This work may offer insight into well controlled design of organic linkers for the synthesis of LMOFs with specified functionality.Luminescent metal–organic frameworks (LMOFs) have been extensively studied for their potential applications in lighting, sensing and biomedicine-related areas due to their high porosity, unlimited structure and composition tunability. However, methodical development in systematically tuning the emission properties of fluorescent organic linker-based LMOFs to facilitate the rational design and synthesis of target-specific materials has remained challenging. Herein we attempt to build an emission library by customized synthesis of LMOFs with targeted absorption and emission properties using donor–acceptor–donor type organic linkers. By tuning the acceptor groups ( An emission library was built for donor–acceptor–donor type linker-based LMOFs, which can be used to rationally design organic linkers to prepare LMOFs with emission from deep blue to near-infrared. To tune the emission properties of LMOFs in a broad energy region (including the visible light and near-infrared range) and to study the structural effect on their emission behaviors, it is essential to build design principles based on the structure–property relationship, which will facilitate the customized synthesis and target-specific applications of LMOFs. However, tuning the emission behaviors of organic linker based LMOFs in a fully controllable manner remains a challenging task.Organic linker-based luminescent metal–organic frameworks (LMOFs)15,16 In these donor–acceptor–donor (D–A–D) based linkers, the emission tunability relies on the changeable electron-withdrawing capacity of acceptor groups. While these acceptors have been used in different D–A–D linkers to prepare LMOFs for various applications,10,17–23 a systematic study to tune the emission properties of D–A–D linker based LMOFs and to develop a related design principle is still lacking.In our previous work, we achieved full-color emissive LMOFs using 2,1,3-benzothiadiazole and its derivative-based dicarboxylic acids and tetratopic carboxylic acids as organic linkers.15,16 and (3) tuning the bridging units between donor and acceptor groups24,25  with strong electron donating or electron withdrawing capacity; (2.2) use of totally different donor groups, for which metals may link to different binding sites, e.g. to oxygen when using carboxylic acid-based donor groups or nitrogen when using pyridine or azole-based donor groups  tuning the electron density of the acceptor groups; (2) tuning the electron density of the donor groupsups24,25 . There ar groups . Note thN , where N is the number for each new MOF. This relatively large database allows us to build an emission library with a broad range of emission energies, including both visible and near infrared (NIR) regions.Based on these strategies, we carry out a systematic study in the present work to tune the emission properties of D–A–D type linker based LMOFs by tailoring the acceptor groups, donor groups and the bridging units, where 2,1,3-benzothiadiazole and its derivative are utilized as the acceptor groups. Among the 23 organic linkers used in this study, 13 are newly synthesized or used to prepare MOFs for the first time, and 8 new MOF structures are obtained. For MOFs with the same structure as UiO-68, we name them UiO-68-L, in which L is the abbreviation of the organic linker; and for MOFs with new structures, we name them HIAM-15 the emission colors of LMOFs can be preciously controlled from blue to red by changing 2,1,3-benzothiadiazole and its derivative based acceptor groups from 2H-benzo[d]triazole, 5,6-dimethylbenzo[c]thiadiazole, benzo[c]thiadiazole, benzo[c]selenadiazole, naphthothiadiazole and naphthoselenadiazole to form six dicarboxylic acids based linkers of 4,4′-bis(3-methoxybenzoic acid) (BAMB), 4,4′-dibenzoic acid (MBTB), 4,4′-bis(3-methoxybenzoic acid) (BTMB), 4,4′-bis(3-methoxybenzoic acid) (BSMB), 4,4′-bis(3-methoxybenzoic acid) (NTMB) and 4,4′-bis(3-methoxybenzoic acid) (NSMB)  (NSMB) . This stH-benzo[d]imidazole and benzo[c]oxadiazole were used to form 4,4′-bis(3-methoxybenzoic acid) (BIMB) and 4,4′-bis(3-methoxybenzoic acid) (BOMB). These two linkers show deep blue and green emissions in N,N-dimethylformamide (DMF) solution . These results further demonstrate that highly tunable emissions are achievable for D–A–D type organic linker based LMOFs by simply changing 2,1,3-benzothiadiazole and its derivative based acceptor groups.As mentioned earlier, two strategies can be used to tune the donor groups: (2.1) Modifying original donor groups, where the linker–metal bond model remains intact; (2.2) Employing different types of donor groups, in which new linker–metal bonds may form, leading to different structures.c]thiadiazole-4,7-diyl)bis(3-(trifluoromethyl)benzoic acid) (BTTB), 4,4′-bis(3-methoxybenzoic acid) (BTMB), 4,4′-bis(3-hydroxybenzoic acid) (BTHB) and 4,4′-bis(3-aminobenzoic acid) (BTAB), were designed and synthesized with single-site modification on the donor groups  with strong electron withdrawing capacity and a significant bathochromic shift was realized when using strong electron donating groups (i.e. OH and NH2).26 These results indicate that single-site modification of original donor groups is indeed a useful strategy to tune the emission properties of the LMOFs without changing the crystal structure.To confirm that the modification of original donor groups indeed has the capacity to tune the emission of LMOFs, four linkers, 4,4′-, 4,9-di(pyridine-4-yl)naphthothiadiazole (PNT) and 4,9-di(pyridine-4-yl)naphthoselenadiazole (PNS), were designed and synthesized benzothiadiazole to naphthothiadiazole or naphthoselenadiazole. As depicted in 24N2) to the 6-c binuclear structure 2(COO)2N4). Two Zn(ii) atoms are bridged by two carboxylate groups, and the remaining coordination sites are occupied by four N atoms from two pairs of PNT (or PNS) ligands and two O atoms from two BDC ligands. Those binuclear SBUs are further connected by BDC ligands and PNT (or PNS) ligands to give the 3D pcu network with 2-fold interpenetration. Strong π–π interactions are found between the naphthothiadiazole rings in HIAM-3002 (centroid-to-centroid distance is 3.5317(3) Å), and naphthoselenadiazole rings in HIAM-3003 (centroid-to-centroid distance is 3.5925(3) Å).HIAM-3002 and HIAM-3003 form a similar 2-fold interpenetrating X (X = 1–3) also exhibits high resistance to heat and stability up to 350 °C for HIAM-3001 and 400 °C for HIAM-3002 and HIAM-3003, respectively benzo[c]thiadiazole (DPBT), 4,7-di(1H-pyrazol-4-yl)benzo[c]selenadiazole (DPBS) and 5,6-dimethyl-4,7-di(1H-pyrazol-4-yl)benzo[c]thiadiazole (DDPBT), were designed and synthesized to study their effect on the emission properties of their corresponding LMOFs  are similar to those used to synthesize HIAM-3001 but without addition of 1,4-dicarboxybenzene. Single-crystal X-ray diffraction analysis reveals that HIAM-3004 crystallizes in the tetragonal crystal system with an I41 space group (ii) cation is fully coordinated in a tetrahedral geometry with four nitrogen atoms from four DPBT ligands. Each DPBT ligand is fully coordinated with four Zn(ii) cations, in which each pyrazolate group of the ligand connects two adjacent Zn(ii) cations. The alternative connection of Zn atoms and pyrazolate groups results in an infinite 41 helical chain along the c axis  Å). An identical crystal structure was formed when DPBS was employed as the luminescent linker. It should be noted that a similar MOF to HIAM-3004 was reported for photocatalytic aerobic oxidation when we prepared our manuscript, which further demonstrates the promising applications of these MOFs.34The