Multiple advantageous properties are achieved simultaneously by the resulting composite material (denoted as IrO2@TaB2), including high electrical conductivity, improved iridium mass activity and enhanced corrosion resistance. As a consequence, the IrO2@TaB2 can be used to fabricate the membrane electrode with a low iridium loading of 0.15 mg cm−2, and to give an excellent catalytic performance (3.06 A cm−2@2.0 V@80 oC) in PEMWE―the one that is usually inaccessible by unsupported Ir-based nanocatalysts and the vast majority of existing supported Ir-based catalysts at such a low iridium loading.The sluggish kinetics of oxygen evolution reaction (OER) and high iridium loading in catalyst coated membrane (CCM) are the key challenges for practical proton exchange membrane water electrolyzer (PEMWE). Herein, we demonstrate high-surface-area nano-metal diborides as promising supports of iridium-based OER nanocatalysts for realizing efficient, low-iridium-loading PEMWE. Nano-metal diborides are prepared by a novel disulphide-to-diboride transition route, in which the entropy contribution to the Gibbs free energy by generation of gaseous sulfur-containing products plays a crucial role. The nano-metal diborides, TaB 2 for realizing efficient, low Ir loading PEMWE.The extreme scarcity of Ir largely limits the wide application of proton exchange membrane water electrolyzer (PEMWE) for hydrogen production. Here, the authors report high-surface area nano-metal diboride as promising support of anode nanocatalyst IrO The heart of PEMWE is a catalyst coated membrane (CCM) composed of solid electrolyte , cathode catalyst layer, and anode catalyst layer  is an advanced hydrogen production technology that can achieve large current density >1 A cm A cm−2, 25. Despite their great potential, boride ceramics have not been investigated as the support of Ir-based OER nanocatalysts for constructing anode catalyst layer of CCM in PEM electrolyzer. In part, this is because some potential borides  prepared with some existing methods are usually composed of micrometer-sized particles with low surface areas, and sometimes contain large amounts of defects and impurities30. Thus, synthesizing high-quality borides with large surface areas is an essential prerequisite for meeting our goal of making efficient boride-supported anode catalysts and applying them in low-iridium-loading PEMWEs.To this end, we turn our attention to boride ceramics, which are generally characterized by high melting points, high hardness, high chemical resistance, and high electrical conductivity. A number of various borides can be produced and used as, for example, high-temperature structural materials, anti-oxidation coating materials and wear resistant materials in industry2, as the effective support material of anode nanocatalyst IrO2 in PEMWE. The TaB2-supported IrO2 nanocatalyst (denoted as IrO2@TaB2) possesses a large electrical conductivity (0.1–0.2 S cm−1) similar to nano-IrO2 itself, shows about 10 times higher iridium mass activity than IrO2, and exhibits much lower iridium leaching than IrO2 during electrocatalysis. When integrated into a PEM single-cell electrolyzer, the IrO2@TaB2 can achieve 3.06 A cm−2 current density at 2.0 V and 80 °C (membrane: NafionTM N115), with a low iridium-loading of 0.15 mg cm−2. The performance of our PEMWE has reached that of US DOE 2023 target  loading of 1 mg cm−2), under a much lower PGM loading (0.15 mgIr cm−2 and 0.27 mgPt cm−2).Herein, we present the entropy-driven synthesis of high-surface-area nano-metal diboride from the corresponding disulphide, and demonstrate the practical usefulness of boride ceramics, especially TaB2 + B → MB2 + Sx) under a KCl–CsCl molten salt condition , while S2 is considered to be the most abundant species31. We calculate the reaction enthalpy and entropic term to determine the Gibbs free enthalpy of the chemical reaction (ΔG = ΔH − TΔS). As exhibited in Fig. S of the gaseous sulfur-containing products is the dominant part of the total Gibbs energy. After reaching a certain temperature, the entropy gain would drive the reaction from a thermodynamically unfeasible process to a thermodynamically spontaneous process. According to the entropy-driven route, we can synthesize nine monometallic diborides, including MB2 . The powder X-ray diffraction (XRD) patterns in Fig. 2 and MoB2 possess a typical nanosheet structure, WB2 shows a nanowire morphology, and other MB2 samples  are composed of nanoparticles with sizes ranging from 50 to 200 nm. Most of these MB2 samples possess large Brunauer–Emmett–Teller (BET) surface areas within the range of 40–60 m2 g−1 , suggesting a fast electron transport property37. Experiments present TaB2 has a high conductivity of 25.2 S cm−1 , TaC (0.65 S cm−1) and TiO2 (1.8 × 10−4 S cm−1). Given its multi-advantages including large BET surface areas, great acid corrosion resistance, and high conductivity, the nano-TaB2 is specially selected to explore the potential as support of IrO2 catalysts. Moreover, the crust abundance of Ta is five orders of magnitude higher than that of Ir . The SEM  of TaB2 nanosheet  is achieved through calcinating a mixture of K2IrCl6 and TaB2 in molten NaNO3 . The loading amounts of IrO2 can be controlled by changing the feeding of the iridium source. When TaB2 is not employed in the synthesis, pure IrO2 nanoparticles with a size of 1.4–2.0 nm are formed . The XRD patterns of these IrO2@TaB2 samples are shown in Fig. 2 and unsupported IrO2 as references. The diffraction peaks of TaB2 supports dominate the XRD patterns of IrO2@TaB2. In addition, a broadened peak at 30-40o can be attributed to IrO2, suggesting the very small size of IrO2. With the increase in IrO2 contents on TaB2 supports, the diffraction intensities of IrO2 increase gradually.The loading of IrO3 i.e., 30 °C. The2@TaB2 samples are further characterized by X-ray photoelectron spectroscopy (XPS) and X-ray absorption spectroscopy (XAS). Figure f XPS spectra of IrO2@TaB2 samples and the reference TaB2. For TaB2, the surface comprises both metallic Ta elements and oxidized Ta elements, indicating slight surface oxidation. Unlike that of TaB2, the surface layers of IrO2@TaB2 samples are fully dominated by oxidized Ta elements, suggesting further surface oxidation of Ta species during the IrO2 loading process. With increasing IrO2 content in the samples, the signal intensities of Ta4f XPS decrease gradually, implying the decrease of surface Ta concentration. Similarly, the surface B concentration also decreases with increasing IrO2 content and is almost undetectable after the 16% Ir content  spectra. We note that we chose 16 wt% IrO2@TaB2 as a representative sample, which exhibits an optimized iridium mass activity toward OER in a standard three-electrode cell. Unless otherwise specified, subsequent IrO2@TaB2 refers to the sample with 16 wt% Ir loading. We also note that we chose Ir L1-edge for the XANES measurements, considering that the Ir L3-edge overlaps with the Ta L2 and Ta L1-edges. Figure 1-edge XANES spectra of IrO2@TaB2 and IrO2. Compared to that of IrO2, the white-line energy position of IrO2@TaB2 has a slightly weaker peak intensity, indicating an increased occupation in 5d electron orbital and a relatively lower Ir oxidation state in IrO2@TaB2. Moreover, extended X-ray absorption fine structure (EXAFS) exhibits similar structural information of IrO2@TaB2 and IrO2, and the corresponding fitting results  the lattice of IrO2 has almost unchanged after loading on TaB2; (ii) however, the interfacial electronic interaction between IrO2 and support leads to the charge redistribution of IrO2.The charge redistribution of IrO2@TaB2 sample. As shown in Fig. 2 nanoparticles are uniformly and tightly dispersed on the TaB2 sheets. The main sizes of IrO2 are about 1.5 nm  was applied to investigate IrOges Fig.  show thaage Fig.  presents2@TaB2 sample is shown in Fig. 2 support tends to form amorphous TaOx layer. Second, the IrO2 nanocrystallites are uniformly and tightly dispersed on the supports to form 5 nm thick IrO2 layers. Third, the IrO2 nanocrystallites with intrinsic conductive properties are spatially interconnected, and the IrO2 is electrically connected to the metallic TaB2 support, ensuring the formation of conductive networks. Fourth, the interfacial electronic coupling in TaOx/IrO2 catalytic layer results in the valence state reduction of Ir atoms.Based on the above electron microscopy, electron diffraction, and spectroscopic evidence, the structural illustration of IrO2@TaB2 were measured using a standard three-electrode configuration in 0.1 M HClO4 electrolyte. As the IrO2 content increases, the electrocatalytic activity for OER increased rapidly  for 16 wt% IrO2@TaB2 is 288 mV, lower than that of unsupported IrO2 (307 mV). The Tafel slope of 16 wt% IrO2@TaB2 is 42.6 mV dec−1 , indicating that the the TaB2 support has no change to OER pathway of IrO2.The electrocatalytic activities toward OER of IrOdly Fig. , and rea2@TaB2 by estimating the accumulated charge amount at the electrode surface. Generally, the ECSA values become larger with increasing Ir content in IrO2@TaB2  at 1.53 V versus reversible hydrogen electrode (RHE) among these IrO2@TaB2 samples, which is 5.2 times larger than that of IrO2. We further compared the iridium mass activities of IrO2@TaB2 and IrO2 by normalizing the measured currents over the mass of iridium (jir). Although 16% IrO2@TaB2 has 70 wt% less iridium relative to IrO2, it displays a high iridium mass activity of 345 A g−1 at 1.53 V versus RHE 47 and those representative supported catalysts 52.Electrochemical double layer capacitance (DLC) measurements were carried out to obtain the electrochemically active surface area (ECSA) of IrOaB2 Fig. . In ordeaB2 Fig. , right. 2@TaB2 displays great catalytic and structural stability for acidic OER. The galvanostatic measurements , suggesting that the dispersion of IrO2 on TaB2 supports markedly improves the structural stability. The stability of IrO2@TaB2 is further evaluated by calculating the stability number 53, which is a good metric that relates the amount of evolved oxygen to the dissolved iridium. As shown in Fig. 2@TaB2 during the acidic OER is 5.2 × 104, which is higher than that of IrO2 (2.9 × 104). In addition, IrO2@TaB2 still maintains the initial morphology and structure after OER, as supported by HRTEM, XRD, and XPS results  scale  process54, and the OER at Ir sites strictly follows the conventional adsorbates evolution mechanism (AEM) pathway. In addition, we applied in situ differential electrochemical mass spectrometry (DEMS) to study the reaction mechanisms. The O source of evolved oxygen product can be identified through labeling of catalysts with 18O isotope. When the 18O-labeled IrO2@TaB2 electrocatalyst works in H216O electrolyte, 34O2 (or 16O18O) to 32O2 ratio in gaseous product is 0.43% , excluding the participation of lattice oxygen during the electrocatalysis.To investigate the catalytic mechanism, OER performance of IrOale Fig. . The pot43% Fig. . It shou2@TaB2 possesses high activity for OER. Given that the crystal lattice and catalytic mechanism of IrO2 have almost no change after loading on TaB2, the enhanced performance of IrO2@TaB2 for OER is interpreted as follows. (i) The average particle sizes of IrO2@TaB2 and IrO2 are estimated to be 1.5 nm and 1.8 nm  The interfacial electronic coupling in TaOx/IrO2 catalytic layer is responsible for the high intrinsic activity of IrO2@TaB2. A metal−semiconductor heterojunction is constructed between TaOx and IrO2 on TaB2 surface, resulting in the formation of a surface electric field and strong electronic interaction , amorphous TaOx layer, and their heterointerface, makes it unrealistic to establish a clear structural model of the catalyst. This limits us from accurately modeling the electrochemical processes of OER and quantitatively describing catalytic activity by advanced calculation methods 57.After determining the catalytic mechanism, we sought to explain why IrO2@TaB2 as an anode catalyst was finally evaluated on a real PEM electrolyzer  to deliver high current densities of several A cm−24. The electrical conductivity of IrO2@TaB2 becomes higher as the IrO2 content increases . The structure and composition characterizations of the 40%IrO2@TaB2 sample by ICP-OES, XRD, XPS  than the DOE 2023 target (1.0 mg cm−2) and even the DOE 2025 target (0.5 mg cm−2)58.The performance of as-prepared IrOzer Fig. . Unlike XPS Fig. , and TEMXPS Fig.  confirm itself 0. S cm−1. −2 with a cell potential of 2.0 V, operating at 80 °C and ambient pressure , under such low PGM loadings (0.15 mgIr cm−2 and 0.27 mgPt cm−2). We note that the traditional CCMs in commercial PEMWE achieve reasonable activity and stability using a high Ir loading of 2–4 mgIr/cm2 to ensure sufficient in-plane conductivity and mechanical stability of catalyst layer59. In fact, the performance of PEMWE using IrO2@TaB2 as anode electrocatalyst exceeds the most recent reports of PEMWEs using novel anode electrocatalysts 67, as shown in Fig. 4C, Ir@Nb2O5-x) have reported to reach the DOE 2023 target, they require several times higher iridium loadings69.Even such low noble metals used in our CCM, the polarization curve of PEMWE reveals a current density of 3.06 A cmure Fig. . It is s2@TaB2 and commercial IrO2 as anode electrocatalysts  at 2. 