, simplexvirus and varicellovirus, which appeared in both CSF DNA and CSF RNA specimens, also appeared in negative controls and skin swab specimens, suggesting that these species potentially originated from skin or other contaminations. Although metagenomic analysis detected the Aspergillus turcosus species in specimens from four individuals , contamination controls (DNA extraction buffer), and positive controls (skin swab specimens). After the subtraction, 8 microbes that were potentially CSF-inhabiting microbes were obtained. Viruses were the most common before and after subtraction. Interestingly, most of these viruses were bacteriophages. Previous studies also found bacteriophages in CSF specimens . HoweverAspergillus turcosus detected in CSF DNA specimens. Aspergillus turcosus is well known as an opportunistic pathogen and can infect individuals with compromised immune systems. However, it is rarely reported in clinical samples  of the body, experiment reagents, and consumables. In addition, during the lumbar puncture, tissues such as skin, muscle, and blood vessels can also be potential sources of contamination. However, due to technical and ethical restrictions in the actual operation process, it is difficult to collect specimens of all kinds of tissues that are exposed during the sampling process. For example, it is very difficult to collect muscle and blood specimens during the collection of CSF, especially for healthy people. Moreover, we need to be very cautious when designing a control, because whether the control itself contains microbes will also be controversial. Thus, strict disinfection measures before the operation and the construction of a database of colonizing microorganisms of these tissues will help filter out noise signals and reduce the false-positive rate.The main challenge of this study is an overall lack of CSF samples from healthy human subjects. Only 23 pregnant females aged from 23 to 40 were enrolled. This is because lumbar puncture to collect CSF is an invasive surgery and will cause pain and damage to the subjects. Usually, only patients with CNS infections will receive lumbar punctures. Lumbar puncture in healthy people is rare. In addition, technically sound sampling, as well as data analysis methods based on different reference databases and taxonomic strategies, can also result in some biases.In conclusion, using metagenomic combined with metatranscriptomic deep sequencing, we found that the microbiome profile in CSF specimens was indistinguishable from that in negative controls, indicating no strong evidence of a colonized microbial community in the CSF of healthy individuals. Such findings will strengthen our understanding of the internal environment of the CSF in healthy people, which has strong implications for human health, especially for neurological disorders and infections, and will help to further disease diagnostics, prevention, and therapeutics in clinical settings.A total of 23 donors who needed intraspinal anesthesia before caesarean section were recruited from the Department of Obstetrics, Xijing Hospital of the Fourth Military Medical University, during the summer of 2018. Subjects who had suffered from central nervous system infectious diseases , systemic infectious diseases (hepatitis and tuberculosis), or autoimmune diseases  or had received antibiotic treatment in the past 6 months were excluded. Subjects with a history of hypertension, diabetes, heart disease, cancer, or neurological diseases  were also excluded. This study was approved by the Ethics Committee of the Xijing Hospital of the Fourth Military Medical University. All procedures were conducted in accordance with the approved guidelines. All donors read and signed the consent form before sample collection.To investigate whether there is a microbiome in CSF, we collected CSF samples from 23 pregnant women aged 23 to 40 years who underwent intraspinal anesthesia via lumbar puncture before caesarean section, and we coupled these samples with normal saline samples collected by syringe as negative controls. For each subject, oral and skin swab specimens were also collected as one-to-one-matched positive controls. Six sterile swabs and 3 DNA extraction buffer specimens were also collected as contamination controls. All samples were then subjected to DNA extraction and metagenomic sequencing. Finally, to validate whether the microbiome, if any, detected in CSF was physiologically active, metatranscriptomic sequencing was performed for 12 of the pregnant women’s CSF specimens. Three RNA extraction buffer specimens were also collected as contamination controls.2 area around the puncture site  was swabbed using a sterile cotton swab before the skin was cleaned with povidone iodine. To maximize the microbial load, no bathing was permitted