synthesis conditions for pyrazolate-based HIAM-300ce group  and S8†.c]thiadiazole to 5,6-dimethylbenzo[c]thiadiazole for pyrazolate-based linkers. For HIAM-3006, a similar connection model to HIAM-3004 was observed where the Zn(ii) cation is fully coordinated in a tetrahedral geometry with four nitrogen atoms from four DDPBT ligands. Each DDPBT ligand is fully coordinated with four Zn(ii) cations, in which each pyrazolate group of the ligand connects two adjacent Zn(ii) cations to give a 1D Zn chain along the a-axis. The Zn chains are further extended by the DDPBT ligand to yield a 3D framework with 1D channels along the a-axis  also exhibits high stability in aqueous solutions from pH 2 to 12 and thermal stability up to 500 °C , HIAM-3001 (499 nm) and HIAM-3004 (563 nm), it is clear that introducing a pyridine group as the donor will induce a blue-shift, while a red-shift is observed when using pyrazolate as the donor. More importantly, with the tunable donor groups, abundant structural diversity can be realized, which might lead to new properties and applications.24,25 Incorporating thiophene groups into fluorophores could also increase the light absorption and emission.35–37 In our previous work, we found that when the acceptor groups change from benzo[c]thiadiazole to naphthothiadiazole, the emission shifts from 520 nm to 585 nm.15 According to these results, we believe that organic linkers with a significant red-shift might be synthesized if these two features are combined in one structure. On the other hand, benzo[c]thiadiazole and naphthothiadiazole based carboxylic compounds have been utilized to prepare LMOFs with tunable emissions, and thus the biggest challenge is how to synthesize carboxylic compounds with a bathochromic shift in their emissions, which may provide the opportunity to obtain organic-linker-based NIR LMOFs.It has been reported previously that different bridging groups show significant effects on the light absorption and emission behavior of D–A–D type compounds. For example, an emission shift from 610 nm, 622 nm to 647 nm was observed upon adding an ethynyl or a vinyl group between the donor and acceptor.c]thiadiazole-4,7-diyl)dibenzoic acid (BTBA)10 and 4,4′-)dibenzoic acid (BTTBA).22 And the other four are newly synthesized linkers, namely 4,4′--2,2′-bisdibenzoic acid (BTEBA), 4,4′--2,2′-bisdibenzoic acid (NTEBA), 4,4′-bis)dibenzoic acid (BTTB) and 4,4′-bis)dibenzoic acid (BTTD). BTBA, BTTBA and BTEBA were chosen to confirm the effect of bridging groups of ethynyl and vinyl. NTEBA was designed to achieve NIR emission. BTTB and BTTD were prepared to investigate the effect of thiophene and the substitution sites on the emission behaviors. The molecular orbitals of BTBA, BTTBA, BTEBA and NTEBA were calculated using density functional theory (DFT). As depicted in c]thiadiazole to naphthothiadiazole.To prove our hypothesis, six organic linkers were utilized in this section . Two of c]thiadiazole-4,9-diyl)dibenzoic acid (NTB), in which a bathochromic-shift of 100 nm was recorded after the addition of vinyl groups to NTB to form NTEBA. (ii) When acceptors were changed from benzo[c]thiadiazole to naphthothiadiazole, a 125 nm red-shift was realized. These results demonstrate that the combination of these two strategies together indeed offers a powerful approach to tune the emission properties and to achieve dicarboxylic acid-based NIR emissive organic linkers.The UV-vis absorption and steady-state emission spectra of the four organic linkers in DMF solution were then measured and plotted in 10,22 A typical synthesis of BTEBA and NTEBA based Zr-MOFs, HIAM-400X  is as follows: a 5 mL vial containing 54.0 mg l-proline and 63.0 μL was placed in a preheated oven at 100 °C for 30 minutes; after cooling down to room temperature, 3 mL DMF, ZrCl4 , and an organic linker (0.094 mmol) were added to the vial, which was placed in a preheated oven at 120 °C for 48 hours. Orange (HIAM-4005) and dark red (HIAM-4006) octagon-shaped single crystals were obtained  were synthesized according to literature procedures Fig. S13.10,22 A obtained . Single-ameworks . Therefoameworks , indicatThe solid-state photoluminescence and UV-vis absorption spectra were measured. As shown in In addition to ethynyl and vinyl bridging units, thiophene was also utilized to link the donor and acceptor groups. Compared with BTBA (480 nm), BTTBA (500 nm) and BTETA (565 nm), the maximum emission peaks of BTTB and BTTD are 553 nm and 638 nm in DMF solution under 365 nm excitation, respectively Fig. S14. This reBased on the aforementioned results, it is clear that the emission properties of D–A–D type organic linker-based LMOFs can be systematically tuned by changing the acceptor groups, modification of the original donor groups, using different donor groups and choosing various bridging units. Therefore, an emission library can thus be built by applying different strategies described in this work .In conclusion, four strategies have been used to methodically tune the emission behaviors of D–A–D type organic linker-based LMOFs, from which a large emission library can be built in a systematic manner. By precisely controlling the acceptor groups, donor groups and the bridging units, emissions of the resultant LMOFs covering the entire visible light spectrum as well as the NIR region can be achieved with abundant structural diversity. A large number of organic linkers can be designed and synthesized with varying emission behaviors from deep blue to the NIR range by gradually decreasing the electron density of acceptor groups or increasing the electron density of donor groups. This work may not only serve as a toolbox to facilitate the development of design principles for the rational design of organic linkers and customized synthesis of target-specific LMOFs but also provide a useful platform to explore NIR emissive LMOFs for biosensing and bioimaging applications.All crystallographic data have been deposited in the CSD. No other data is present.X.-Y. L. conceived the idea and designed the experiment; H.-L. Xia, S. Wu and D. Ren worked on the synthesis and the characterization studies of all materials. K. Zhou and K. Xing carried out the analysis of the single crystal structures. X. Wang and J. Guo did the density functional theory calculations. X.-Y. L. and J. Li wrote the paper with help from all authors. H.-L. Xia and K. Zhou contributed equally to this work.The authors declare no competing financial interests.SC-013-D2SC02267B-s001SC-013-D2SC02267B-s002


We found that all (100%) patients had CSF antibodies when the in house IIF-CBA was used, but only 88 (83%) were positive if the commercial test was used. In contrast, sera positivity rate was higher with the commercial test (94%) than with the in house assay (86%). If both serum and CSF were examined with the commercial IIFA-CBA, 69/70 (98.5%) patients were positive in at least one of the samples. These findings are clinically important for centers in which rat brain IIHC and in house IIFA-CBA are not available. Moreover, the observation that all patients with anti-LGI1 encephalitis have antibodies in CSF is in line with the concept that these antibodies are pathogenic.Detection of Leucine-rich glioma inactivated 1 (LGI1) antibodies in patients with suspected autoimmune encephalitis is important for diagnostic confirmation and prompt implementation of immunomodulatory treatment. However, the clinical laboratory diagnosis can be challenging. Previous reports have suggested that the type of test and patient’s sample (serum or CSF) have different clinical performances, however, there are no studies comparing different diagnostic tests on paired serum/CSF samples of patients with anti-LGI1 