0 V cell potential. To evaluate catalyst stability, the IrO2 and IrO2@TaB2 anode cells were tested at a constant current density of 1 A cm−2. The PEMWE using IrO2 anode undergoes a severe deactivation under the low Ir loading of 0.2 mg cm−2, and the IrO2 anode cell exhibits reasonable stability when the Ir loading increases to 2 mg cm−2  The Rcell denotes the cell ohmic resistance, which is related to ohmic losses of all components including membrane, catalyst layers, porous transport layers (PTL), bipolar plate, and their interfacial resistances. The RCell is almost unaffected at different current densities for IrO2@TaB2 anode cell, which can be reflected by high frequency resistance  The Rac denotes the activation resistance to determine the reaction kinetics of anode and cathode electrocatalysts. Rac is sensitive to current density and is mainly contributed from the anode. The Rac of IrO2@TaB2 anode cell is significantly reduced compared with that of IrO2 anode cell, due to the much better catalytic performance IrO2@TaB2 relative to IrO2. (iii) The Rd denotes the diffusion resistance, as reflected by the Warburg diffusion element (Wd). Rd is negligible at a low current density and increases with the increase of current density. The IrO2@TaB2 anode cell presents much lower transport loss compared with the IrO2 anode cell. Taken overall, it can be concluded that the IrO2@TaB2 anode cell exhibits simultaneous decreases in both ohmic, activation, and diffusion losses relative to the IrO2 anode cell.To deduce the origin of performance differences, we carry out chemical impedance spectroscopy (EIS) experiments on PEMWEs using IrOely Fig. . We furtely Fig. , inset, 2 nanocatalysts. As a demonstration, we have prepared TaB2-supported IrO2 nanocatalyst , which displays uniformly distributed IrO2 nanoparticles (∼1.5 nm), highly conductive networks, and great acidic corrosion resistance. Impressively, the as-prepared IrO2@TaB2 catalysts with an optimized IrO2 content exhibit a 10 times higher iridium mass activity than IrO2, while showing lower iridium leaching during acidic OER. When integrated into a PEMWE, the IrO2@TaB2 anode cell requires 2.0 V to attain 3.06 A cm−2 (reaching the US DOE 2023 target), under a much lower PGM loadings of 0.15 mgIr cm−2 and 0.27 mgPt cm−2 than DOE 2023 target (PGM loading of 1 mg cm−2). Our findings highlight the rational design of highly conductive, corrosion resistance, and large surface area materials as IrO2 supports for acidic OER, and stimulate practical applications of low-iridium-loading anode catalyst layers in industrial PEMWE.In conclusion, we have demonstrated an entropy-driven disulphide-to-diboride transition route strategy that can be operated to synthesize large-surface-area metal diborides as the promising support of IrO2H6O), isopropyl alcohol ((CH3)2CHOH) and NaNO3 were purchased from Sinopharm Chemical Reagent Co., Ltd. TiS2, ZrS2, HfS2, VS2, NbS2, TaS2 and ReS2 were purchased from Nanjing MKNANO Tech. Co., Ltd. MoS2 and WS2 were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. HClO4, was purchased from Tianjin Xinyuan Chemical Co., Ltd. Nafion® perfluorinated resin solution was purchased from Sigma-Aldrich. K2IrCl6 and IrO2 were purchased from Shanghai Macklin Biochemical Co., Ltd. Pt/C (Pt 40 wt%) was purchased from Johnson Matthey Company. All the chemicals and reagents were used without further purification. Highly purified water (>18 MΩ cm resistivity) was obtained from a PALL PURELAB Plus system.Absolute ethanol  with an equal molar ratio. The metal disulfide, boron powder, and molten salt are ground thoroughly in a mortar for 0.5 h. Then the mixture was transferred into a corundum boat and heated at 900–1100 °C under Ar atmosphere in a tubular furnace, and the heating rate was 10 °C/ min  were collected at BL14W beamline in Shanghai Synchrotron Radiation Facility (SSRF). The storage rings of SSRF were operated at 3.5 GeV with a stable current of 200 mA. Using Si (111) double-crystal monochromator, the data collection was carried out in fluorescence mode using Lytle detector. All spectra were collected in ambient conditions. Data reduction, data analysis, and EXAFS fitting were performed with the Athena and Artemis software packages. The energy calibration of the sample was conducted through a standard IrO2, which as a reference was simultaneously measured. For EXAFS modeling, EXAFS of the IrO2 is fitted and the obtained amplitude reduction factor S02 value (0.900) was set in the EXAFS analysis to determine the coordination numbers (CNs) in the Ir-O/Ir/Ta scattering path in the sample.XRD patterns were conducted by a Rigaku D/Max 2550 X-ray diffractometer with Cu Kα radiation (λ = 1.5418 Å). SEM images were obtained with field emission scanning electron microscopy  at an accelerating voltage of 5 kV. EDX analysis was obtained with an EDX system attached to JEOL JSM7800F SEM. Low-magnification transmission electron microscope (TEM) images were obtained with a Philips-FEI Tecnai G2S-Twin microscope equipped with a field emission gun operating at 200 kV. High-resolution TEM images were recorded on a JEM-2100F electron microscope . XPS measurements were carried out using a Thermo Fisher Scientific ESCALAB 250Xi with photoelectron spectroscopy system using a monochromatic Al Ka (1486.6 eV) X-ray source. ICP-OES was performed on a PerkinElmer Optima 3300DV ICP spectrometer. XAS (Ir L4 solution bubbled with O2 gas. Linear sweep voltammetry (LSV) measurements were performed with the scan rate of 1 mV s−1 and compensated by 85% iR-drop. The chronopotentiometric curve was measured without iR-drop compensation. The counter electrode was a Pt wire and the reference electrode was a saturated calomel electrode (SCE). The potential was normalized with respect to the reversible hydrogen electrode (RHE), according to Eq.  and the electrolyte was 0.1 M HClO2) and dried in air. The catalyst loading on the working electrode was 0.28 mg cm−2. To avoid the passivation of GCE, a piece of carbon paper (Active area = 0.09 cm2) loaded with catalysts was used as the working electrode for long-term stability test, and the catalyst loading was 0.3 mg cm−2. In order to accurately evaluate the ion leaching and structural evolution behavior of the catalyst during the OER process, the catalyst ink was dropped onto the surface of a Ti plate (Active area = 1 cm2) with a catalyst loading of 10 mg cm−2. OER was catalyzed for 10 h at 10 mA cmgeo−2, and a portion of the electrolyte was taken out every 2 h for ICP-OES. After the catalysis, the catalyst sample was obtained by eluting the electrode with ethanol.The working electrode was a glassy carbon electrode (GCE) loaded with catalysts. 4 mg catalyst powder was ultrasonically dispersed in 400 μL isopropyl alcohol containing 10 μL Nafion solution (5 wt.%). Then 2 μL catalyst ink was dropped on a GCE . A linear trend could be obtained via plotting the currents difference (Δi) between the anodic and cathodic sweeps (ianodic − icathodic) at 0.88 V against the scan rate. The slope of the fitting line was equal to twice the geometric double layer capacitance (Cdl). The ECSA of the catalyst was estimated according to the Eq. .To calculate ECSA of catalysts, we performed CV tests in the non-faradaic current region between 0.83 V and 0.93 V vs. RHE at different scan rates  The pristine IrO2@TaB2 was labeled with 18O isotope in 0.1 M HClO4 solution using H218O as solvent at 1.6 V for 10 min. (ii) The labeled electrode was washed with H216O to remove the residual H218O that was physically attached to the catalyst layer. Subsequently, CV tests were carried out at the rate of 50 mV s−1 in the potential range of 0.6 V and 1.2 V vs. RHE for removing the adsorbed 18O species on the surface of the catalyst. (iii) Three LSV cycles were applied for the labeled electrode in 0.1 M HClO4 solution using H216O as a solvent in the potential range of 1.2–1.7 V vs. RHE. The gaseous products including 36O2, 34O2, and 32O2 were monitored by the mass spectrometer. The fraction of 36O2, 34O2, and 32O2 can be estimated from the integral areas of corresponding mass signals of these gaseous products.In situ DEMS test was performed in a three-electrode cell with 0.1 M HClO2O2, deionized water, and 0.5 M H2SO4 at 80 °C for 1 h. Then the treated N115 membrane was rinsed with deionized water. Commercial Pt/C (40 wt%) was used as a cathode electrocatalyst and commercial IrO2 or IrO2@TaB2 (Ir 40 wt%) was used as an anode electrocatalyst. In order to prepare the catalyst ink, the catalyst was dispersed into a mixed solution of isopropyl alcohol and distilled water . Subsequently, Nafion with an ionomer mass fraction of 10 wt% at the anode or 35 wt% at the cathode was added into the solution. The suspension was ultrasonically treated in an ice water bath for 1 h to obtain the catalyst ink. The anode catalyst ink and cathode catalyst ink were sprayed on polytetrafluoroethylene (PTFE) film respectively. Then the PTFE films supported with catalysts and a N115 membrane were hot pressed under 10 Mpa at 135 °C for 10 min. After cooling, the PTFE films were stripped to obtain CCM. The loading of the cathode was 0.27 mgpt cm−2 and the loading of the anode was 0.15 mgIr cm−2 for IrO2@TaB2 or 0.2 mgIr cm−2 for IrO2. The actual catalyst loading was determined by ICP-OES test. In order to construct a PEM electrolyzer, a well-defined pore Ti plate  coated with Pt was used as the porous transport layer (PTL) of the anode, and a piece of carbon paper was used as the PTL of the cathode. The active area was 5 cm2. The PEM electrolyzer was operated at 80 °C and the reactant was deionized water, which was circulated through a peristaltic pump. The polarization curve of the PEM electrolyzer was collected at the cell voltage of 1.4–2.0 V, and the stability was tested by chronopotentiometry at 1 A cm−2.Before the construction of catalysis coated membrane (CCM), the N115 membrane was successively treated with 3 wt% H74. The Generalized gradient approximation (GGA) with the Perdew−Burke−Ernzerhof (PBE) exchange correlation functional was employed with 500 eV cut-off energy75. All crystal structures were obtained from the ICSD  database. During the structural optimization, we used k-point separation length of 0.04 2π A−1, while the k-point separation length was promoted to 0.03 2π A−1 for DOS calculation. For all calculations, we apply an energy convergence criterion of 10−5 eV with the force convergence criterion of 0.02 eV A−1.We performed all DFT calculations using the Vienna ab initio simulation package (VASP 5.4.4)zpe is the zero-point energy difference between product and reactant, T is the absolute temperature, and ΔS is the entropy difference between product and reactant. For the reaction:For the Gibbs free energy change of the reaction, we use the Eq.  to calcu2 is a gas, we only need to consider the zero-point energy and entropy of S2. The former can be calculated as 4.47 kJ mol−1, and the latter can be obtained as 228.2 J mol−1 K−1 from the database.Since only SSupplementary InformationPeer Review File