within the 24 h before sample collection. For oral swab samples, the surfaces of the tongue, buccal fold, hard palate, soft palate, teeth, gingiva, and saliva  were swabbed with sterile swabs. All subjects fasted for at least 6 h before the operation. Unused sterile swabs were also collected as contamination controls. Details of collected samples are described in Fig. S1 and Table S2.Lumbar puncture was performed in the 23 subjects enrolled in this study. CSF was collected into a 4-ml centrifuge tube and then transferred to a −80°C freezer for metagenomics sequencing. Twelve CSF specimens were randomly selected from the 23 pregnant women for metatranscriptomic studies , and RNA protection reagent was added to the CSF immediately after collection. The skin of the back of each individual in the 5- by 5-cmDNA was extracted using the QIAamp DNA minikit  according to the manufacturer’s instructions. Swab tips were cut into a 2-ml microcentrifuge tube. A total of 400 μl of phosphate-buffered saline (PBS), 20 μl of proteinase K, and 400 μl of buffer AL was added into the tube. After eddying for 10 s, swab samples were incubated at 56°C for 15 min, and 400 μl ethanol (100%) was mixed again with each sample. Samples were then transferred into QIAamp mini-spin columns. After washing with buffer AW1 and AW2, DNA of each sample was stored in 35 μl of buffer EB at −20°C. From the CSF and normal saline samples, 200 μl of each sample was mixed with 20 μl of proteinase K and 200 μl of buffer AL. After incubating at 56°C for 15 min, 200 μl ethanol (100%) was added and mixed in. DNA was then extracted as described above.The QIAseq FX DNA library kit  was used to construct DNA libraries. From each sample, 32.5 μl purified DNA was fragmented into 200- to 300-bp segments by incubation with 5 μl of FX buffer, 2.5 μl of FX enhancer, and 10 µl of FX enzyme mixture in cycles of 4°C for 1 min, 32°C for 12 min, and 65°C for 30 min. Five microliters of adaptor, 20 μl of ligation buffer, 10 μl of DNA ligase, and 15 μl of nuclease-free water were added, and the mixture incubated at 20°C for 15 min to initiate adapter ligation. Adapter ligation cleanup was performed immediately using 80 µl of resuspended AMPure XP beads (0.8×). After incubating at room temperature for 5 min, the beads were pelleted on a magnetic stand (Invitrogen) for 2 min. The supernatant was discarded, the pellet was washed twice with 200 µl of 80% ethanol, and then the beads were eluted with 52.5 µl of buffer EB. Subsequently, the supernatant was transferred into a new 1.5-ml microcentrifuge tube for a second purification using 50 µl AMPure XP beads (1×). The purified DNA was then used to construct the DNA library using the QIAseq FX DNA library kit . After mixing the library product with 25 μl of HiFi PCR master mix and 1.5 μl of primer mix, PCR enrichment was performed under cycling conditions of 2 min at 94°C, 12 cycles of 20 s at 98°C, 30 s at 60°C, and 30 s at 72°C, and 1 min at 72°C. The PCR products were finally purified with AMPure XP beads as described above.Total RNA was extracted using the RNeasy minikit  according to the manufacturer’s instructions. The pellet of each sample that had been treated with RNA protection reagent as described above was resuspended in 100 μl Tris-EDTA (TE) buffer containing lysozyme. Proteinase K was added, and the mixture incubated at room temperature for 10 min. After mixing in 350 μl of buffer RLT, RNA isolation and purification were performed with buffers AW1 and RPE, respectively, using the RNeasy mini-spin column, followed by elution with RNase-free water.2O sc, and 10 μl of the diluted DNA and FX enzyme mix was used to obtain 300-bp library sequences with the following reaction conditions: 4°C for 1 min, 32°C for 15 min, 65°C for 30 min, and a 4°C hold. Five microliters of adapter and 45 μl of ligation master mix were added into each sample, and the mixture incubated at 20°C for 15 min. Subsequently, the adapter ligation cleanup was performed with AMPure XP beads as described above. The purified libraries were finally obtained for sequencing without further PCR amplification.The QIAseq FX single-cell RNA library kit  was used to construct RNA libraries. A total of 8 μl of purified RNA and 3 μl of NA denaturation buffer were added into a sterile PCR tube and incubated at 95°C for 3 min. To remove genomic DNA (gDNA), 2 μl of gDNA wipeout buffer was added and the mixture incubated at 42°C for 10 min. Four microliters of RT buffer, 1 μl of random primer, 1 μl of oligo(dT) primer and 1 μl of Quantiscript RT enzyme mix were added to each sample prior to RT at 42°C for 60 min. Eight microliters of ligase buffer and 2 μl of ligase mix were added into the reverse transcription reaction mixture and incubated at 24°C for 30 min. Then, 1 μl of REPLI-g SensiPhi DNA polymerase