encephalitis. Here, we assessed the clinical performance of a commercial and an  All patients with anti-LGI1 encephalitis harbor LGI1 antibodies in CSF. These antibodies are detectable by indirect immunofluorescence cell based assay (IIF-CBA) on cells co-expressing LGI1 and ADAM23.In patients with suspected autoimmune encephalitis, both serum and CSF should be examined if a commercial IIF-CBA is used for antibody determination.Autoimmune Encephalitis constitutes a group of inflammatory brain diseases that are characterized by prominent neuropsychiatric symptoms associated with autoantibodies against neuronal cell-surface proteins, ion channels or neurotransmitter receptors . Anti-LGLeucine-rich glioma inactivated 1 (LGI1) is a neuronal secreted synaptic linker protein that interacts with the presynaptic disintegrin and metalloproteinase domain-containing protein 23 (ADAM23) and with the postsynaptic protein ADAM22, that are associated with Kv1.1 voltage-gated potassium channels and AMPA receptors, respectively , 6. AntiAnti-LGI1 antibody detection is important for the diagnosis of the encephalitis and prompt treatment with immunotherapy . Most clin-house cell-based IIF in which cells are co-transfected with LGI1 and one of its natural synaptic ligands (ADAM23). All studies were examined by two independent observers.We retrospectively examined 70 consecutive patients with available paired serum and CSF known to be LGI1 antibody positive by IIHC in at least one of the samples and a clinical suspicion of autoimmune encephalitis according to Graus et al.  a commercial assay, Autoimmune Encephalitis Mosaic 6 kit , following manufacturer’s instructions and recommended dilutions (undiluted CSF and 1:10 serum), to test IgG antibodies against N-methyl-D-aspartate (NMDA) receptor (GluN1), a-amino-3-hydroxyl-5-methyl-4- isoxazole-propionate (AMPA) receptor , gamma-aminobutyric (GABA) B receptor (B1 and B2 subunits), contactin-associated protein-like 2 (CASPR2), leucine-rich glioma-inactivated protein 1 (LGI1) and dipeptidyl-peptidase 6 (DPPX), and 2) in-house IIF-CBA in which Human Embryonic Kidney 293 (HEK293) cells were transfected with DNA constructs to express LGI1 together with ADAM23 . BrieflyThe study was approved by the ethics committee of Hospital Clínic of Barcelona. Patients’ samples were coded and clinical information was anonymized prior to analysis.B receptor and another against CASPR2).The median age of the patients was 62 years , and 27 (40%) were women (no demographic information available from 3 patients). From the 66 patients with available information, the most common clinical findings were seizures (70%), followed by cognitive impairment (62%), memory loss (59%) and behavioral changes (38%)  were positive by brain indirect immunohistochemistry, 58 (83%) by commercial IIF-CBA, and 70 (100%) by in-house IIF-CBA.Among the 70 sera examined, 69 (98.5%) were positive by brain indirect immunohistochemistry, 66 (94%) by commercial IIF-CBA, and 60 (86%) by in-house IIF (concordant patients). The other 23 (33%) patients showed discordant findings (discordant patients) that resulted from the technique employed  or the sample tested (serum or CSF) (in-house assay missed LGI1 antibodies in serum more frequently than in CSF (10 [43%] vs. 0 [0%]). All samples negative by one of the assays were positive by the other. No clinical differences were found between concordant and discordant groups of patients (47 (67%) patients were positive for LGI1 antibodies in both samples (CSF and serum) with the three indicated techniques, brain immunohistochemistry, commercial IIF, and  or CSF) Table 1.3. CSF testing using the Autoimmune Encephalitis Mosaic 6 kit (Euroimmun) was negative for all antibodies . Given the suspicion for autoimmune encephalitis, a sample of CSF was sent to our center for further analysis. The CSF sample was tested by rat brain IIHC, showing robust immunostaining of the hippocampus in a pattern highly suggestive of LGI1 antibodies . At admission, the MRI showed increased FLAIR/T2 signal predominantly involving the left hippocampus, and the EEG showed slow activity. Blood chemistry was normal except for hyponatraemia (128 mEq/L), and the CSF showed mild pleocytosis with 7 leukocytes/mmIn this study, we show that using an IIF-CBA co-expressing LGI1 and ADAM23 all patients with LGI1 autoimmune encephalitis had LGI1 antibodies in CSF. Thus, the concept that in this type of autoimmune encephalitis the antibodies predominantly occur in serum should be reconsidered. The reason why one IIF-CBA performs better than the other depending on the type of sample (serum or CSF) is unclear. We postulate that the repertoires of antibodies in serum and CSF are different and that when using CSF, the expression of LGI1 with its natural ligand facilitates the detection of antibodies. Irrespective of these or other unsuspected reasons the findings have important diagnostic implications.Previous studies suggested that up to 47% of patients with anti-LGI1 encephalitis only have detectable LGI1 antibodies in serum , 8, 9 Table 2A receptor or glycine receptor antibodies may occur in patients with a wide variety of diseases or symptoms that are not immune-mediated or do not respond to immunotherapy  by IIHC and subsequent confirmation of antibodies in CSF with the IIF-CBA that co-expresses LGI1 with ADAM23 is the best approach to detect LGI1 antibodies. All patients included in our cohort (N=70) were positive with this approach. Although the commercial IIF-CBA with LGI1 alone was more sensitive in serum than our IIF-CBA expressing LGI1-ADAM23, the commercial assay performed substantially worse with CSF. Moreover, the diagnosis of 4 patients would have been missed using serum with the commercial IIF-CBA, which is the standard diagnostic test for LGI1 antibodies in most clinical laboratories. In practice, some of these cases are later diagnosed in research laboratories due to the persistence of symptoms and high level of clinical suspicion, but delays in treatment can affect outcome , 20. TheIn our experience, brain tissue IIHC is a useful complementary test for antibody detection in autoimmune encephalitis. As shown here, this is particularly important for LGI1 antibodies because the brain tissue IIHC with either serum or CSF is highly sensitive and produces a pattern of hippocampus and cerebellum immunostaining that is very suggestive of these antibodies .A task for the future is to determine the repertoire of LGI1 antibody specificities in serum and CSF. We postulate that autorreactive B cells present in CNS are in prolonged contact with the antigen and undergo somatic hypermutation and affinity maturation, facilitating the selection and expansion of auto-reactive B cell clones that specifically recognize LGI1 linked to its natural ligands. Our study suggests heterogeneity among patients’ antibodies regarding LGI1 epitope recognition and provides the best strategy to reduce false negative results with tests currently available.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the ethics committee of Hospital Clínic of Barcelona. Patients’ samples were coded and clinical information was anonymized prior to analysis. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. The animal study was reviewed and approved in accordance with European (2010/63/UE) and Spanish (RD 53/2013) regulations by the ethics committee of Hospital Clínic of Barcelona.JD, RR-G, EM-H, JP, GM-S and FG designed the study and wrote the manuscript. RR-G, JP, GM-S, LN, LS and RC performed laboratory work and/or data analysis. JP, EM-H and MG collected clinical information from the LGI-1 patients. All authors contributed to the article and approved the submitted version.