Genu valgum is considered to be a risk factor for patellar instability. Correction of valgus alone or combined with a tibial tuberosity medialization has been described. However, changes occurring in the tibial tuberosity-trochlear groove (TT-TG) distance after a lateral distal femoral opening wedge osteotomy (LDF-OWO) are not known.This study aimed to define changes in TT-TG distance with increasing amounts of valgus correction after LDF-OWO.® software. An oblique lateral opening wedge osteotomy of the distal femur was simulated in 2-degree increments up to 12 degrees of varus opening. Change in TT-TG distance was measured in mm on 3D models of the knee.Three-Dimensional Computed Tomography (3D-CT) scans of six patients  aged between 19 and 35 years with genu valgum and patellar instability were analyzed. 3D models of femoral, tibial, and patellar bones were created with the advanced segment option of Mimics 21Compared to baseline without osteotomy, the TT-TG distance decreased significantly (p < 0.05) for all corrections from 2 to 12 degrees in 2° steps. The TT-TG distance decreased by an average of 1.7 mm for every 2 degrees of varus opening.Lateral opening wedge distal femoral osteotomy causes a decrease in TT-TG distance. The surgeon should be aware of the magnitude of this change .The online version contains supplementary material available at 10.1186/s12891-023-06832-w. Genu valgum is considered a risk factor for recurrent patellar instability in adolescents and adults . AlthougThe Tibial Tuberosity-Trochlear Groove (TT-TG) Distance is widely used to objectively measure the increased lateral quadriceps vector in patients with patellar instability. First described by Dejour, the TT-TG distance was found to be 19.8 mm in patients with patellar instability compared to 12.7 mm in normal knees. A tibial tuberosity medialization osteotomy is indicated for patients with > 20 mm TT-TG, while 15–20 mm range is considered to be the “grey zone” . The effThis study aimed to analyze changes in the TT-TG distance after a Lateral Distal Femoral Opening Wedge Osteotomy (LDF-OWO) in a computer simulation study using 3 Dimentional Computed Tomography (3D-CT) scans of patients with patellar instability and genu valgum. We hypothesized that the TT-TG distance would decrease after LDF-OWO.Supine 3D CT scans of six adults  with patellar instability and genu valgum between 19 and 35 years of age were analyzed. We excluded patients with prior deformity correction or fracture fixation, incomplete imaging of the knee, and knee arthritis. A whole leg standing orthoroentgenogram was performed to evaluate the mechanical axis of the lower leg and the degree of genu valgum. CT scans were performed on a Siemens SOMATOM Emotion  . Anonymization of the patients’ demographic data was performed with the software Mimics 21 .3D models of femoral, tibial, and patellar bones were created with the advanced segment option of Mimics 21. Thresholding limits were determined as 200–2150 Hounsfield Unit to isolate the bone tissue from the medullary canal and soft tissues as previously described . An incoThe trochlea’s deepest point and the tibial tuberosity’s midpoint were marked using create point option of 3-Matic. The most posterior points of the lateral tibial condyle, medial, and lateral femoral condyles were marked on 3D models. The posterior condylar line in the XY axis, referencing the most posterior points of medial and lateral femoral condyles, was created . A planeUsing the interactive rotate option of 3-Matic, the distal part of the femur below the osteotomy line was rotated 2 degrees in the coronal plane to perform a varus opening. The pivot point of opening was selected as 1 cm from the medial cortex at the apex of the osteotomy. The posterior condylar line and the posterior plane with a perpendicular midplane were created again using the same method. Midplanes were created and translated as passing through the previously defined points. The distances were measured using the automated ruler of 3-Matic. This procedure was repeated for all TT-TG measurements performed in 2-degree increments from 2 to 12 degrees varus opening Fig. .A single observer, experienced in the use of the 3D simulation software performed all measurements.Mean, standard deviation, median, minimum, maximum value frequency, and percentage were used for descriptive statistics. The distribution of variables was checked with the Shapiro-Wilk normality test. Wilcoxon test was used for the repeated measurement analysis. All statistical analyzes were performed using SPSS 28.0 . A p-value < 0.05 was considered statistically significant.We determined the threshold probability for rejecting the null hypothesis as α (two-tailed) = 0.05 (Type I error rate), probability of failing to reject the null hypothesis under the alternative hypothesis as a β = 0.20 (Type II error rate), and after determining expected correlation coefficient r ≥ 0.9, we found the minimum number of cases to be 6.This observational study was carried out with the approval of the Institutional Review Board of Ankara City Hospital No. 1 clinical research ethics committee . All experimental protocols were approved by this ethics committee. All patients gave informed consent to their anonymized data being used in the study. All methods were carried out in accordance with relevant guidelines and regulations.The mean pre-osteotomy HKA of the patients was 3,2° . Mean LDFA and MPTA were 82,2° and 87,7° respectively. All patients reported at least one episode of patellar instability.The mean TT-TG distance was 21 mm. All patients had a TT-TG distance above 15 mm, and three patients had a TT-TG > 20 mm.Following simulated LDF-OWO, the TT-TG distance demonstrated a significant decrease for every 2° increase in distal femoral varus opening from 2° to 12° compared to baseline (p < 0.05). The differences in increments of 2 degrees were statistically significant for all values except for the differences between 6 and 8 degrees and 10 and 12 degrees  are distal to the rotation point. Thus, they remain in line no matter the amount of proximal correction. However, due to the so-called pendulum effect, the distance between the two points changes depending on the perspective. Fig. Linear correlation of these two data points using the Pearson correlation coefficient was . A very high negative correlation between varization and decrease in TT-TG distance  was found.The most important finding of this study was that the TT-TG distance decreased incrementally in patients with valgus knees undergoing 3D-CT simulated LDF-OWO, and for every 2° of varus opening up to 12°, the TT-TG distance would decrease by 1.7 mm due to the pendulum effect described in Fig. Correction of valgus alignment of the distal femur has been recognized as an important factor in treating patellar instability, and both lateral open and medial closed wedge osteotomies have been described. A recent meta-analysis has shown favorable clinical outcomes and low recurrent instability rates using both types of osteotomies .Lateral opening wedge techniques may increase patello-femoral contact pressure, necessitate bone grafting, and have a risk of hinge fracture, collapse, and loss of correction. Lateral opening wedge osteotomies decrease patellar height and may help correct patella alta and allow for an easier trochleoplasty . In contIncreased TT–TG distance is a well-known risk factor for acute patellar dislocations and has also been implicated in the failure of soft tissue reconstructions for patellar instability . ChangesFlury et al. performed a comparative (pre vs. post-operative) 3D computer mapping study of 10 knees undergoing biplanar medial closed wedge osteotomy and found that Q angle decreased after surgery and was correlated with the degree of varization . HoweverGuided growth techniques with temporary or permanent hemiepiphysiodesis have been described in skeletally immature patients to correct valgus and patellar instability. Most of these studies do not utilize soft tissue procedures on the medial soft tissues and rely on gradual valgus correction to treat patellar instability . Ceroni We are not aware of a study analyzing the changes in TT-TG distance after a lateral opening wedge distal femoral osteotomy. Swarup et al. reported on eight patients undergoing lateral open wedge DFO combined with lateral retinacular release for patellar instability. Good clinical outcomes and correction of coronal alignment were reported, but pre- and postoperative TT-TG distance measurements were not performed . Wilson This study has several limitations. Only six knees were available for analysis due to the low prevalence of valgus knees requiring correction in adults with patellar instability; therefore although the study was adequately powered for correlation analysis, it may be underpowered for other parameters. Furthermore, the study was performed in patients with underlying bone and soft tissue risk factors for patellar instability and not sawbones, reflecting the actual clinical situation. It should be kept in mind that this is a pilot study analyzing feasibility and will pave the way to adequately powered larger studies.Only the situation after LDF-OWO was analyzed, and this may not reflect the changes occurring in TT-TG distance using guided growth techniques or medial closing wedge osteotomies. Changes in the ligaments and soft tissues around the knee could not be considered using a 3D-CT simulation. However, no effect of soft tissue tension is expected on TT-TG distance.The varization was performed with strict avoidance of unintended rotation in this computer simulation study. This might not be the case during surgery where unintended rotation may occur during opening of the osteotomy site.We used standing orthoroentgenograms to evaluate the mechanical axis and supine CT scans for simulation of osteotomies. A weight bearing CT scan of the whole extremity could have better replicated the actual clinical situation, however this imaging technique was not available. A single observer performed the measurements, therefore the reliability and reproducibility of the measurements were not verified.Lateral opening wedge distal femoral osteotomy causes a decrease in TT-TG distance.The surgeon should be aware of the magnitude of this change (1.7 mm decrease for every 2° increment of varization) and consider modifying the tibial tuberosity medialization osteotomy in patients with patellar instability if indicated.Below is the link to the electronic supplementary material.Supplementary Material 1


Currently there are no established fertility preservation options for pre-pubertal boys facing cancer treatment. Granulocyte-colony stimulating factor (G-CSF) treatment has been proposed to be chemoprotective against spermatogonial cell loss in an alkylating chemotherapy model of busulfan treated adult mice. Having previously shown that exposure to the alkylating-like chemotherapy cisplatin resulted in a reduction in germ cell numbers in immature human testicular tissues, we here investigate whether G-CSF would prevent cisplatin-induced germ cell loss in immature human and mouse  testicular tissues.n = 14 fetuses) and mouse pre-pubertal (n = 4 litters) testicular tissue pieces were cultured and exposed to cisplatin or vehicle control for 24 hrs and analysed at 72 and 240 hrs post-exposure. Combined G-CSF and cisplatin exposure groups explored varying concentrations and duration of G-CSF supplementation to the culture medium . In addition, effects of G-CSF supplementation alone were investigated. Survival of total germ cell and sub-populations were identified by expression of AP2γ and MAGE-A4 for human gonocytes and (pre)spermatogonia, respectively, and MVH and PLZF, for mouse germ cells and putative spermatogonial stem cells (SSCs) respectively, were quantified.Organotypic in vitro culture systems were utilised to determine the effects of clinically-relevant concentrations of G-CSF in cisplatin-exposed immature testicular tissues. Human fetal . Continuous supplementation with G-CSF alone for 14 days did not change the germ cell composition in either human or mouse immature testicular tissues.This study demonstrates that exposure to G-CSF does not prevent cisplatin-induced germ cell loss in immature human and mouse testicular tissues in an in vitro system.The online version contains supplementary material available at 10.1186/s12885-023-10702-y. Improvements in chemotherapeutic drug development have resulted in more than 80% of children with cancer surviving for more than 5 years . Increas+ve cells) and (pre)spermatogonia, also known as (pro)spermatogonia or fetal spermatogonia (MAGE-A4+ve) [The key germ cell populations found in the human fetal testes are gonocytes (AP2γE-A4+ve) . GonocytE-A4+ve) , whilst E-A4+ve) , 14, makThe impact of chemotherapy treatment on immature testis is clear, however there are no established fertility preservation strategies for male pre-pubertal cancer patients. Current strategies are experimental and involve testicular tissue biopsy prior to chemotherapy treatment and then either maturing tissue or extracted cells in vitro or transplanting these back into the patient . HoweverThere are no human studies investigating whether G-CSF could be used as a chemoprotective agent for pre-pubertal boys. Therefore, this study aimed to investigate whether exposure to one of several regimens of combined G-CSF and cisplatin could protect the key germ cell populations from cisplatin-induced damage in immature human