and 29 μl of reaction buffer were used for multiple displacement amplification (MDA) at 30°C for 2 h. A length of approximately 2,000 to 70,000 bp of amplified cDNA was finally produced. The amplified cDNA was diluted 1:3 in HShotgun sequencing was performed on the Illumina HiSeq platform for all samples (paired-end library with 150-bp read length). Approximately 25 Gb and 5 Gb of raw paired-end reads were obtained per sample in CSF DNA-positive and -negative samples, respectively.To reduce the impact of host reads, we needed to remove human reads from the raw sequencing data before bioinformatics analysis. KneadData (version 0.7.4) , a widelMetaPhlAn (version 3.0.1)  is a comβ-Diversity (between-sample diversity) was estimated by Bray-Curtis dissimilarity in the vegan package of R (version 4.1.0). All figures were also plotted using R.https://ngdc.cncb.ac.cn/gsa.The clean sequence data reported in this paper have been deposited in the Genome Sequence Archive in the BIG Data Center , 36, Chi


The cerebrospinal fluid (CSF) flows through and around the central nervous system to nourish, cleanse, and support the brain and spinal cord. Though abnormalities of this CSF flow have been linked to multiple human neural diseases, little is known about the underlying mechanics of CSF flow. This study was designed to test the hypothesis that movement of the body’s trunk could cause CSF flow; hence, the study was conducted on a snake, an animal with prominent trunk movement. The results demonstrate that the resting snake has a CSF pressure profile that is very similar to what is seen in humans and other mammals, and that the CSF dynamics are changed during either artificial  or natural (locomotor) movement of the snake’s bodyCandoia aspera), the cerebrospinal fluid (CSF) shows two stable overlapping patterns of pulsations: low-frequency (0.08 Hz) pulses with a mean amplitude of 4.1 mmHg that correspond to the ventilatory cycle, and higher-frequency (0.66 Hz) pulses with a mean amplitude of 1.2 mmHg that correspond to the cardiac cycle. Manual oscillations of anesthetized C. aspera induced propagating sinusoidal body waves. These waves resulted in a different pattern of CSF pulsations with frequencies corresponding to the displacement frequency of the body and with amplitudes greater than those of the cardiac or ventilatory cycles. After recovery from anesthesia, the snakes moved independently using lateral undulation and concertina locomotion. The episodes of lateral undulation produced similar influences on the CSF pressure as were observed during the manual oscillations, though the induced CSF pulsations were of lower amplitude during lateral undulation. No impact on the CSF was found while C. aspera was performing concertina locomotion. The relationship between the propagation of the body and the CSF pulsations suggests that the body movements produce an impulse on the spinal CSF.In the viper boa ( Alligator mississippiensis) revealed that this mammalian pattern is not universal; in the alligator, there are both cardiac and ventilatory influences on the CSF, but they are far more variable than what has been reported from any mammal [Perspectives on the cerebrospinal fluid (CSF) have changed; what once was seen as an almost “passive” supporting substance is now generally seen as a dynamic system, perturbations of which can cause a number of neural disorders ,2. Despiy mammal . The basy mammal ,8 and vey mammal ,10 of thAlligator influences central venous pressure or CSF flow dynamics.Relating the alligator and mammalian CSF dynamics is challenging not only because of the greater plasticity of the alligator systems, but also because of the limits of our understanding of the underlying mechanics of CSF flow. Multiple studies have documented the relationship between the ventilatory cycle and CSF flow in mammals ,12,13, bWhile the cardiac and ventilatory cycles may have the greatest effect on CSF dynamics, other influences have been documented. Postural shifts create orthostatic gradients that influence CSF pressure in humans , other mInsight into these issues of CSF propulsion might be gained through an examination of CSF flow dynamics in snakes (Order Serpentes). Snakes have some of the same cardiac specializations that occur in crocodilians ,31, thouCandoia aspera. This is a terrestrial species [Candoia aspera is a relatively heavy-bodied species [C. aspera, particularly as they relate to the axial deflection typical of serpentine locomotion.The purpose of this study is to explore the CSF dynamics in the viper boa,  species  and so p species . Candoia species  and so w species . Among o species . Herein,Candoia aspera were obtained commercially. The snakes were housed in (60 × 120 × 35 cm) terraria within a special reptile-holding facility with a 12:12 