The use of two-dimensional (2D) printing technologies of drugs on orodispersible films (ODF) can promote dose individualization and facilitate drug delivery in vulnerable patients, including children. We investigated midazolam pharmacokinetics after the administration of 2D-printed ODF.Midazolam doses of 0.03 and 3 mg were printed on an ODF using a 2D drug printer. We investigated the bioavailability of the two midazolam doses with ODF swallowed immediately (ODF-IS) or delayed after 2 min (ODF-DS) by comparing their pharmacokinetics with intravenous and oral midazolam solution in 12 healthy volunteers.Cmax of ODF-IS 0.03 mg was 95.5% (83.2–110) compared to oral solution and 94.3% (78.2–114) after 3 mg. Absolute bioavailability of ODF-IS 0.03 mg was 24.9% (21.2–29.2) and for 3 mg 28.1% (23.4–33.8) ; 3 mg: 28.0% (25.0–31.2)). Absolute bioavailability of ODF-DS was significantly larger than for ODF-IS .The relative bioavailability of ODF-IS 0.03 mg was 102%  compared to oral solution and for 3 mg 101% (86.8–116). 0–∞ in both doses was bioequivalent to the administration of an oral solution. However, Cmax of the therapeutic dose of ODF-IS missed bioequivalence by a clinically not relevant extent. Prolonged mucosal exposure increased bioavailability. .This trial demonstrates the tolerability and unchanged bioavailability of midazolam printed on ODF over a 100-fold dose range, proving the suitability of ODF for dose individualization. Midazolam ODF-IS AUC The broad use of medicines in very diverse and often vulnerable populations such as older patients or children, the substantial variability of drug clearance  and thus individual dose requirements, and the development of targeted medicines with a narrow therapeutic index require flexibly adaptable and precise dosing regimens for successful and safe drug therapy. Within days, dose requirements can change by several orders of magnitude , 2, makiCurrently, most oral dosage forms are solid, but it is often difficult for vulnerable patient groups, such as older patients and young children, to swallow them –7. In adIn recent years, several steps have been taken to develop personalized oral dosage forms that meet the challenges of variable dose requirements while facilitating the administration process. One solution may be the use of 2-dimensional (2D) inkjet printing technologies on orodispersible films (ODF) to overcome the challenges of oral drug administration , 13. Dig0–∞) [Midazolam is a sedative frequently used in children before interventions and—at therapeutic and microdoses—a marker substrate of cytochrome P450 (CYP) 3A4 activity , 20. The0–∞) . In healThis was a single-center, open-label, fixed-sequence, phase I clinical trial in 12 healthy volunteers, which was started after approval by the competent regulatory authority  and favorable assessment by the responsible Ethics Committee of the Medical Faculty of Heidelberg University (AFmo-012/2021). All procedures were carried out in accordance with the good clinical practice guideline, the pertinent version of the Declaration of Helsinki, and all specific legal requirements in Germany. The study was conducted between 12 April 2021 and 09 July 2021 at the Early Clinical Trial Unit of the Department of Clinical Pharmacology and Pharmacoepidemiology at Heidelberg University Hospital, which is certified according to DIN EN ISO9001. After having received detailed written and oral information, participants (aged 18–50 years) had to give their written informed consent prior to any study-related procedures.® II white . Midazolam-containing ink was also manufactured at Gen-Plus GmbH and consisted of midazolam hydrochloride , purified water, absolute ethanol, and Kollidon® 12 PF (polyvinyl pyrrolidone). Ink containing midazolam was then printed using a 2D printer , which dynamically passes ODF under a stable print head that applies small droplets of ink containing the API and optically measures the size of the individual droplets. The printed ODF were dried on an integrated heating plate, packed air-tight in polypropylene screw top tubes, and stored at room temperature. The ODF contained 3.03 mg midazolam hydrochloride, separated into doses of 3 mg and 30 µg each, corresponding to 2.7 and 0.027 mg midazolam base. The ODF had an area of 10.2 cm2 (3.5 × 2.9 cm), and a transverse perforation line divided the rectangular film into an area of about 2.61 cm2 containing the 30-µg dose and a larger area (7.54 cm2) containing the therapeutic dose. The doses could easily be divided at the perforated line to administer each dose separately.ODF were manufactured by Gen-Plus GmbH  using purified water, absolute ethanol, sorbitol, and Opadry® V 5 mg/5 ml, CHEPLAPHARM Arzneimittel GmbH, Greifswald, Germany), an oral therapeutic dose  (3 mg Dormicum® diluted in 100 ml of bottle water), and an oral microdose solution (30 µg Dormicum® diluted in 100 ml of bottle water) were administered.For comparison, an intravenous (i.v.) microdose of 30 µg midazolam , and screening for illicit drugs. Any intake of drugs or substances known to interact with CYP metabolism, as well as alcohol and grapefruit, were prohibited 2 weeks before and throughout the trial.The trial was designed to assess the absolute and relative bioavailability of different midazolam oral administration forms, the impact of the oral residence time of the ODF, and dose linearity. In a fixed-sequence design, the participants received the following treatments: 30 µg i.v. as a short infusion over 10 min, 30 µg as an oral solution, 30 µg immediately swallowed ODF (ODF-IS), 30 µg delayed swallowed ODF (ODF-DS), 3 mg as an oral solution, 3 mg ODF-IS, and 3 mg ODF-DS , and food intake was not allowed until 4 h after midazolam administration. To study the effects of the oral residence time of the ODF on midazolam pharmacokinetics, the ODF was either swallowed immediately followed by 200 ml of bottle water (ODF-IS) without waiting for dissolution, or delayed (ODF-DS), where participants kept the ODF beneath their tongue for exactly 2 min followed by drinking 200 ml of bottled water. After ODF administration, the local tolerance of the ODF was asked and the time after the ODF was reported to have completely dissolved was measured.Blood samples were collected via a peripheral venous catheter on the forearm before and 5, 10, 20, 30, and 45 min, and at 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, and 24 h after IMP administration. Participants were monitored for adverse events (AEs) for 10 h after administration and then discharged. Within 14 d after the last IMP administration, they underwent an end-of-treatment examination to confirm their health status. AEs were documented and evaluated for seriousness, relationship to the IMP, and severity based on the US National Cancer Institute’s Common Terminology Criteria for Adverse Events (CTCAE) Version 5.0.Blood samples for midazolam quantification were centrifuged within 60 min at 2500 g at room temperature for 10 min and frozen in 600 µl aliquots at -20 °C. Midazolam concentrations in plasma were quantified in the department’s analytical chemistry laboratory by tandem mass spectrometry after prior separation by ultra-performance liquid chromatography (UPLC-MS/MS) for samples after administration of 30 µg doses  and by hH0: geometric mean ratio (GMR) of the area under the plasma concentration–time curve extrapolated to infinity (AUC0–∞) ≤ 0.8 or ≥ 1.25. In a sample size of 12, a GMR of 1 could be detected with an α-error of 0.05 and a power of 0.82. Standard pharmacokinetics parameters , and apparent volume of distribution: Vz/F) were calculated using Phoenix WinNonlin Software 8.2  using noncompartmental analyses. Because of the short washout period between the microdose treatments, 9 predose samples showed residual midazolam concentrations. In order to derive a single-dose curve, these concentration profiles were transformed via the reverse superposition principle published by Bauer and co-workers [0–∞ and Cmax must be within 80–125% to be considered bioequivalent [p-value < 0.05 was considered statistically significant.Sample size calculation was conducted based on the assumption of -workers . Statistuivalent . A one-w2 were included, and all completed the trial. After ODF administration, the subjectively perceived dissolution time for the 30 µg dose with an ODF area of 2.61 cm2 was 30.4 s (20.4–45.3 s) and 55.9 s for the 3 mg dose with an ODF area of 7.54 cm2 , but participants reported difficulty in determining the exact time of complete dissolution. Swallowing was reported to be comfortable after complete dissolution of ODF-DS, except for a bitter taste after therapeutic-dose ODF-DS. When