testicular tissues, and to identify species-dependent effects of G-CSF exposure with and without cisplatin by comparing findings in human and mouse immature testicular tissues.Given that exposure to cisplatin results in germ cell loss in human fetal testicular tissues  and thatIn the initial set of experiments, human fetal testicular tissue pieces were cultured in a hanging drop system and exposed to a clinically-relevant concentration of cisplatin (0.5 μg/ml) or vehicle control on day 3 for 24 hrs. In combined G-CSF and cisplatin groups, tissue pieces were exposed to clinically-relevant doses of G-CSF (5 and 20 ng/ml) once daily for 7 days  at the beginning of the culture. The tissue culture was ended at 240 hrs post-cisplatin exposure (14-day culture). This set of experiments is referred to as Regimen 1.For the second set of experiments, Regimen 2, in combined G-CSF and cisplatin groups, human fetal testicular tissue pieces were cultured in regular culture medium for 3 days prior to cisplatin treatment and exposed to a range of G-CSF concentrations  daily starting on the day of cisplatin exposure until 10 days after chemotherapy exposure .In the third set of experiments (Regimen 3), human fetal testicular tissue pieces were exposed to G-CSF throughout the whole 14-day culture period .In addition, where sufficient tissue was obtained, human testicular tissue pieces were exposed to G-CSF alone  daily for a period of 14-day culture.In the final set of experiments, Regimen 3, mouse pre-pubertal testicular tissue pieces were cultured at a liquid-air interphase on a floating membrane. Tissue pieces were exposed to cisplatin alone or vehicle control and combined 100 ng/ml G-CSF and cisplatin in all three regimens as described above for human fetal testicular tissue experiments. Exposure to 100 ng/ml G-CSF alone was performed daily for a 14-day culture period.2) with assessor blinded to treatment details.In all experiments, each individual fetus or mouse litter were considered the experimental unit (n), whilst each tissue fragment taken from an individual fetus or litter was considered an experimental replicate. Two tissue pieces were included per treatment per fetus/litter. Two tissue sections per tissue piece (3 slides in from either end of the tissue block) were subjected to immunostaining and each immunostaining run included all treatment groups, positive and negative controls  from the same fetus/litter. Sample sizes were based on the results of previous studies. Positively stained cells were manually counted per seminiferous area , Newcastle: NRES committee North East – Newcastle and North Tyneside 1 (08/H0906/21 + 5) and London: NRES Committee London – Fulham (18/10/0822).For studies involving animals, specific approval, including ethical approval, was given by the UK Home Office. All procedures were carried out by the University of Edinburgh Bioresearch and Veterinary Services and performed in accordance with the Animal (Scientific procedures) Act 1986.n = 14; ages: 17–22 GW) were obtained from elective termination of pregnancy. This was undertaken at the Royal Infirmary of Edinburgh and additional tissue samples were obtained from Human Developmental Biology Resource (HDBR) facilities in Newcastle and London. None of the samples were from termination of pregnancy due to fetal abnormalities. Gestational age was determined by ultrasound and direct measurement of foot length. A small piece of skin or limb was taken to confirm the male sex of each fetus by presence of sex-determining region Y (SRY) gene by qPCR.Human fetal testicular tissues from 2nd trimester  were collected from post-natal day 5 male pups (C57/B6 strain), which were bred within the University of Edinburgh animal facility. Male pups were euthanised by cervical dislocation. Testes were removed and placed in cold dissection medium consisting of Leibovitz L-15 supplemented with 3 mg/ml of bovine serum albumin  and kept on ice during transportation.Pre-pubertal mouse testicular tissues (3) and randomly placed into 30 μl droplets of pre-warmed media on the upturned lid of Petri dish. The lid was then inverted and placed back on the dish, which contained a small volume (10 ml) of Phosphate Buffered Saline  at the bottom to keep the humidity inside the dish. The dish was placed in an incubator set to 5% CO2 at 37 °C.Hanging drop culture was set up as previously described . This cuRegular culture medium composition for human fetal testicular tissues consisted of: Minimum Essential Medium α , 1x MEM non-essential amino acids (Thermo Fisher Scientific), 2 mM sodium pyruvate (Thermo Fisher Scientific), 2 mM L-glutamine (Life Technologies), 1x Insulin-Transferrin-Selenium , 1x penicillin/streptavidin (Thermo Fisher Scientific) and supplemented with 10% fetal bovine serum . Treatment medium was prepared by adding an appropriate volume of stock solution into regular medium. Tissue culture medium was replaced every 24 hrs.3 pieces and placed on a Whatman membrane  floating on pre-warmed 1 ml treatment culture medium consisting of MEM (Gibco), 10% Knockout Serum Replacement (KSR) and appropriate concentration of drug stock per well on a 24-well plate. Fragments were randomly allocated to treatment groups. The plate was incubated at 35 °C, 5% CO2. Tissue culture medium was changed every 24 hrs.Membrane culture was set up as previously described . BrieflyCisplatin concentration (0.5 μg/ml) was based on publications reporting plasma serum levels in paediatric cancer patients after administration of cisplatin . Stock sConcentrations of G-CSF used were selected based on reported plasma levels in paediatric patients after G-CSF administration . To prepAt the conclusion of each experiment, tissue pieces were fixed in Bouin’s fluid (Clin-Tech) for 1 hr. and transferred to 70% ethanol. Post fixation, samples were paraffin-embedded, sectioned (5 μm) and assessed by H&E using standard protocol or used for immunohistochemistry as described below. Only samples that showed healthy tissue morphology  in pre-culture and vehicle controls were included in the analyses. Two sections from two replicates for each treatment were stained and analysed.Double colourimetric immunohistochemistry and double immunofluorescence were performed as previously described . Staininp < 0.05.Statistical analysis was performed using GraphPad Prism 8 software . Data are presented as mean ± standard error of mean (SEM). For any of the tests used, statistical significance was considered where Two-way analysis of variance (ANOVA) was performed as treatment group and individual sample were considered as two individual variables to assess for overall significance. To compare for differences of means between treatment groups, multiple comparisons were tested using Bonferroni’s post hoc test.Data sets were tested for normality using Shapiro-Wilk test, confirming a normal distribution of the data. As each litter of pups was genetically similar, differences of means between treatment groups were analysed using unpaired two tailed t-test (when two treatment groups were compared) or one-way ANOVA (when three or more treatment groups were compared). Bonferroni’s post hoc test was performed to assess for significance between treatment groups.CSF3R. Data are presented as t-SNE plots.Datasets from single-cell RNA sequencing of human fetal and pre-pubertal testis , 14 were2), p < 0.0001; Fig. p < 0.0001, Fig. p < 0.0001; Fig. 2), p < 0.05; Fig. In Regimen 1 Fig. , human f2), p < 0.01; Fig. 2), p < 0.001; Fig. p < 0.0001) or 20 ng/ml G-CSF and cisplatin  but not in 5 or 100 ng/ml G-CSF and cisplatin exposure groups, compared to control  in combined exposure experiments were investigated Fig. . In Regi2), p < 0.05; Fig. S2), p < 0.01), but no differences were seen between any other exposure groups , p < 0.05) or 10 ng/ml G-CSF and cisplatin compared to control (338.6 ± 30.6 vs 492.7 ± 57.2 cells/cord area (mm2), p < 0.01) but no changes were observed in any combined G-CSF and cisplatin groups compared to cisplatin alone  showed daily exposure to a range of G-CSF concentrations (5–100 ng/ml) for 14 days had no effect on total germ cell, gonocyte or (pre)spermatogonial cell numbers , p < 0.0001; Fig. 2), p < 0.0001; Fig. Sections from tissue pieces exposed to control, cisplatin alone or combined 100 ng/ml G-CSF and cisplatin at 240 hrs post-exposure (14-day culture) using 3 different regimens were subjected to germ cell analysis based on the expression of MVH  and PLZF (putative SSCs) Fig. A-C. At 2Previous animal studies suggested that exposure to G-CSF alone without chemotherapy exposure increased steady-state spermatogenesis in adult mice . Tissue The main aim of this study was to understand whether exposure to G-CSF had a protective effect against cisplatin-induced reduction of germ cell numbers in immature testicular tissues, in both rodent and human. The results confirmed that cisplatin alone resulted in a significant reduction in total germ cell number but showed that this was not prevented by various regimens of G-CSF supplementation in combination, e.g. short-term supplementation with G-CSF or continuous exposure to G-CSF throughout the culture period. In addition, short supplementation with 5 ng/ml G-CSF in combined treatment in Regimen 1 resulted in a reduction in gonocyte number compared to cisplatin alone which could suggest that gonocyte sensitivity to cisplatin exposure may even be enhanced by G-CSF supplementation. However, it should also be pointed out that the cisplatin-only treatment group contained an outlier that may have influenced this result, which would also explain why a similar result was not seen with 20 ng/ml G-CSF. The Food and Drug Administration (FDA) datasheet for Filgrastim (G-CSF) states that exposure to G-CSF is thought to increase the sensitivity of immune cells to chemotherapy treatment in patients, thus G-CSF is not recommended to be given prior to chemotherapy treatment and that G-CSF administration should commence 24 hrs post-exposure to chemotherapy [+ve cells) and (pre)spermatogonia, also known as (pro)spermatogonia or fetal spermatogonia (MAGE-A4+ve) [Exposure to cisplatin alone did not always result in a significant reduction in numbers of separate germ cell sub-populations, thus it cannot be clearly concluded whether supplementation with G-CSF in Regimens 2 and 3 were able to protect against cisplatin-induced damage in separate germ cell sub-populations. This could be explained by changes in germ cell composition during the development of human fetal testis. With increasing gestational age, the number of gonocytes decreases whilst the (pre)spermatogonial number increases , 25. In E-A4+ve) . GonocytE-A4+ve) , whilst E-A4+ve) , 14. The+veUTF1+ve cells) was observed in tissues exposed to cisplatin compared to controls [Whilst this study was not intended to try and replicate the previous animal studies that reported chemoprotective effects of G-CSF in adult male mice exposed to busulfan , 17, it controls . Taken tAn alternative explanation for the differing results relates to species difference between findings reported in the animal studies and in this study. A recent publication investigated the effects of G-CSF on the reproductive function of male pre-pubertal guinea pigs and ram lambs . Animals+ve spermatogonia. However, single-cell sequencing of human fetal and post-natal testis has shown the expression of CSF3R in testicular macrophages and not in the SSC population , indicating that G-CSF exposure did not protect the testis from cisplatin-induced germ cell loss. In in vivo mouse experiments, the extent of the protective effects of G-CSF co-exposure with busulfan was minimal , 17 and This study has demonstrated in vitro exposure to G-CSF did not confer direct protection of immature mouse or human testicular tissues from cisplatin-induced germ cell loss. Future work to determine the role of G-CSF in protecting germ cells in the pre-pubertal testis from chemotherapy-induced damage should focus on other experimental models  capable of identifying indirect mechanisms that may result in protection of germ cells in the testis.Additional file 1: Fig. S1. Effects of exposure to combined G-CSF and cisplatin (Regimen 2&3) compared to cisplatin alone or control on gonocyte and (pre)spermatogonial numbers in human fetal testicular tissues at 240 hrs post-exposure. Fig. S2. Effects of exposure to G-CSF alone for 14 days on germ cell numbers in mouse pre-pubertal testicular tissue. Fig. S3. Expression of CSF3R in human fetal and post-natal testicular tissues.Additional file 2: Table S1. Summary of immunohistochemistry (IHC) and immunofluorescent (IF) protocols.