hour light cycle, water ad libitum, and a temperature range of 28 to 32 °C. The animals were maintained on a diet of live and/or previously frozen rodents . Seven adult specimens  of The snakes were placed  in an induction chamber and exposed to isoflurane until their righting reflex was extinguished and they no longer responded to tactile stimulation. The snakes were removed from the induction chamber, given an analgesic , then intubated. The snake was then placed on a stiff board (244 × 28 × 3.8 cm thick). The endotracheal tube was connected to a custom anesthesia system that included a ventilator pump , Vaporstick anesthesia machine , isoflurane vaporizer (Surgivet), and Capnomac Ultima respiratory gas monitor . The snakes were maintained on a ventilatory pattern of 5 breaths per minute; the tidal volume, pressure, and peripheral resistance of the anesthesia machine were adjusted for each snake so that the airflow entering the snake’s respiratory system was just enough to cause body movement over the vascular lung. Once it was determined that a surgical plane of anesthesia was established, the isoflurane was reduced to 1.5–2%.The snakes’ EKGs were recorded using two silver chloride surface cup electrodes , coated with a layer of conducting gel . The electrodes were placed on the lateral surface of the snake on either side of the heart. The electrodes were connected to a P511 preamplifier (GRASS). A stainless steel surgical burr was used to bore an approximately 1.5 mm-diameter portal through the skull, slightly parasagittal and just caudal to the parietal–frontal suture. In snakes, as in all terrestrial vertebrates, the outer surface of the brain is bathed in CSF, which is isolated from the skull by the dura matter. Pre-surgical dissections indicated that at the location of the surgical portal through the skull, the dura was further away from the surface of the brain, resulting in a localized increase in the volume of CSF (a cistern). The surgical portal allowed for direct exposure of the dura mater surrounding this cistern; a small incision in the dura was used to inset a segment of PE tubing into the CSF-filled subarachnoid space. The PE tubing was connected to a P23AA fluid pressure transducer , both of which were filled with reptilian Ringer’s solution. The pressure transducer was mounted to the board at a fixed site immediately adjacent to, and level with, the snake’s head, so that rotation of the snake did not produce a pressure head between the PE tubing and the transducer. The implanted PE tubing was snug, and the attachment was further secured by applying epoxy cement to the PE tubing and surrounding bone. The pressure transducer was coupled to a P122 preamplifier (GRASS). The CSF pressure, EKG, and ventilatory pattern  were recorded simultaneously (at 2 kHz) using the MiDas data acquisition system . Ninety second data records were gathered while the snake was anesthetized and being ventilated at 5 breaths/min; at least one recording per snake was taken in which the ventilator was turned off, forcing the snake into apnea. The CSF pressure transducers were individually calibrated following each experiment.The board holding the snake was anchored to a rotating spindle machined to have, in addition to a stable horizontal stop, fixed “stops” at 30° above and below the horizon. The snake’s head and tail were taped to the board so that the snake did not shift relative to the board, then 90 s rotation trials were performed. Each trial consisted of a 30 s horizontal baseline, a 30 s period in the rotated posture, then a 30 s return phase. To avoid artifacts (vibration in the board), none of the “transitional” periods were analyzed. Following the orthostatic trials, the EKG leads and the tape holding the snake’s tail to the board were removed. Hypo-allergenic fingernail polish was used to place visible markers at intervals (approximately every 5 cm) along the dorsal midline of the snake. A digital camera  recording at 480 fps was positioned over the snake. The snake was still anesthetized, and thus had little body tone; by manually wiggling the tail back-and-forth, it was possible to create oscillatory waves that propagated along the snake’s body (to the fixed head). Care was taken not to elevate the tail or any portion of the snake’s body. An LED flash was used to synchronize the video and CSF pressure recordings. https://www.kinovea.org/download.html, accessed on 22 May 2021), which was used to quantify the kinematics of the marker points on the snake’s body. For statistical comparison, the kinematic traces and CSF pulses were rectified and normalized. Kinematic and CSF traces were imported into Spectra-Plus  for FFT and Power Spectrum analysis.Following these axial deflection trials, the snake was allowed to fully recover from the anesthesia, with