swallowing the ODF immediately, some participants reported that the ODF stuck to the palate and, therefore, swallowing felt uncomfortable.Twelve healthy volunteers  with a median age of 24.0 years (range 22.0–30.8) and a mean (± standard deviation) body mass index of 24.1 ± 5.3 kg/mThe midazolam plasma concentration–time profiles after administration of a microdose are shown in Fig. t1/2 of all administration forms and doses was comparable, and the oral bioavailability of ODF-IS was comparable to the corresponding oral administration of midazolam solution (both p > 0.9), oral bioavailability increased significantly . To assess dose proportionality, we compared the dose-normalized AUC0–∞ after administration of 3 and 0.03 mg as an ODF-IS, which revealed no statistically significant difference.The microdose administration of midazolam as ODF-IS was bioequivalent to the oral solution Table . After an = 7), diarrhea (1) (assessed as unrelated), and pain at the injection site (1).The trial medication and study procedures were well tolerated. In total, 9 AEs occurred, and all were mild (Grade I according to CTCAE 5.0) and transient. The observed AEs were headaches .All ODF used were well tolerated and handling was easy. The two doses (therapeutic and microdose) were well separated and easy to divide by a perforation line, confirming that different doses can be applied to a single ODF. This may be useful to achieve the dose variety that is otherwise only possible with liquid oral dosage forms. Different doses printed on ODF with a sophisticated and well-thought-out system may be easily divided into the required dose, and among other things, counting of drops is no longer necessary. The ODF dissolved completely and rapidly in saliva in less than one minute. The therapeutic dose required a longer dissolution time (55.9 vs. 30.4 s), which may be related to the larger ODF area of the therapeutic dose , midazolam administered as a microdose was bioequivalent to an oral solution. Also, the AUCCmax and AUC0–∞ increased considerably. T1/2 remained unchanged with prolonged buccal exposure.As expected , the bioBuccal administration could therefore help to achieve rapid and strong drug effects as desired, for example, before interventions, even when they are to be performed in a fasting state or also in epileptic crises, which are often treated with administration of buccal midazolam, which is faster and easier to administer than many other routes of administration . In addiThe limitation of this study is that we examined healthy volunteers in a single age group, as is common for phase I studies. Although this middle age group should be well represented in a future patient population, the intended use for older and very young patients needs to be further evaluated in terms of handling and oral environmental conditions in these age groups. For example, dry mouth could influence the variability of midazolam pharmacokinetics through differential dissolution, and mucosal permeability could be higher in neonates and infants than in older people.In conclusion, this trial confirmed that midazolam can be printed on ODF and that the new oral formulation makes this benzodiazepine readily absorbable. The immediate swallowing of the ODF provides already exposure comparable to oral midazolam solutions and, by increasing the residence time of the ODF in the oral cavity, oral bioavailability can be substantially increased. 2D-printing on ODF could be a promising novel technology for the oral delivery of flexible doses over a wide dose range (100-fold) with considerable potential to facilitate drug administration in vulnerable patient groups, which are less capable of swallowing and could potentially benefit from this novel technology.


Genomic profiling of cerebrospinal fluid (CSF) can be used to detect actionable mutations and guide clinical treatment of non-small cell lung cancer (NSCLC) patients with central nervous system (CNS) metastases. Examining the performance of CSF samples in real-world settings can confirm the potential of CSF genotyping for guiding therapy in clinical practice.We included 1,396 samples from 970 NSCLC patients with CNS metastases in real-world settings. All samples underwent targeted next-generation sequencing of 1,021 cancer-relevant genes. In total, 100 CSF samples from 77 patients who had previously received targeted treatment were retrospectively analyzed to explore the mechanisms of TKI-resistance.EGFR in CSF were higher than those in plasma samples of treated patients . For concordance between paired CSF and plasma samples that were simultaneously tested, the MSAF of the CSF was significantly higher than that of matched plasma cfDNA (p <0.001). From multiple comparisons, it can be seen that CSF better detects alterations compared to plasma, especially CNV and structural variant (SV) alterations. CSF cfDNA in identifying mutations can confer the reason for the limited efficacy of EGFR-TKIs for 56 patients .For NSCLC patients with CNS metastases, CSF samples were slightly more often used for genomic sequencing in treated patients with only distant CNS metastases compared to other patients (10.96% vs. 0.81–9.61%). Alteration rates in CSF samples were significantly higher than those in plasma, especially for copy number variants (CNV). The MSAFs of CSF samples were significantly higher than those of plasma and tumor tissues . Remarkably, detection rates of all actionable mutations and This real-world large cohort study confirmed that CSF had higher sensitivity than plasma in identifying actionable mutations and showed high potential in exploring underlying resistance mechanisms. CSF can be used in genomics profiling to facilitate the broad exploration of potential resistance mechanisms for NSCLC patients with CNS metastases. Lung cancer, one of the most common cancers, remains the most common cause of cancer-related deaths, with high global morbidity and mortality . Non-smaEGFR), anaplastic lymphoma kinase (ALK), and ROS proto-oncogene 1 (ROS1), for NSCLC (Next-generation sequencing (NGS)-based genetic testing, which provides abundant genetic information about cancer, has developed therapeutic strategies against driver mutations such as epidermal growth factor receptor , which is used in various body effluents, namely, blood, cerebrospinal fluid, ascitic fluid, pleural fluid, and urine instead of tumor tissue, has been widely used in clinical practice to detect genomic alterations in NSCLC –19. It cTo provide more implications for the clinical application of liquid biopsy using CSF and explore its potential to identify actionable mutations and explore underlying resistance mechanisms for NSCLC patients with CNS metastases, we analyzed 1,396 samples from 970 NSCLC patients with CNS metastases  who underwent NGS in real-world settings.In this retrospective cohort study, a cohort of 970 NSCLC patients with CNS metastases (BM and LM) was enrolled at the Geneplus Medical Laboratory  from May 2019 to July 2021. The diagnosis criteria for BM were based on metastatic lesions detected on brain magnetic resonance imaging, while the diagnostic criteria for LM were based on tumor cells detected in CSF samples or leptomeningeal enhancement on brain magnetic resonance imaging. To analyze the real-world efficacy of CSF in detecting actionable mutations, all patients underwent NGS in a laboratory accredited by the College of American Pathologists. Demographic, clinicopathological, and tumor histopathological results, such as TNM stag, metastatic sites, and cellular differentiation grade, were obtained for each patient. This study was approved by the Institutional Review Board of Shaanxi Provincial People’s Hospital. All subjects provided written informed consent before undergoing any study-related procedures. This study was conducted in accordance with the principles of the Declaration of Helsinki.Within 72 h of collection, peripheral blood samples were centrifuged to obtain plasma and white blood cells (WBCs). The CSF supernatant was centrifuged to separate it from the cell sediment. All tissue samples, including fresh and formalin-fixed paraffin-embedded (FFPE) tissue samples, underwent pathological assessment to confirm histologic classification and adequacy of the tumor tissues, which required a minimum of 20% tumor content. Circulating cell-free DNA (cfDNA) was isolated from the CSF supernatant and plasma using a QIAamp Circulating Nucleic Acid Kit . Genomic DNA (gDNA) from WBCs and tumor tissues was extracted using the DNeasy Blood & Tissue Kit . Circulating cfDNA from other body fluids were processed into indexed libraries, as discussed in previous studies –28.