Historically, long term intravenous (IV) antimicrobial therapy has been considered superior to oral (PO) for the management of BJIs. In recent years, this concept has been challenged with studies showing similar efficacy and safety between PO and IV therapy. The purpose of this study is to evaluate areas for improvement within Johns Hopkins Medicine (JHM) in utilizing oral antibiotics for the treatment of BJIs as outlined in the OVIVA trial.A multicenter, retrospective study was performed of adult patients with BJIs discharged from The Johns Hopkins Hospital and Johns Hopkins Bayview Medical Center followed by the JHM OPAT Service between February 1, 2021 and February 1, 2022. The following was collected on all patients: demographics, comorbidities, site of infection, OPAT-related adverse events (AEs), 30-day readmissions, emergency department (ED) visits, source control, microbiological data, antibiotic regimen, and treatment outcomes. The primary outcome is the proportion of patients treated for BJIs with OPAT who would have qualified for oral antibiotics according to OVIVA trial criteria. Secondary outcomes include the rate of OPAT-related AEs and treatment outcomes . Descriptive statistics were used to summarize demographics and the primary and secondary outcomes.Among the 350 patients with BJIs, 221 patients met inclusion criteria. For the primary outcome, 169 (76.4%) met oral eligibility criteria with sulfamethoxazole-trimethoprim, tetracyclines, and cephalosporins being the most common PO options. There were 35 total OPAT-related AEs (24 [14.2%] in the PO eligible group and 11 [21.2%] in the PO ineligible group). There were 15 readmissions (11 [6.5%] in the PO eligible group and 4 [7.7%] in the PO ineligible group) and 17 ED visits (13 [7.7%] in the PO eligible group and 4 [7.7%] in the PO ineligible group). For 6-month treatment failure, there were 29 total occurrences (23 [13.6%] in the PO eligible group and 6 [11.5%] in the PO ineligible group).Based on OVIVA trial criteria, the majority of evaluated patients could have utilized oral antibiotics, which could have resulted in fewer OPAT-related AEs, readmissions, and ED visits.Sara C. Keller, MD, MPH, MSPH, Pfizer: Advisor/Consultant


The 3D structures consist of an anionic inorganic tetrel halide framework stabilized by the cesium cations (Cs+). We use computational methods to show that the geometrical connectivity between the inorganic monoanions, [TtX3−]∞, that leads to the formation of the TtX64− octahedra and the 3D inorganic perovskite architecture is the result of the joint effect of polarization and coulombic forces driven by alkali and tetrel bonds. Depending on the nature and temperature phase of these perovskite systems, the Tt···X tetrel bonds are either indistinguishable or somehow distinguishable from Tt–X coordinate bonds. The calculation of the potential on the electrostatic surface of the Tt atom in molecular [Cs+•TtX3−] provides physical insight into why the negative anions [TtX3−] attract each other when in close proximity, leading to the formation of the CsTtX3 tetrel halide perovskites in the solid state. The inter-molecular (and inter-ionic) geometries, binding energies, and charge density-based topological properties of sixteen [Cs+•TtX3−] ion pairs, as well as some selected oligomers [Cs+•PbI3−]n , are discussed.The ion pairs [Cs The all-inorganic counterparts are obtained when the A-site cation is replaced by an inorganic moiety, such as Cs+, Rb+, or K+, for example. The first type has been somewhat misleadingly referred to as organo-metal halide perovskites ) and these are linked to each other in an infinite array in the presence of the inorganic or organic cations. Why these negatively charged units are repeated in an infinite crystal appears not to have been discussed in the perovskite literature, nor have their bond-specific roles. A number of studies have shown that the extrema of the valence and conduction bands in these 3D materials are driven by orbital contributions arising from the inorganic framework formed by the M and X atoms, and the A-site cation is not involved in contributing to or elevating the functionality of these materials   ion pairs, except when Tt = Si; these are the building blocks of the 3D structure of CsTtX3. The strength of these positive sites on Tt is sufficient to attract nucleophiles on the surfaces of the halogen derivative on another similar interacting unit of TtX3−, and a repetition of this process results in the formation of an infinite inorganic framework in the presence of Cs+ ions. We show that the attractive interaction between a pair of TtX3− units in a pair of ion pairs is nothing other than a “tetrel bond” [+•TtX3−] ion pairs and in the [Cs+•PbI3−]n  oligomers is discussed.The aim of this study is to shed light on one of the pressing issues mentioned above. In particular, we shall demonstrate that when a TtXel bond” . The phyel bond” ), in a vA tetrel bond occurs in chemical systems when there is evidence of a net attractive interaction between an electrophilic region associated with a covalently or coordinately bonded tetrel atom (or atoms) in a molecular entity and a nucleophilic region in another, or the same, molecular entity . In othe+•PbI3−]n , but Quantum Theory of Atoms in Molecules (QTAIM) and Molecular Electrostatic Surface Potential (MESP) calculations are also performed on [Cs+•TtX3−]  ion pairs that are supposedly the building blocks in 3D tetrel halide perovskites. The study shows that the formation of the inorganic framework and the geometric stability of cesium tetrel halide perovskite systems arise from the same underlying interactions, tetrel and alkali bonds. Crystalline cesium tetrel halide perovskites were found by searching the Inorganic Crystal Structure Database (ICSD) , when periodically expanded, reveals a cage-like structure. The formation of the latter is driven by Tt···I tetrel bonds, which are equivalent and linear when Tt = Pb and Sn and quasi-linear and non-equivalent when Tt = Ge. For instance, the distances associated with the Tt–I coordinate bonds within the [TtI3−] fragment and Tt···I tetrel bonds between a [TtI3−] pair are 3.145 and 3.110 Å in cubic (Pm-m) CsPbI3  and cage-like (bottom) structures of cesium tetrel iodide perovskites, CsTtI) CsPbI3 a and CsSsPbI3 , are shown in 3 as a representative example of this series, each PbI3− unit, which is a face of the PbI64− octahedron, is involved in an attractive coulombic interaction with Cs+, thereby forming the ion pair [Cs+•PbI3−]. As shown in 3− faces of eight PbI64− octahedra, forming a cage-like structure, with Cs+ trapped inside the cage formed by the eight PbI64− octahedra. In other words, the arrangement between the anion and the cation in CsPbI3 is such that each face of the corner-shared PbI64− octahedra hosts a Cs+ cation  along the outermost extension of each I–Tt bond. In specific, S,maxV > 0 in [Cs+•PbI3−], [Cs+•SnI3−], and [Cs+•GeI3−], becomes progressively less positive, and switches to S,maxV < 0 in [Cs+•SiI3−]. This is the result of the polarizing field induced by Cs+ when it is placed close to the [TtI3−] anion. The polarization of the surface charge density of Tt in [TtI3−] is accompanied by an appreciable transfer of charge from the iodide to Cs+ cation, varying between 0.085 and 0.116 e. This causes the charge density on the surface of Tt to be rearranged in such a manner as to produce depleted regions of charge density on its surface. The charge density-depleted regions appear along the outermost extensions of the I–Tt bonds when Tt = Pb, Sn, or Ge, but not when Tt = Si. These charge density regions on the tetrel atoms along the extension of the three I–Tt bonds are characteristic of σ-holes since they appear opposite to the I–Tt σ coordinate bonds; thus, σ-holes on Tt are electrophilic in [Cs+•PbI3−], [Cs+•SnI3−], and [Cs+•GeI3−]  < Sn  < Ge  < Si . This indicates that Pb and Sn are entirely electrophilic, unlike Ge and Si. The MESP graphs also suggest that the stereochemically active lone pair of the Tt sites are squeezed onto the surfaces of the iodides when Tt = Pb and Sn, but not when Tt = Ge and Si. This conclusion is in accordance with a previous study where it was suggested that “a stereochemically active lone pair of electrons of the Pb atom may lie between the two I atoms in the plane” , [Cs+•SnI3−], [Cs+•GeI3−], and [Cs+•SiI3−]. This is expected, as Si is relatively more electronegative in the tetrel series, hence, the ability of Cs+ to polarize electron density of small-sized Tt decreases. In all cases, both the lateral and axial portions of the halide atoms in [TtI3−] are entirely negative, with S,minV < 0, as seen in The local most maximum potential on the surface of Cs+•TtI3−] have a more ionic character. The Si–I bonds are characterized by negative values of both ∇2ρb and Hb at the Si–I bcps. Although the Tt–I  bonds possess ionic character, the negative Hb values at the Tt–I bcps are indicative of some measure of covalency. These coordinate bonds clearly have mixed bonding character. On the other hand, the cesium-centered charge-assisted alkali bonds, Cs···I, possess closed-shell character (∇2ρb > 0 and Hb > 0) and the charge density at the Cs···I bcps is significantly smaller than that at the Tt–I bcps.Our QTAM results in (b) Cesium Tetrel Bromide Perovskites3] , have been reported in different temperature crystalline phases, except for [CsSiBr3]; the structures of the high-temperature cubic phase are shown in +•SiBr3−] ion pair is unable to coulombically attract the nucleophilic bromide in a neighboring unit. This is supported by the MESP plots of [Cs+•TtBr3−] shown in +•PbBr3−] is entirely positive along and around the outermost extension of the Br–Pb bonds . That of Sn in [Cs+•SnBr3−] is appreciably positive  along and weakly negative  around the outermost extension of the Br–Sn bonds. In [Cs+•GeBr3−], the surface of Ge is weakly positive along and appreciably negative around the outermost extension of the Br–Ge bonds. However, in the case of [Cs+•SiBr3−], the S,maxV and S,minV on Si are all negative. For all four ion pairs, both the axial and equatorial portions of the Br atom along and around the Tt–Br extensions are nucleophilic .The cesium tetrel bromide perovskites, [CsTtBr+•TtBr3−]   bcps, as seen in e.The topological charge density characteristics of the Tt–Br and Cs···Br bcps in the ion pairs  . However(c) Cesium Tetrel Chloride Perovskites3  are known, whereas that of [CsSiCl3] has not been reported. The connectivity between the [TtCl3−] units that lead to the formation of the TtCl64− octahedra in the solid state are evident in all three structures shown in 3 and CsSiCl3 , showing that there is very little difference between the three Tt···Cl tetrel and three Tt–Cl coordinate bonds in these systems. In the case of CsGeCl3, three of the coordinate bonds are different to the other three; the three Ge–Cl coordinate bond distances are equivalent  = 3.036 Å and ∠Cl–Ge···Cl = 172.1°).The solid-state structures of CsTtCl3−] units, which leads to the development of the cage-like inorganic framework, is evident from the results of the MESP analysis shown in +•TtCl3−]. Each of the three Cl–Tt bonds in each ion pair in [Cs+•TtCl3−]  contains electron density-deficient regions along the Cl–Tt bond extensions (σ-holes) with positive electrostatic potentials. However, the potential is weakly negative in [Cs+•GeCl3−] along the three Cl–Ge bond extensions . It is clear from these results that the formation of CsTtCl3  perovskite systems in 3D is expected when repeating units of [Cs+•TtCl3−] ion pairs are in close proximity. This kind of assembly is unlikely when [Cs+•TtCl3−] (Tt = Ge and Si) pairs are in close proximity because of coulombic repulsion between the halogen of an ion pair in close proximity to the negative tetrel site in a neighboring unit.The fact that tetrel bonding plays a vital role in assembling the [TtCl+•GeCl3−]. When the 0.001 a.u. isoelectronic density was used