the CSF pressure catheter still in place. When the snake was fully recovered from the anesthesia  the snake was placed in a 120 × 47 cm filming cage which was lined with a soft material . Synchronized high-speed digital video and CSF pressure recordings were made while the snake was locomoting (unrestrained). All video records were exported to motion analysis software  heart rate of 40.1 beats per minute (bpm), with a standard deviation of 8.9. The standard deviation reflects that each snake had a distinct heart rate, with means ranging from 25.3 to 49.3 bpm. The specimens ranged in mass from 182 to 460 g; linear regression revealed that heart rate decreased by 0.5 bpm for every 10 g of snake body mass.The seven specimens of t-test found no significant difference  in the changes in heart rate resulting from head-up or head-down rotations.When the snakes were rotated 30° head-up, the mean heart rate decreased by 0.6% (s.d. = 4.4%); this decrease changed the mean heart rate from 40.1 to 39.86 bpm. Rotating the snakes to 30° head-down increased the mean heart rate by 0.04% (s.d. = 3.9%); this increase changed the mean heart rate from 40.1 to 40.12 bpm. A paired Candoia, there were two different frequencies evident in the pressure pulsation of the CSF .The CSF had a mean resting pressure of 4.2 mmHg (s.e. = 0.3). In  the CSF A. The hip = 1.11 × 10−16, df = 6). Linear regression demonstrated that the amplitude of the ventilator-related pulses decreased with increasing body size . The low-frequency pulses (which occur at approximately 0.08 Hz) had an amplitude that was some 3.5× greater than those of the higher-frequency pulses (which occurred at approximately 0.66 Hz).The lower-frequency pressure pulsations were temporally correlated to the ventilator cycle, and disappeared during periods of induced apnea A. PoolinPower spectrum analyses of 90 s recordings of CSF pressure from horizontal anesthetized snakes showed a similar pattern; the frequencies associated with the ventilator cycle consistently had greater power than the frequencies associated with the cardiac cycle B. If thet = 5.88, p < 0.00001, df = 19) than were the decreases in CSF pressure caused by head-up rotation; and (3) the amplitude of the high-frequency CSF pulses increased following head-down rotation, and decreased following head-up rotation  the CSF pressure changed rapidly upon rotation, and then held stable until the snake was returned to horizontal ; (2) therotation . The 30°The manual axial deflections were performed as the snake was recovering from anesthesia, but prior to the snake developing muscle tone. The EKG leads were removed, and to preserve the integrity of the CSF pressure catheter the snake’s head was taped to the board (those forming a stationary node for the undulations). The endotracheal tube was left in place, and was connected to the ventilator during some of the trials; in other trials (where the snake exhibited more signs of recovery), the endotracheal tube was not connected to the ventilator. By moving the snake’s tail rapidly horizontally it was possible to produce a series of lateral undulations which propagated towards the snake’s head . PoolingThese manual oscillations were not intended to duplicate locomotion; rather, the goal was to induce vertebral deflection and rapid but realistic changes in direction . Every t2 = 0.48; this slope was significantly different from zero, t = 4.15, p = 0.0005, df = 19).As shown in The CSF pressure traces recorded during the manual oscillations were all characterized by the presence of pulsations A. These T-test revealed that the dominant frequencies from the CSF pressure were significantly higher than those from the kinematic data . Regression analysis revealed that increasing the dominant frequency of the kinematic displacement resulted in an increase in the dominant frequency of the CSF traces that was significantly different from zero , and that there was considerable variation around the regression line . The kinematic displacement and CSF pressure curves have the same duration. After the curves are rectified and normalized, there is a positive regression relationship  between the areas under the curves which is significantly  greater than zero (so increasing kinematic displacement increases CSF pressure). FFT and power spectral analyses were performed on the kinematic data of displacement of the snake’s body during the manual oscillations. The results B show a 2 = 0.37; this slope was significantly different from zero, t = 3.35, p = 0.003, df = 19).As shown in p = 5.21 × 10−8, n = 31); Tukey’s post hoc analysis found significant differences (using a threshold of p = 0.05) among all three kinematic categories . ANOVA of the maximum velocities found significant differences among the different kinematics ; Tukey’s