+Seq-2000 Sequencing System , according to the instructions of the manufacturer.DNA concentration was measured using the Qubit fluorometer  and the Qubit dsDNA HS (High Sensitivity) Assay Kit . The size distribution of circulating cfDNA was assessed using the Agilent 2100 BioAnalyzer and DNA HS kit . The SeqCap EZ Library system  was used for target enrichment. In total, 1,386 libraries from 970 patients were hybridized to custom-designed biotinylated oligonucleotide probes  covering 1.6 Mbp of the genome, and the captured genomic regions included 1,021 cancer-related genes Table S1Sequencing data were analyzed using default parameters. After the removal of terminal adaptor sequences and low-quality reads, the clean reads were aligned to the reference human genome (hg19) using the Burrows–Wheel Aligner  with default parameters. Base quality recalibration and local realignment were performed using the Gene Analysis Toolkit . Somatic single nucleotide variations (SNVs) and insertion or deletion of small fragments (Indels) were determined by the MuTect2 algorithm. The Contra algorithm (version 2.0.8) was used to detect somatic copy number alterations. All candidate fusion genes were manually mapped to the initial cfDNA fragments using unique barcoding and alignment information. The minimal mean effective depth was 300×, 1000×, and 1,000× in tissue, CSF, and plasma samples, respectively.p-values <0.05 represented statistical significance.Associations between two or more categorical variables were analyzed using Fisher’s exact or Chi-square tests. The comparison of means among three or more groups was performed using one-way ANOVA tests. All statistical analyses and presentations were performed using R v3.6.3. All tests were two-sided, and In total, 970 patients  with stage IV NSCLC and CNS metastases were enrolled in this study. Patient characteristics are presented in In this real-world setting, 119 CSF, 416 tumor tissue, 791 plasma, and 70 other samples were collected. The 416 tissue samples included 269 primary tissues, 46 intracranial metastatic tissues, 39 lymph node tissues, 26 other metastatic tissues, and 36 tissues of unknown origin, while the 70 other samples included 59 pleural effusion samples, 5 already extracted DNA, 4 pericardial fluid samples, one brushing washing fluid, and one ascitic fluid. Most patients  had a single CSF, tissue, plasma, other body fluid sample, or DNA tested, while 282 patients had multiple samples tested simultaneously or consequently. Meanwhile, 240 samples were treatment-naive, containing 2 (0.83%) CSF, 129 (53.75%) tissue, 98 (40.83%) plasma, and 11 (4.58%) other samples; and 1,156 were treated, comprising 117 (10.12%) CSF, 287 (24.83%) tissue, 693 (59.95%) plasma, and 59 (5.10%) other samples Figure 1Among 970 patients included in this study, 428 (44.12%) had distant metastases only from the CNS (CNSM group), and 542 (55.88%) patients had other organ involvement and distant metastases other than CNS metastases (CNSM+ group). Based on whether they were treated samples or distant metastases other than CNS metastases, the 1,396 samples from these 970 NSCLC patients with CNS metastases were divided into four groups: the treatment-naive CNSM group (n = 117), the treatment-naive CNSM+ group (n = 123), the treated CNSM group (n = 438), and the treated CNSM+ group (n = 718).Comparisons between the four groups revealed the choice of specimen type for genetic profiling in NSCLC patients with CNS metastases in real-world settings Figure 2Moreover, plasma samples were slightly more often used in the treated CNSM+ group than in other groups (62.95% vs. 40.17–55.02%). And the treated CNSM group (10.96%) had the largest proportion of CSF samples Figure 2To further analyze the efficacy of CSF samples in real-world settings, 970 NSCLC patients with CNS metastases were retrospectively analyzed. All samples were subjected to targeted NGS of 1,021 cancer-relevant genes. Alterations were identified in 114 (95.80%) CSF samples and 416 (100%) tumor tissue samples, compared to 684 (86.47%) plasma and 67 (95.71%) other samples. The detection rate of all alterations in CSF was lower than that in tumor tissues  but higher than that in the plasma   of the CSF was compared to that of other types of samples  types. Among treated-tumor tissues, the most recurrent mutations were observed in TP53, followed by EGFR  in patients with different metastatic sites in real-world settings. Due to the small number of treatment-naive patients who opted for CSF testing, this analysis was performed on treated patients. On the basis of distant metastases other than the CNS metastases, samples were divided into two groups as follows: distant metastases only from the CNS group (CNSM group) and distant metastases other than CNS metastases group (CNSM+ group). We analyzed mutation rates and MSAF of CSF and plasma in the two groups of treated patients.The mutation rate in CSF samples was significantly higher than that in the plasma in both the treated CNSM and CNSM+ groups   and CNSM+ (distant metastases other than CNS metastases) groups. The actionable mutation detection rates of the different groups are shown in To further analyze the detective capability of actionable mutations of CSF samples in a real-world setting, 1,156 treated samples from NSCLC patients with CNS metastases were retrospectively analyzed. Thus, we compared all actionable mutations and EGFR in CSF were significantly higher than those in tissue samples in both the CNSM and CNSM+ groups     . The MSAF of 32 CSF cfDNAs was compared to that of plasma cfDNA from 28 patients. The MSAF of the CSF was significantly higher than that of the matched plasma cfDNA (p <0.001)  were tested for NGS using CSF samples. We successfully tested 100 CSF samples obtained from 77 patients, of which 22 patients were resistant to first or second-generation EGFR-TKIs, 49 were resistant to osimertinib or almonertinib, and 6 were resistant to ALK- or ROS1-TKIs Table 2.EGFR resistance mutation, activation of bypass signaling pathways, EGFR amplification, TP53 exon8 mutation, PI3K-AKT-mTOR gene alterations, and cell cycle gene alterations. In total, known EGFR-TKIs resistance mechanisms, such as PI3K-AKT-mTOR signaling-related genomic alteration, MET amplification, and absence of sensitizing mutations were detected in 1, 3, and 1 patient(s), respectively, who were resistant to first- or second-generation EGFR-TKIs. EGFR C797S/L792X/G724S/L718Q, PI3K-AKT-mTOR signaling-related genomic alteration, KRAS mutation, ERBB2 amplification, MET amplification, and absence of sensitizing mutations were detected in 8, 7, 1, 4, 9, and 3 patients, respectively, who were resistant to osimertinib or almonertinib. Cell cycle gene alterations, EGFR amplification, and TP53 exon8 mutations were identified in 11, 43, and 13 patients with EGFR-TKI resistance, respectively. However, determining whether it would result in EGFR-TKI resistance remains controversial. Co-occurrence of resistance mechanisms was observed in 21 patients, including one patient without EGFR-TKIs sensitizing mutations.While exploring mechanisms of TKI-resistance, EGFR-TKI sensitizing mutations were undetected in CSF cfDNA in 4.30% (4/93) of patients with EGFR-TKI resistance. Fifty-six patients  harbored concurrent alterations that might limit the efficacy of EGFR-TKIs, namely, ALK or ROS1 fusions. Known ALK-TK resistance mechanisms, such as ALK G1269A and absent sensitizing mutations, were detected in one and two of five patients with ALK-TKIs resistance. TP53 mutations that may limit the efficacy of ALK-TKIs were identified in two patients with ALK-TKI resistance.Four patients with ALK- or ROS1-TKI resistance were identified as having This confirmed that liquid biopsy using CSF showed high potential in exploring underlying resistance mechanisms in NSCLC patients with CNS metastases.NSCLC patients with CNS metastases usually have a poor prognosis and limited treatment options. However, following the development of cancer genomics and more effective targeted therapies, new treatments are emerging –7, 9. GeAmong NSCLC patients with CNS metastasis, ctDNA in CSF, which circulates throughout the CNS, can reveal genomic alterations in intracranial lesions –32. PrevTo date, this real-world study recruited the largest cohort of NSCLC patients with CNS metastases who had CSF or other samples available for NGS testing. All CSF samples in this study were tested using the CSF supernatant, as it was reported that more mutations could be detected in cfDNA from the CSF supernatant than in paired CSF cells because CSF cfDNA was less affected by non-tumor cell components , 33. ComEGFR and TP53 were the two most frequently mutated