for mapping, the potential associated with each of the three σ-hole holes on Ge was weakly negative . However, when a 0.0015 a.u. isoelectron density was used, the S,maxV of the σ-holes on the same atom was positive, S,maxV = 4.4 kcal mol−1. The positive nature of the σ-hole on Ge explains why Ge in [Cs+•GeCl3−] is capable of attracting the negative portion on the Cl atoms in a neighboring interacting ion-pair, thus leading to the formation of CsGeCl3 perovskite crystals in the crystalline phase  , as well as the formation of three equivalent Cs···Cl alkali bonds. The surface of Cs is strongly positive relative to that of Tt in each [Cs+•TtCl3−] , which rationalizes why the cation lies at the center of the inorganic tetrel halide cage, thus interacting simultaneously with the lone-pair dominant regions of coordinate halides on each of the eight faces of eight octahedra , is evident in the molecular graphs shown in 2ρb > 0 and Hb < 0 .The formation of the alkali bonds in each ion pair, which is expected to mimic what occurs in the crystal ((d) Cesium Tetrel Fluoride Perovskites+•TtF3−] are similar to the other cesium halide perovskite ion pairs discussed above. While the formation of these ion-pair systems is likely in the gas phase, they are not all stable in the crystalline phase. The instability of these perovskite structures is arguably due to the mismatch between the cavity of the fluoride-based inorganic perovskite cage formed from the repeating units of [TtF3−] and the radial size of Cs+. This is not the case for CsPbF3, as seen in Pmm, ICSD ref: 30739  (Tt = Pb and Sn) structures in the solid state since the surfaces of the Tt site in the [Cs+•TtF3−] ion-pair systems are highly electrophilic, with the former more so than the latter. Specifically, the surface of Pb in [Cs+•PbF3−] is entirely positive along and around the F–Pb bond extensions  ion pairs is also evident in the QTAIM-based molecular graphs shown in 2ρb at the Tt–F bcps are positive, showing that they are closed-shell interactions and appreciably ionic. The values of ∇2ρb at Tt–F bcps across the series follow the order [Cs+•SiF3−] > [Cs+•GeF3−] > [Cs+•SnF3−] > [Cs+•PbF3−], which parallels the trend in the negative Hb values for the same bonds  γ-phase, the PbI64− octahedra tilted around all three pseudocubic axes, −a−c+a, which is different to the tilt observed in the tetragonal (4/mbmP), 0a0c+a, β-phase 2 a and 166•PbI3−]3 b. The phstigated ,63,64. I β-phase , and the+•PbI3−]2 dimer (and [Cs+•PbI3−]3 trimer) are given in +•PbI3−]2 dimer (and [Cs+•PbI3−]3 trimer) are characterized by ρb, ∇2ρb and −Hb values in the ranges of 0.0411–0.0472 (0.0379–0.0474), 0.0511–0.0116 (0.0509–0.0549), and 0.0071–0.007 (0.0058–0.0097) a.u., respectively. The corresponding values for the Cs···I alkali bonds were 0.0075–0.0116 (0.0069–0.0103), 0.0020–0.0301 (0.0186–0.0269), and −0.0008 (−0.0008) a.u., respectively. Although the former bonds possess a non-negligible amount of covalent character, the latter are purely electrostatic interactions. Their characteristics are comparable to those of I–Pb···I tetrel bonds . The [Cs+•PbI3−]3 trimer has two non-equivalent I–Pb···I tetrel bonds  a.u., ∇2ρb = 0.0279 (0.0265) a.u., and Hb ≈ 0.00001 a.u for the shorter (longer) bonds. The detailed nature of ∇2ρb at various bcps is shown in +•PbI3−]2 dimer and [Cs+•PbI3−]3 trimer, respectively.The results of our QTAIM analysis for the [Cs+•PbI3−]2 and [Cs+•PbI3−]3 oligomers using MESP results, +•PbI3−] ion pair . Again, this is the result of the gas phase, where the role of the periodic boundary condition is nullified and no packing forces act on the system. The [Cs+•PbI3−] ion pairs are free to interact with each other in the gas phase at 0 K, causing the linear Pb–I–Pb bonds found in the cubic structure of CsPbI3 to change appreciably in a manner so as to adopt a significantly distorted geometry very similar to that observed in the low-temperature orthorhombic phase of the system (vide supra).An interesting feature of the 4 tetramer are shown in Vb) and 2ρb at the bcps). Regardless of the nature of the bonding interactions involved, the sign of Vb is always negative and, hence, stabilizing. Values of ∇2ρb are all positive, indicating that the bonding interactions are of the closed-shell type. While the relationship Eb(QTAIM) = −½ Vb may be empirical, it suggests that the I–Pb···I tetrel bond is stronger than the Cs···I alkali bond (Eb(QTAIM) values of 4.0 kcal mol−1 vs. 1.5 kcal mol−1).The molecular graphs of two different orientations of the [Cs+•PbI3−] ion pairs in the [Cs+•PbI3−]4 tetramer can also be understood from the MESP plots shown in 3−] anions in the presence of Cs+. The physical chemistry of tetrel bonds also plays a significant role in stabilizing 1D CsPbI3, a material suitable for stable X-ray detection .+] and [TtX3−] for all the sixteen [Cs+•TtX3−] ion pairs are summarized in −1) as large as −93.43 [I4Ge···F−], –112.15 ([I4Si···F−]), and −84.05 ([I4Pb···F−]) with [CCSD/def2-TZVPPD], which were very close to those calculated with [ωB97X-D/def2-TZVPPD]  . Large i-TZVPPD] , halogen-TZVPPD] , hydroge-TZVPPD] , and pni-TZVPPD]  in other+•TtX3−], the interaction energies increase as the atomic size of the tetrel derivative increases (Si < Ge < Sn < Pb). This trend agrees well with the increasing strength of the σ-hole on Tt across the series for a given type of halogen derivative .For a series with a given halogen derivative [Cs+•TtF3−] > [Cs+•TtCl3−] > [Cs+•TtBr3−] > [Cs+•TtI3−]). For each series shown in +•PbF3−], is the strongest. This is expected since fluorine in the inorganic moiety [PbF3−] is the most electronegative and electron-withdrawing of the halogens; hence, it strongly interacts with Cs+, which, therefore, cannot create strong σ-holes on Pb in [Cs+•PbF3−]. All these trends remain valid regardless of whether Eb or Eb(BSSE) is considered since the BSSE is calculated to be very small.For a given tetrel derivative, the interaction energy increases as the atomic size of the halogen increases ([Cs+] and [TtX3−] increases when increasing the intermolecular distance between them (+•TtF3−] > [Cs+•TtCl3−] > [Cs+•TtBr3−] > [Cs+•TtI3−]) when decreasing the intermolecular bond distance ([Cs+•TtF3−] < [Cs+•TtCl3−] < [Cs+•TtBr3−] < [Cs+•TtI3−]), as seen in +•TtX3−] is shown in As a rule of thumb, it is expected that the interaction energy increases as the intermolecular distance between interacting moieties decreases. However, this is not the case with the ion pairs explored in this study, where we find the opposite trend. The interaction energy for a given halogen derivative between [Cseen them . This is+•PbX3−]2 dimer, [Cs+•PbX3−]3 trimer, and [Cs+•PbX3−]4 tetramer shown in −1, respectively. These were obtained by subtracting the total electronic energy of the oligomer from two, three, and four times the total electronic energy of the [Cs+•PbX3−] ion pair, indicating that the binding energy is nearly additive. However, these may not be solely due to the tetrel bonds since each Cs+ ion in each [Cs+•PbX3−] ion pair also contributes to the binding of the resulting oligomer through Cs···I alkali bonds. From these results, it is apparent that the empirical relationship Eb(QTAIM) = −½Vb is not applicable to tetrel bond energies since it largely underestimates the magnitudes. The relationship might be useful for some hydrogen-bonded systems ∞, is driven by the inorganic cation, Cs+, through the effects of both electrostatic polarization and coulombic attraction. The joint involvement of electrostatic polarization and coulombic attraction causes redistribution of the charge density profile on the electrostatic surfaces of the molecular tetrel halide perovskites, resulting in the development of positive σ-holes on the Tt atom in the TtX3− anions in the ion pairs [Cs+•TtX3−] . This is accompanied by appreciable amount of charge transfer from the halides of the TtX3− anions to Cs+ when they are in close proximity. The σ-holes formed on the Tt atom, therefore, are able to simultaneously attract the negative halogens from three interacting TtX3− anions, thus leading to the formation of TtX64− octahedra, the underlying framework of the 3D CsTtX3 cesium tetrel halide perovskites. The tetrel bonds formed are hidden between the anion moieties in the solid state structures, and to demonstrate their presence requires appropriate theoretical methods such as the QTAIM and MESP models.This study was undertaken to reveal the underlying reasons why the TtX+•SiX3−]. Therefore, Si is incapable of engaging with the negative halogen in a neighboring unit because of the columbic repulsion between them.There has been a failure to experimentally produce 3D tetrel halide perovskites when Tt = Si. We have explained this failure using the results of the MESP model. The underlying reason is the lack of an appreciable positive potential on the Si atom along the X–Si bond extensions in the molecular ion pair, [Cs3−] units, a cavity that cannot accommodate the inorganic cation. This might explain why CsSnF3 crystalizes in low-dimension. By contrast, the results of the MESP model have showed that CsGeF3 and CsSiF3 perovskites cannot be formed in the crystalline phase because the Tt along the F–Tt bond extensions in the [Cs+•SiF3−]  ion pair has negative σ-holes. These negative σ-holes would repel the entirely negative fluorine atom(s) in a neighboring interacting unit(s), preventing the formation of Tt···F  tetrel bonds. This also explains why the crystal structures of CsSiF3  are unknown.Although the fluorinated ion-pair systems displayed electrophilic regions when Tt = Pb and Sn, the latter does not form a perovskite structure because of the small size of the cavity formed by repeating [SnF+•TtX3−] , were fully energy-minimized, followed by frequency calculations. The most stable conformer was considered. The ωB97XD functional , which we refer to as the [Cs+•PbI3−]n  oligomers, to demonstrate the charge density topologies of alkali and tetrel bonding interactions responsible for the formation of the 3D network of CsPbI3. The nature of physical chemistry revealed for these systems might be transferable to other oligomers when Pb in [Cs+•PbI3−]n  is replaced, for example, by Sn and Ge.Similar calculations, as above, were also performed for a dimer, a trimer, and a tetramer of  were calculated using Equations (1) and (2), respectively. BSSE refers to the basis set superposition error, evaluated using the counterpoise procedure of Boys and Bernardi [ET [Cs+•TtX3−], ET [Cs+] and ET [TtX3−] are the total electronic energies of the respective species.The uncorrected and BSSE-corrected interaction energies , the Laplacian of the charge density (∇2ρb), and the total energy density (Hb) at the  bond critical points (bcps), critical points where the gradients of ρ(r) vanish, were analyzed. The latter two properties at bcps provide insight into the closed-shell and/or open-shell nature of an interaction between a pair of atomic basins in molecular and intermolecular entities. For instance, the positive and negative signs of ∇2ρb were utilized to demonstrate the closed- and open-shell interactions, respectively, which were identified between the inorganic anion and cation and between the anions , utilizing the fully relaxed geometries of the ion pairs and oligomers. The signs and magnitudes of the potential extrema were computed using the 0.001 a.u. isoelectronic density envelope of the ion pairs. The magnitude of potential is a measure of the strength, whereas its (positive and negative) signs were used to provide insight into regions of charge density depletion and concentration on the electrostatic surfaces of the ion pairs, respectively. That is, th corresponding signs of the local most maxima and minima of potential  were utilized to arrive at these conclusions. For instance, the sign of both S,maxV and S,minV can either be positive or negative, or sometimes even neutral. When positive, it is generally assumed that the region on the surface that accompanies this is electrophilic and, hence, may be suitable for accepting electron density from an interacting electron donor in close proximity. When it is negative, the region on the surface of the molecular entity that features this is nucleophilic and, hence, may be capable of donating electron density to an interacting electrophile when in close vicinity. However, it should be kept in mind that all negative or positive sites on the surface of the molecular entity may or may not always be capable of engaging in attractive interaction with a region that features the opposite reactive profile. The MESP calculations ,97,98,99S,maxV is positive  [S,maxV is negative  [3C–F is negative, whereas that on X in H3C–X, X3C–X, and F5CX  is positive. The underlying equation details and applicability of the MESP model to understand non-covalent interactions have appeared in several studies [An electrophilic σ-hole on atom A lying opposite to the R–A covalent bond is characterized when the sign max > 0) ,100. Simmax < 0) ,100. For studies ,106,107,AIMAll  and Mult