post hoc analysis found that concertina locomotion was significantly slower than the other two modes, which were not significantly different. A t-test found that the lateral undulation had significantly  slower propagation velocity than the manual oscillations.As soon as the snake appeared to be fully recovered, it was placed into a filming cage. The pressure catheter, which was still attached to the snake’s skull, was draped over a central support wire in an attempt to reduce signal noise and provide some structural support. The intention was to record CSF pressure from freely undulating snakes. While this worked , it was p = 0.018, n = 31) greater than the areas of the CSF traces recorded during either of the locomotor trials.Compared to the CSF traces recorded during the manual oscillations, the traces recorded during lateral undulation lacked a clear baseline elevation and had smaller, less frequent CSF pulses A. No cleCandoia typically exhibited a single harmonic, with a marked decrease over higher frequencies in the displacement traces . We hypr snakes , or by cr snakes .Alligator mississippiensis reported CSF pressure pulses that closely resembled the “typical” mammalian pattern [Alligator, the ventilatory and cardiac cycles had highly variable contributions to the CSF pressure profile, both in terms of temporal pattern and amplitude. Young et al. [Candoia. Previous work on  pattern , with log et al.  could noCandoia aspera  sta aspera A; and  for each specimen so that there was just enough tidal volume to cause discernible movement of the body wall. Without a discrete intrathoracic space, and pressure, would expansion of the lung (which fills most of the body cavity [The stability of the ventilatory influence on CSF pressure in y cavity ) have alCandoia, which lacks a diaphragm, has a clear functional coupling between ventilation and CSF pulsations, argues that the functional complex between ventilatory movement and CSF pulsations is not a simple one. Further complicating this is the horizontal posture of both Alligator and Candoia, which presumably minimizes the collapsing of the jugular vein, as is seen in humans during upright posture [There is good evidence for a functional connection between jugular venous pressure and CSF pressure in mammals ,52,53, b posture .Candoia aspera established traveling waves of displacement from the tail to the head , potentially inducing differential CSF flow on either side of the spinal cord. Surface concertina locomotion does not involve propagating loops or waves of axial deflection ; when C.lsations . The latifferent . The resThere is an extensive body of work modeling the effect of impulses on the displacement and/or deformation of the human brain , and ineRana catesbeiana) withstand the repeated impulses associated with saltatory locomotion?We presume that the prominent CSF response to movement reported herein reflects the artificial nature of this experiment, i.e., manual oscillation of an anesthetized snake. Nevertheless, we do think this study supports the findings of Xu et al.  in demon


Swallowing dysfunction is related to major cause of adverse events and an indicator of shorter survival among patients with neuromuscular disorders (NMD). It is critical to assess the swallowing function during disease progression, however, there are limited tools that can easily evaluate swallowing function without using videofluoroscopic or videoendoscopic examination. Here, we evaluated the longitudinal changes in tongue thickness (TT) and maximum tongue pressure (MTP) among patients with amyotrophic lateral sclerosis (ALS), myotonic dystrophy type 1 (DM1), and Duchenne muscular dystrophy (DMD).Between 2010 and 2020, TT and MTP were measured from 21 ALS, 30 DM1, and 14 DMD patients  at intervals of more than half a year. TT was measured, by ultrasonography, as the distance from the mylohyoid muscle raphe to the tongue dorsum, and MTP was determined by measuring the maximum compression on a small balloon when pressing the tongue against the palate. Then we examined the relationship between these evaluations and patient background and swallowing function.Mean follow-up periods were 24.0 months in the ALS group, 47.2 months in the DM1group, and 61.1 months in the DMD group. The DMD group demonstrated larger first TT than the other groups, while the DM1 group had lower first MTP than the ALS group. The ALS group showed a greater average monthly reduction in mean TT than the DM1 group and greater monthly reductions in mean body weight (BW) and MTP than the other groups. Significant differences between the first and last BW, TT, and MTP measures were found only in the ALS group.This study suggests that ALS is associated with more rapid degeneration of tongue function over several years compared to DMD and