genes in all sample types, which were the same as previously reported , especially CNV alterations, in the CSF samples were significantly higher than those in plasma , which corresponded to the findings of other reports . The MSAreported . These rEGFR, ALK, ROS1, BRAF, KRAS, RET, MET, and ERBB2 alterations in the tissues was consistent with previous reports . Thus, for treated NSCLC patients with CNS metastases, CSF outperformed plasma in detecting actionable mutations. For concordance between paired CSF and plasma samples, the MSAF of the CSF was significantly higher than that of the matched plasma cfDNA (p <0.001). From multiple comparisons, it was seen that CSF is better than plasma in detecting alterations, especially CNV and SV alterations.For driver gene alterations, the detection rate of  reports . RemarkaEGFR-mutated NSCLC and found that the detection rate of EGFR mutations using CSF genotyping was 97.1%, compared to 95.5% in our study . A recent study evaluated the role of CSF-NGS in osimertinib-treated ur study . It alsour study . A previur study . In thisPrecision medicine has led to improvements in the prognosis of patients with advanced NSCLC. Adoption of NGS is time-saving and cost-efficient . For NSCThis study has some limitations, which include its retrospective snapshot study design, without consideration of additional clinical factors and therapeutic efficacy. Hence, we could not precisely determine optimal specimens for patients in different clusters. The number of treatment-naive patients included in this study was small, and the role of CSF in this subset of patients is limited. Furthermore, the paired CSF and plasma samples were small, and no tissue samples were included. Therefore, we could not investigate concordance between tissue, plasma, and CSF at the same time point. The clinical implications of CSF genotyping on treatment outcomes were not analyzed in this study.This large-scale, real-world study confirmed that liquid biopsy using CSF showed high potential for identifying actionable mutations and exploring the underlying resistance mechanisms in NSCLC patients with CNS metastases. CSF can be used as a liquid biopsy source to facilitate the broad exploration of potential resistance mechanisms in clinical practice.https://ngdc.cncb.ac.cn/gvm/getProjectDetail?project=GVM000345.The original contributions presented in the study are publicly available. This data can be found in the GVM in National Genomics Data Center, China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences, under accession number GVM000345, The studies involving human participants were reviewed and approved by the Institutional Review Board of Shaanxi Provincial People’s Hospital. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.FS, JB, NL, WG, and ZF contributed to conception and design of the study. JB, JZ, WG, GD, MZ, and QH organized the database. KW and MY performed the statistical analysis. KW and RC wrote the first draft of the manuscript. KJ, XW, YM, and GD wrote sections of the manuscript. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This work was supported by the National Natural Science Foundation of China (82072583), the Beijing Municipal Administration of Hospitals Incubating Program (PX2020044), and the Beijing Municipal Science & Technology Commission (No. Z171100000417029).KW, MY, and RC are current employees of Geneplus-Beijing.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Left bundle branch pacing (LBBP) is a novel physiological pacing technology. We aim to explore the relation between LBB potential (LBB Po) and left ventricular (LV) electrical/mechanical synchrony in bradycardia patients without heart failure (HF) receiving LBBP.A total of 62 patients undergoing LBBP were categorized by LBB Po: the LBB Po positive (+) group and the LBB Po negative (−) group. The perioperative electrocardiographic and echocardiography parameters related to cardiac synchrony were analyzed.There were 42 (67.74%) patients in the LBB Po (+) group and 20 patients in the LBB Po (−) group. Paced QRS duration  and stimulus left ventricular activation time  were shorter in the LBB Po (+) group than in the LBB Po (−) group. No significant differences in the LV mechanical synchrony  between the two groups. There was not statistically difference in ventricular lead parameters measured intraoperative between the two groups. Compared with the LBB Po (−) group, the LBB Po (+) group showed a dramatically higher total procedure duration time  and fluoroscopy time for ventricle lead implantation .The appearance of LBB Po may suggest better electrical synchrony during LBBP, but similar in LV mechanical synchrony. However, the total operation duration and fluoroscopy time of ventricular lead implantation in the LBB Po (+) group were longer. Therefore, it may be unnecessary to deliberately recognize the LBB Po when it is difficult to detect LBB Po and meet the LBBP criterion. Left bundle branch pacing (LBBP), defined as the capture of left bundle branch (LBB) via trans-ventricular septal approach, has been shown to be a novel physiological pacing modality with potential for application in both heart failure(HF) and conventional pacing patients –4. DurinLeft bundle branch potential (LBB Po), which is recognized as a high-frequency signal before the QRS onset and the interval between LBB Po and ventricular potential is usually 20–30 ms, was identified when the lead was implanted at left bundle trunk or proximal branch or left posterior fascicle or left anterior fascicle during intrinsic rhythm or LBBB correction by HBP . The preThe aim of our study was to explore the relationship between LBB Po and LV electrical/mechanical synchrony in bradycardia patients receiving LBBP. To this purpose, we evaluated electrocardiography (ECG) characteristics, two-dimensional Tissue Doppler imaging (2D-TDI) and two-dimensional speckle-tracking echocardiography (2D-STE) parameters in patients with and without LBB Po undergoing LBBP implantation.The study prospectively enrolled pacemaker-indicated patients for symptomatic bradycardia without HF receiving LBBP at Shanghai Chest Hospital from March 2019 to August 2020 (ChiCTR1900020817) . PatientLBBP was performed by screwing in a non-retractable active helix pacing lead from the right ventricular (RV) aspect into the interventricular septum as our previously described . The C31A transition from non-selective LBB capture to selective LBB or LV septal capture was distinguished while descending pacing output.Abrupt shortening of stimulated left ventricular activation time (S-LVAT) > 10 ms was observed in lead V5 during the change of output.If LBB Po could be recorded, the difference between S-LVAT and the interval from intrinsic LBB Po to R wave peak was ≤ 5 ms in V5. When LBB Po could not be recognized, the difference in V5 between the interval from the onset of paced QRS to R wave peak and the interval from intrinsic QRS onset to R wave peak was ≤ 5 ms.Based on the electrical characteristics described by Huang and Chen et al. , LBBP waWe defined the time from first incision to last suture as the total procedure duration. The time from insertion of the 3830 lead and C315His sheath across the tricuspid valve to the appropriate lead final fixation site was called “fluoroscopy time for ventricle lead implantation”.During the operation, an intracardiac electrogram (EGM) was recorded from the 3830 pacing lead tip in a unipolar pacing mode to identify LBB Po. Intrinsic LBB Po has been considered to be a high-frequency signal about 20–30 ms before the surface QRS complex . AmplituThe single-chamber pacemaker was used in patients with atrial fibrillation (AF) and programmed in VVI mode. The lower rate of the single-chamber pacemaker was routinely set at 75 beats per minute (bpm) and avoided fusion with intrinsic wave. For patients with sinus rhythm, the dual-chamber pacemaker was used and adjusted in DDD mode with short atrioventricular delay to ensure complete LBBP capture and avoid fusion with intrinsic conduction. To avoid septal anodal pacing, all ventricular electrodes were programmed in tip unipolar pacing manner with an output of 3.5 V/0.4 ms in the process of synchronization research. After the analysis of LV mechanical synchrony, all pacemakers were programmed according to the patients' specific condition to avoid the influence of Purkinje fiber conduction on RV activation.Cardiac electrical synchrony between the ventricles was assessed using QRSd of 12-lead simultaneous body surface ECG recordings in lead V5 before and after the operation . LV elecEchocardiographic examinations were performed before LBBP operation using color Doppler ultrasonic