All AIE+ patients exhibited intrathecal synthesis of FLCK compared to only 39% of AIE– patients and 81% of patients in the INE group. No intrathecal synthesis of FLCK was found in DGE and NIND patients. While intrathecal FLCK was equally specific for an inflammatory etiology as oligoclonal bands (OCB) in the cerebrospinal fluid (CSF), the sensitivity of intrathecal FLCK for any inflammatory intrathecal process was higher than that of OCB (83% vs. 38%). Intrathecal FLCK synthesis was found to discriminate AIE+ from non-inflammatory encephalopathies and AIE– when the CSF cell count was normal , while it failed to differentiate between AIE+ and INE in the presence of CSF pleocytosis . In conclusion, in the absence of CSF pleocytosis, intrathecal FLCK discriminated AIE+ from competing diagnoses in our cohort of subacute onset neuropsychiatric syndromes. In addition to established markers of CSF inflammation, intrathecal FLCK might support clinical decision-making and contribute to selecting patients for (repeated) antibody testing.Intrathecal synthesis of free light chains kappa (FLCK) is increasingly recognized as a marker of inflammatory CNS pathologies. Here, we tested the performance of FLCK in differentiating autoimmune encephalitis (AIE) from non-inflammatory etiologies in subacute onset neuropsychiatric syndromes. Patients undergoing diagnostic work-up for suspected autoimmune encephalitis at our department between 2015 and  Infectious causes of encephalitis tend to be reliably detected, while the diagnosis of autoimmune encephalitis (AIE) can be challenging2. The published diagnostic criteria have facilitated clinical decision-making and complemented the gold standard of antibody detection3. However, antibody test results return delayed (or negative) in an often dramatic clinical setting4. Conversely, studies and real-world evidence found that timely diagnosis and initiation of immunosuppressive therapy foster recovery and long-term independence in AIE patients5. While standard cerebrospinal fluid (CSF) parameters and polymerase chain reaction often allow a swift identification of infectious etiologies, the need for biomarkers to disentangle subtypes of AIE and guide management in the early phase of the diagnostic process has not been met6.Neuropsychiatric symptoms are among the most common reasons for neurologic consultation. Among many underlying disorders, encephalitis—if recognized timely—represents a treatable diagnosis7. Intrathecal synthesis of FLCK is increasingly recognized as a marker of inflammatory CNS pathologies such as multiple sclerosis11. Like immunoglobulins, FLCK can be measured in CSF and serum by nephelometry. Standardized, commercially available nephelometric assays provide a fast, easy-to-analyze, and quantitative measure of intrathecal FLCK, which is relatively resilient to blood contamination, storage time, temperature, as well as some types of immunomodulation13. While stand-alone serum measures of FLCK do not faithfully reflect inflammation in the intrathecal space due to the blood-CSF barrier and various confounding factors in the systemic compartment, pairwise measures of FLCK in serum and CSF are used to calculate intrathecal synthesis, taking into account the blood-CSF barrier function. The Reiber diagram for the determination of the intrathecal fraction of FLCK (IF-FLCK) has been widely accepted in German-speaking countries and has gained some attention throughout Europe as it considers blood-CSF barrier function and diffusion of serum proteins. Therefore, it provides superior diagnostic accuracy as compared to linear ratio calculations of intrathecal FLCK15.In addition to intact immunoglobulins, plasma cells in the systemic and CSF compartment produce excess monomeric free light chains (FLC) of either kappa (FLCK) or lambda (FLCL) subtypeHere, we tested whether intrathecal FLCK synthesis (as defined by the Reiber diagram) can aid in discriminating AIE from relevant differential diagnoses in patients with subacute neuropsychiatric syndromes.In neuropsychiatric syndromes with subacute onset, a diverse spectrum of differential diagnoses must be considered. CSF antibody testing (as the gold standard) for the diagnosis of AIE returns with a latency that may delay treatment and thus negatively affect the neurological outcome. In a cohort of suspected AIE patients, we tested whether FLCK could be an early marker of AIE to narrow the gap for timely intervention.We screened 371 patients undergoing diagnostic work-up for suspected AIE between 2015 and 2020. One of the known anti-neural  antibodies or antibodies against intracellular antigens was detected in 46 patients (12%). Thirty-three patients (9%) had a syndrome suggestive of AIE but no detectable antibody. Nineteen patients (5%) had encephalitis due to an infectious cause. Thirty-five patients (9%) were diagnosed with a degenerative disorder accounting for their encephalopathic syndrome, and 63 patients (17%) received autochthonous psychiatric diagnoses. The remainder of the syndromes (47%) were assigned to metabolic, neoplastic, toxic, or genetic etiologies, delirium, and unknown or pending causes at the time of the study . Three patients were excluded owing to competing inflammatory diseases  and to a syndrome incompatible with AIE. One patient in the INE group was excluded due to sterile CSF. Eventually, 62 patients remained in the cohort and were stratified according to their diagnosis into AIE+, AIE–, INE, and DGE  allows for psychiatric diagnoses to be made with a reasonable degree of accuracyA control group of 25 patients with non-inflammatory neurological diseases  was established, leading to a total number of 87 patients that were further analyzed.+ group (n = 10/19), 44% of the AIE– group (n = 8/18), and 90% of the INE group (n = 9/10). CSF pleocytosis was absent in the DGE and NIND groups. CSF cell counts differed significantly between the groups, with the highest white cell counts in the INE group , intermediate counts in the AIE– group , and lowest counts in the AIE+ group .CSF pleocytosis was found in 53% of the AIE+ [n = 8/19 (42%)] and AIE– [n = 4/18 (22%) while least frequent in DGE (n = 3/15 (20%)] and NIND [n = 3/25 (12%)]. Intrathecal synthesis of either IgG, IgA, or IgM was present in 40% of INE (n = 4/10), 16% of AIE+ (n = 3/19), 11% of AIE– (n = 2/18), and in none of either the DGE or NIND patients.Blood-CSF barrier dysfunction (corrected for age) was most frequent in INE [n = 6/10 (60%)], followed by AIE+ patients (n = 6/19), in 33% of AIE– patients (n = 6/18), in 50% of INE patients (n = 5/10), and in none of the individuals in the DGE and NIND groups.The presence of CSF-specific OCB was determined in all patients for whom paired CSF/serum samples were still available (n = 84/87). We found CSF-specific OCB in 32% of AIE2) at the time of sample acquisition: two patients in the AIE+ group , two patients in the AIE– group (both with IF-FLCK < Qlim), one patient in the NIND group (IF-FLCK < Qlim), and none in the INE and DGE groups.Renal function was not found to be divergent across groups Table . Overall+ B cell fractions were higher in the inflammatory group as compared to the non-inflammatory groups (p ≤ 0.001). We found the highest CSF B cell fractions in the AIE+ group [mean (SD): 4.6% (4.9)] and the AIE– group [mean (SD): 3.2% (3.1)] and the lowest fraction in the INE group [mean (SD): 1.4% (1.4)]. Here, the group difference between AIE+ and INE reached statistical significance, providing a potential means to distinguish early infectious  from autoimmune encephalitis when the remainder of the CSF parameters were consistent with an inflammatory etiology , indicating intrathecal synthesis of FLCK . In contrast, only 39% of the individuals in the AIE– group (n = 7/18) showed intrathecal FLCK synthesis  of FLCK ratios in the AIE5/19 of Fsis Fig. . In the mean), we observed significant differences in intergroup comparisons between inflammatory  and non-inflammatory  conditions  and one arm with CSF pleocytosis  in this cohort of newly onset neuropsychiatric syndromes .In a cohort of 62 patients with a new onset (< 3 months) neuropsychiatric syndrome, we found only patients with retrospectively validated AIE and patients with infectious encephalitis to exhibit intrathecal FLCK synthesis at first admission. While the CSF white cell count tended to be higher in infectious encephalitis, the intrathecal fractions of FLCK did not discriminate between infectious encephalitis and AIE. However, intrathecal FLCK synthesis allowed us to stratify patients with neuropsychiatric syndromes into inflammatory vs. non-inflammatory etiologies. Here, intrathecal FLCK synthesis performed equally well as CSF pleocytosis. Moreover, in therapy-naive patients with neuropsychiatric syndromes that had a normal cell count in the CSF, elevated intrathecal FLCK indicated an inflammatory etiology , 33% in AIE– (n = 2/6), and 80% in INE (n = 4/5). An even smaller fraction of all FLCK-IF+ individuals showed intrathecal Ig synthesis, i.e. 20% in AIE+ (n = 3/15), 29% in AIE– (n = 2/7), and 44% in INE (n = 4/9). The overall presence of OCB in our diagnostic groups—50% in INE, 33% in AIE–, 32% in AIE+, and none in DGN and NIND control individuals—was consistent with previous reports19. In particular in AIE, the presence of OCB varies substantially, with the highest incidences in anti-GABA(b) and anti-NMDA antibody-positive AIE and the lowest incidences in anti-LGI-1 and anti-IgLON5 antibody-positive AIE19. When comparing intrathecal FLCK synthesis with CSF-specific OCB, intrathecal FLCK synthesis detected inflammatory etiologies in our cohorts with equal specificity but higher sensitivity than OCB  had intrathecal FLCK synthesis at first admission, very similar to INE patients. This is consistent with previous studies reporting elevated levels of intrathecal FLCK in tick-borne encephalitis, Epstein-Barr-Virus-related encephalitis, Varicella-Zoster-Virus-related encephalitis, as well as bacterial encephalitis21.Inflammatory CSF transformation is widely considered as a key feature of AIE. Nevertheless, the degree of inflammatory CSF transformation can differ substantially across the subtypes of AIE. For instance, anti-NMDAR antibody encephalitis presents with CSF pleocytosis in about 80% of the cases, while anti-LGI-1 and anti-CASPR2 antibody positive encephalitis often exhibit normal CSF standard parameters+, only about 40% of AIE– patients showed intrathecal FLCK synthesis. It is possible that pathogenic events other than autoantibody-mediated functional or structural perturbations (such as T cell-mediated autoimmune pathology) might be operational in AIE– patients without intrathecal FLCK synthesis. However, due to the retrospective design of our study, many patients may also have been misclassified as AIE in their medical records. In fact, only 9 of the 18 individuals in the AIE– group fulfilled the