DM1. Patients with neuromuscular disorders (NMD) often experience problems with swallowing during the course of the illness. They require a periodic video fluoroscopic swallowing study (VFSS) or fiberoptic endoscopic evaluation of swallowing (FEES) to reduce the risk of aspiration pneumonia and assess the need for appropriate nutritional support. However, these tests require equipment and can only be evaluated in specialized facilities, and it is difficult to assess tongue function, using VFSS or FEES, quantitatively. Clinically, tongue function is determined by multiple characteristics, including mobility, shape, and posture. In the oral phase, tongue dysfunction may induce inadequate “bolus formation”, “mastication”, and “premature bolus loss” which cause pharyngeal residue or penetration/ aspiration. In NMD, dysphagia may result from tongue muscle weakness as well morphological changes due to muscle hypertrophy or atrophy. For instance, an enlarged tongue is frequently observed in Duchenne muscular dystrophy (DMD) while tongue atrophy is common in amyotrophic lateral sclerosis (ALS). Tongue hypertrophy which causes chewing and swallowing difficulties needs adequate nutritional support in DMD , 2, and In our previous study, we evaluated the relationship between tongue thickness (TT) and strength in NMD patients at a single time point  and founBetween 2010 January to and 2020 December, TT and MTP were measured from 21 ALS patients , 30 DM1 patients , and 14 male DMD patients  at the Department of Neurology, Neuro-Muscular Center, NHO Omuta National Hospital, Omuta City, Japan. Patients received multiple TT and TP measurements at intervals of more than half a year. All DMD and DM1 cases were confirmed by genetic analysis after clinical diagnosis, and all ALS patients were clinically diagnosed as probable or definite by neurologists specializing in ALS, based on Awaji criteria . FurtherThe multidisciplinary team classified diet type into seven levels based on the functional oral intake scale (FOIS) : Level 0TT was measured from the mylohyoid muscle raphe to the upper surface of the tongue Fig.  using a Tongue strength was measured as MTP using a handy probe consisting of a small balloon pressurized with air to 19.6 kPa  . Each pap < 0.05 (two tailed) was considered statistically significant for all tests.Mean BW, TT, and MTP were compared among groups using Steel-Dwass test. Pearson correlation coefficients were calculated to assess the relationships among BW, TT, and MTP. The first and last BW, MTP, and TT measurements were compared by the Wilcoxon signed-rank sum tests. All statistical analyses were performed using SPSS 13.0 J for Windows . p < 0.05, p < 0.01, respectively). The mean first TT was larger in the DMD group than the other groups (p < 0.01), while MTP was lower in the DM1 group than the ALS group (p < 0.05).Patients clinical characteristics are summarized in Table p < 0.01, p < 0.05, respectively), and the greater average monthly reduction in mean MTP compared to both the DM1 and DMD groups (p < 0.01). The ALS group marked the greater average monthly reduction in mean TT but no significant difference from the other two groups. Moreover, only the ALS group showed significant differences in BW, TT and MTP between the first and last measurements   Fig. .Fig. 2aAnalysis of individual subjects revealed TT and MTP were reduced in 15 of 21 ALS patients Fig. . SubgrouIn our previous cross-sectional study, we evaluated the relationship between TT and MTP as a strength metric in DMD, DM1, and ALS patients. There was higher TT in the DMD group, lower MTP in the DM1 group, and a significant correlation between TT and MTP in the ALS group . ThirteeVan Den Engel-Hoek et al. reported that TT can be assessed, conveniently and reproducibly, in DMD patients using ultrasound , 14. MorOn the other hand, progressive bulbar palsy is rapidly linked to dysphagia, malnutrition, and BW loss which leads to poor survival time in ALS, but BW loss could be caused by rising energy consumption due to impairment of respiratory function . Poor toThis study has several limitations, most notably the differences in mean age among groups, which is known to influence MTP . HoweverThis longitudinal study revealed more rapid loss of tongue thickness and strength in ALS patients than DMD and DM1. Only the ALS patients also exhibited substantial weight loss over the observation period, a change strongly associated with shorter survival . On the ALS is associated with a more rapid progression of tongue dysfunction over several years than DMD and DM1. We can conveniently identify the progression of tongue dysfunction based on temporal changes of TT and MTP. TT and MTP are more helpful to examine the relationships between the progress of tongue dysfunction and NMD than VFSS and FESS.