recently published criteria for AIE, which we applied retrospectively to our cohort based on medical records3. Seven of these 9 individuals (78%) presented with intrathecal FLCK synthesis, and 6 (67%) showed a response to first-line immunotherapy (corticosteroids or plasmapheresis) in terms of functional improvement or structural response in follow-up MRI imaging. Interestingly, the post-hoc categorization as probable or possible AIE by clinical criteria and the response to immunotherapy corresponded well with the presence of intrathecal FLCK synthesis  showed intrathecal FLCK synthesis, even if the CSF white cell count was elevated. Patients with encephalopathies due to neurodegenerative disease  and non-inflammatory neurological disease controls exhibited no intrathecal FLCK synthesis. This argues for an accurate selection of our NIND controls. In fact, in other NIND cohorts, intrathecal FLCK synthesis was reported in about 6.5% of cases6. Here, in the search for group-specific cell fraction patterns within the inflammatory groups , only the B cell fraction differed substantially between AIE+ and INE, likely indicating diverse cellular response patterns at symptom onset in viral and autoimmune encephalitis. Notably, IF-FLCK correlated moderately with the intrathecal B cell/plasma blast fractions and with the CSF white cell count.While FLCK production rather reflects B-cell-mediated inflammatory events, other (more T cell/macrophage-mediated inflammatory processes) might not translate to similarly high concentrations of FLCK, which could explain variable findings of IF-FLCK in heterogenous inflammatory neurologic diseases. As recently published, cellular peripheral blood and CSF profiling improve the differential diagnosis of neuroinflammatory disordersBased on our findings in this cohort of suspected AIE cases, the presence of intrathecal FLCK synthesis should raise suspicion of an inflammatory etiology, favoring autoimmune over infectious encephalitis in the absence of CSF pleocytosis. In the presence of CSF pleocytosis, intrathecal FLCK has no added value to the diagnosis of AIE. In the absence of both CSF pleocytosis and intrathecal FLCK synthesis, non-inflammatory etiologies should be considered. Thus, measuring intrathecal FLCK may prevent unnecessary antibody testing, and conversely, when AIE is suspected, elevated IF-FLCK but normal CSF cell counts should encourage a continued search for autoantibodies.FLCK, which is well accepted in German-speaking countries, whereas previous international studies relied on the linear FLCK index or exponential curves19. More importantly, potential pre-analytic confounders of the determination of intrathecal FLCK synthesis are immunomodulatory or immunosuppressive therapies and renal function and must be considered when interpreting FLCK measurements in CSF and serum.Intrathecal FLCK synthesis has been considered a methodologically robust parameter. In the present study, we used the hyperbolic reference range for Q9. Here, we excluded all individuals on steroids or other immunomodulatory agents from the analysis. This strategy might explain some differences in the fraction of intrathecal FLCK synthesis in our patients with inflammatory etiologies as compared to the fraction of intrathecal FLCK synthesis in inflammatory CNS diseases reported by other investigators8.Corticosteroid therapy decreases FLCK concentrations in the serum+, two AIE-, and one NIND). Therefore, we do not consider our interpretation to be substantially biased due to renal function impairment, in particular since this would rather produce a false negative than a false positive classification of intrathecal FLCK synthesis.The likelihood of intrathecal FLCK synthesis decreases with declining renal function due to an increase in serum concentrations of FLCK. Even though Reiber's method is more resilient to the influence of impaired renal function than the linear FLCK index, renal function has to be taken into consideration. We did not observe an unbalanced distribution of individuals with renal failure in our diagnostic groups  shared the initial clinical suspicion of AIE—it also stresses the need to strictly adhere to the diagnostic criteria. Besides the retrospective design, further limitations of our study are due to the small group sizes and single measurements of FLCK in blood and CSF due to the limited availability of material. Even though our study was adequately powered to detect large differences between the groups and the results were significant, our findings will need to be replicated in larger-scale multicenter approaches before any implementation into clinical practice can be considered.First, the post-hoc assignment of patients to the diagnostic groups based on medical records might have introduced a bias. As mentioned above, the retrospective application of the published AIE diagnostic criteria+ appears unlikely in the event of both normal CSF standard parameters and the absence of intrathecal FLCK synthesis. We propose that intrathecal FLCK may be exploited as a predictor of possible AIE in patients with neuropsychiatric syndromes without CSF pleocytosis. Larger cross-sectional studies are required to confirm this hypothesis.In the setting of a new onset neuropsychiatric syndrome and otherwise missing inflammatory CSF transformation, the intrathecal synthesis of FLCK may predict an inflammatory etiology such as antibody-positive autoimmune encephalitis with high sensitivity and specificity. Conversely, AIEWe retrospectively included patients with first-episode subacute-onset neuropsychiatric syndromes undergoing diagnostic work-up for AIE at the Department of Neurology of the Technical University of Munich School of Medicine between 2015 and 2020. This study was approved by the ethics committee of the Technical University of Munich School of Medicine . All methods were carried out in accordance with relevant guidelines and regulations. Informed consent was obtained from all study subjects or their legal guardians.As part of routine diagnostics at our department, freshly drawn serum and CSF samples were frozen within 30 min and stored at − 80 °C in our biobank. Flow cytometric analysis of immune cell subsets (FACS) was routinely performed on fresh CSF samples. Paired blood and CSF samples dating to the first admission were taken from our biobank. Course of disease and discharge diagnoses were extracted from medical records.Patients with insufficient clinical data or incomplete biosamples were excluded from the evaluation, as were patients on immunosuppressive therapies. We also excluded cases with metabolic, toxic, neoplastic, and genetic etiologies of neuropsychiatric syndromes as well as delirium. Of note, cases with retrospectively assigned autochthonous psychiatric diagnoses as a large group of differential diagnoses were also excluded from the evaluation.A/B receptors, anti-LGI-1, anti-CASPR2, anti-ZIC4, anti-DPPX, anti-glycin receptors, anti-AGNA, anti-mGluR1, anti-mGluR5, anti-Rho-GTPase activating protein 26, anti-ITPR1, anti-Homer 3, anti-myelin oligodendrocyte glycoprotein (MOG), anti-neurochondrin, anti-GluRD2, anti-flotillin 1/2, anti-IgLON5, anti-CV2, anti-PNMA2, anti-VGCC, anti-recoverin, and anti-SOX1. The cohort was then stratified according to the following criteria:Eventually, our cohort was defined as a cohort of patients with an encephalopathic syndrome suggestive of or compatible with AIE, characterized by rapid progression of fewer than 3 months, working memory deficits, changes in behavior or mental status , or psychiatric symptoms. All enrolled patients (except patients from the control group) were tested for CSF autoantibodies, including anti-Hu, anti-Yo, anti-Ri, anti-ANNA-3, anti-Tr/DNER, anti-myelin, anti-Ma/Ta, anti-GAD65, anti-amphiphysin, anti-aquaporin-4 (AQP4), anti-glutamate receptors , anti-GABA+—detectable autoantibody (cell-surface or intracellular antigens) in the CSF and absence of an infectious agent; AIE–—absence of autoantibodies and infectious agents in the CSF, also not meeting diagnostic criteria for DGE; INE—absence of autoantibodies and detection of an infectious agent in the CSF by PCR; DGE—patients with a syndrome fulfilling diagnostic criteria for neurodegenerative diseases31 without CSF autoantibodies. The control group, NIND , was defined by the consensus definitions for control groups in CSF biomarker studies32. After pseudonymization, the investigators were blinded to the diagnoses until the end of the analysis.AIEIF = (1 − Qlim/QKappa) × 100 [%]) with QKappa as the CSF/serum quotient of FLCK using the CSF-Tool software from Albaum IT solutions )14. Immunoglobulins and albumin were measured by immunonephelometry on the same Siemens BN Prospec nephelometer according to the manufacturer’s protocol. Quantitative expressions of the intrathecal humoral immune response were based on the calculation of the CSF/serum ratios of IgG, IgM, and IgA with QIg = IgCSF [mg/L]/Igserum [g/L]. The upper limits of the respective reference ranges, Qlim(IgG), Qlim(IgM), and Qlim(IgA), were calculated against QAlb according to Reiber’s revised hyperbolic function. Values for Q (Ig) exceeding Qlim (Ig) were considered to indicate an intrathecal immunoglobulin synthesis33. The CSF/serum albumin ratio, QAlb = AlbCSF [mg/L]/Albserum [g/L], was used to assess blood-CSF-barrier function. Blood-CSF-barrier dysfunction was defined as QAlb > 10 × 10–3 for an age of 60 years or older, QAlb > 8 × 10–3 for an age of 40–59 years, and QAlb > 6.5 × 10–3 for an age of 39 years or younger. Oligoclonal Ig bands (OCB) were assessed by isoelectric focussing and consecutive silver staining in polyacrylamide gels as described previously34. Following the European consensus standards on CSF analysis in Multiple Sclerosis (MS), we classified positive OCB as pattern type 2 or type 335.Paired CSF and serum samples stored at – 80 °C were analyzed for FLCK levels using the N Latex FLC kappa kit  according to the manufacturer’s protocol on a Siemens BN Prospec nephelometer. CSF predilution was set to 1:1; serum predilution was set to 1:100. According to the manufacturer, the lower limit of detection was 0.034 mg/L. The hyperbolic reference range and the intrathecal fraction of FLCK (IF-FLCK) were calculated according to the formula by Reiber et al. (KAntibody testing was performed using cell-based assays (CBAs) and consequently confirmed using immunofluorescence in commercial test kit panels .36. Briefly, fresh CSF was immediately spun down (300 g for 10 min), the supernatant removed, and the pellet resuspended in phosphate-buffered saline (PBS)  with 2% fetal calf serum (FCS) . After incubating with an antibody mix (20 min at 4 °C), cells were spun down, washed, and resuspended in PBS wash solution (including 2% FCS) for flow cytometric analysis . The following antibodies were used for staining: CD4 PerCP, CD3 APC-Cy7, CD45 VM , CD19 ECD, CD56 APC, CD14 FITC, and CD138 PE . This allowed differentiating between CD4+ T cells (CD45+CD3+CD4+), CD8+ T cells (CD45+CD3+CD8+), monocytes (CD45+CD14+), NK cells (CD45+CD56+), B cells (CD45+CD19+CD138−), and plasma blasts (CD45+CD19+CD138+).CSF white cell counts > 4/µL were classified as pleocytosis. FACS (Fluorescence activated cell sorting) was performed at the time of sampling as described previously2) according to the CKD-EPI equation37.Renal function was assessed as part of routine diagnostics for all patients under investigation at the time of sampling and estimated by eGFR (mL/min/1.73 mhttps://www.python.org/). To compare two or more groups, the Mann–Whitney-U test or the Kruskal–Wallis test was used. The locally synthesized absolute amount of FLCK and IF-FLCK were calculated with respect to the Qmean  for statistical group comparison. For ROC (Receiver Operating Characteristics)-analysis, the IF-FLCK was calculated in relation to Qlim .Statistical analysis was performed using GraphPad Prism  and Python 3.10 .Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.