diagnostic apparatus . Echocardiographic measurements were performed by the same specialists according to the recommendations of American Society of Echocardiography . All exaTransthoracic echocardiography (TTE) was performed within 72 h after LBBP by the GE Vivid system. The echocardiographic data were stored and analyzed offline using an independent workstation . LV mechanical synchrony was assessed by 2D-TSI and 2D-STE as described previously , 19.In short, measurement of regional desynchrony was obtained from 2D-TDI images of the four-chamber apical view, and the time to peak myocardial systolic velocity (Ts) suggests the time to reach regional peak systolic tissue velocity. The time from QRS onset to Ts was recorded at the six basal and six midventricular level, including: (1) Ts-SD-12: standard deviation of Ts of the 12 LV segments; (2) Ts-12: maximal difference in Ts between any 2 of the 12 LV segments; (3) Ts-SD-6: standard deviation of Ts of the 6 basal LV segments; (4) Ts-6: maximal difference in Ts between any of the 6 basal LV segments.Images from 2D-STE were obtained by an M5Sc transducer according to current consensus for all participants. An advanced quantitative analysis EchoPAC workstation was used for 2D-STE analyses. After 17 segments of the LV were successfully tracked, LV peak strain dispersion (PSD) was automatically analyzed and obtained.Continuous variables were expressed as mean ± standard deviation (SD) and classified variables were presented as percentages. Differences in mean values between two groups were compared by Student’s t-test for continuous variables. Classified variables were analyzed by the Fisher exact test, Chi-square test and Wilcoxon signed-rank test. Software SPSS 22.0  was used to statistical analysis. A two-tailed P value < 0.05 was considered statistically significant.Between March 2019 to August 2020, there were a total of 62 pacemaker-indicated patients for symptomatic bradycardia without HF accepted LBBP therapy in our hospital. According to the defined criteria for LBB Po, there were 42 (67.74%) patients with LBB Po and 20 (32.26%) patients without LBB Po. Baseline characteristics of the two groups in pacemaker indication and echocardiography had no significant differences. The general information of the patients is shown in Table LBB Po amplitude in LBB Po (+) cases were measured as 0.22 ± 0.05 mV, and mean PV interval was 25.40 ± 3.76 ms. Intraoperative measurement of the 3830 lead parameters demonstrated that there was no dramatically difference between LBB Po (+) and LBB Po (−) groups with regard to capture threshold , R wave amplitude  and pacing impedance  , QTc interval  and QRS axis (P = 0.055) did not significantly differ between LBB Po (+) and LBB Po (−) groups. In addition, patients in the LBB Po (+) group recorded a notably narrower mean paced QRSd than those in the LBB Po (−) group . Meanwhile, in the LBB Po (−) group, paced QTc interval  was prominently wider than those in the LBB Po (+) group. According to the paced QRS axis, patients were divided into three different types as follows: axis between − 30° and 90° , axis between − 30° and − 90°  and axis within 90° and 120° . There was obviously significantly difference between LBB Po (+) group and LBB Po (−) group in paced QRS axis (P = 0.011). More patients belong to normal paced QRS axis in the LBB Po (+) group than in the LBB Po (−) group , Ts-SD-6 , Ts-12 , Ts-SD-12  or PSD  between the LBB Po (+) and LBB Po (−) group , capture threshold  or pacing impedance  between the LBB Po (+) and LBB Po (−) groups. Mean duration of unipolar paced QRSd  in LBB Po (+) group was significantly shorter than that of LBB Po (−) group. Two patients in the LBB Po (+) group were asymptomatic, in whom lead perforation was discovered incidentally on pacemaker interrogation. They cannot be paced at unipolar 10 V while the bipolar threshold increased abnormally. Echocardiogram showed that the 3830 pacing leads perforated through the interventricular septum (IVS), and the perforating leads were removed and replaced in the right ventricular apex by 5076 leads. No other serious complications such as lead dislocation, septal hematoma, coronary artery injury, LV thrombus, and stroke, or procedure-related death were observed during the follow-up  electrical synchronization with satisfactory pacing parameters . CompareIn our research, the incidence of LBBP with LBB Po was about 67.74%, suggesting that LBBP patients accounted for about two-thirds of the total LBBP. The appearance of LBB Po suggested that the lead was close to the LBB and was a possible clue for the capture of the LBB. Therefore, it is generally believed that LBB Po is not a direct sign of LBB capture, but a good indication of LBB capture. Previous studies believe that in participants with an intrinsic non-LBBB rhythm, LBB Po always should be recognized when LBB capture is confirmed . In subjPrevious studies have shown different LBBP characteristics due to the lack of unified criterion for evaluating LBB capture. LBB capture was usually identified by observing a transition of QRS complex from non-selective LBBP (NSLBBP) to selective LBBP (SLBBP) or LV septal pacing while descending output . SLBBP nElectrical asynchrony is defined by an asynchronous electrical activation of the LV leading to a prolonged QRSd > 120 ms) on the ECG. QRSd has been considered as an powerful indirect marker of electrical synchrony, and a wide QRS complex indicated greater ventricular dyssynchrony . In the 0 ms on tIn the present study, the mean S-LVAT were shorter than 75 ms in all patients of LBBP. LBBP patients without LBB Po had longer S-LVAT than those with LBB Po, although the difference in the absolute value is small. In Hou’s study, LBBP patients with recorded LBB Po almost had a shorter S-LVAT and a better LV electrical synchrony compared with those without LBB Po. But there are still 66.7% of patients with long S-LVAT had worse LV mechanical synchrony than those with short S-LVAT in LBB Po negative cases . As we kAs shown in our study, LBBP patients with LBB Po had obvious advantages over LBBP without LBB Po in S-LVAT and electrical synchronization, but there was no significant difference in LV mechanical synchronization. This result was in agreement with Wang's finding . Our stuThe frontal QRS axis represents the main direction of ventricular electrical depolarization on frontal plane. In pacemaker-dependent patients, normal paced QRS axis could provide relatively physiological ventricular activation associating with the preservation of LV function . Recent Prolongation of QTc interval was associated with an increased risk of severe arrhythmia and cardiac death . MeanwhiWe followed up the lead parameters and only two patients in LBB Po (+) group suffered from lead perforation, while all other LBBP patients had stable sensing amplitude, capture threshold and lead impedance. There are no other serious complications reported at three-months follow-up in the two groups. However, the total operation duration and fluoroscopy time of ventricular lead implantation in the LBB Po (+) group were longer. We think that the excessive pursuit of the short S-LVAT and the capture of the LBB may lead to excessive lead rotation and increase the risk of lead perforation. Therefore, it may be unnecessary to deliberately recognize the LBB Po when it is difficult to detect LBB Po and meet the LBBP criterion.This study is a small sample size, single-center and short-term study, possibly causing an underestimation of the actual effects. Noise signals may affect the acquisition of intracavity electrical signal, thus affecting the judgment of LBB Po. The parameters used in this paper to describe the electrical and mechanical synchronization are not comprehensive and need to be further improved. There may be inevitable errors in the measurement parameters of ECG and echocardiography. Whether the long-term clinical effects of LBBP mechanical synchronization change after echocardiography in a short time needs to be further investigated. More detailed analysis may be based on parameter differences between spontaneous and paced status and relation between them. For future analysis may be important the longer follow up and the analysis of prolonged QTc interval. The validity of the conclusion needs to be further demonstrated by using more accurate techniques and expanding the sample size in the future.LBB potential is commonly observed during LBBP and can be associated with a better LV electrical synchronicity in bradycardia patients with normal cardiac function. However, LBBP with and without LBB Po resulted in similar cardiac mechanical synchrony, indicating that it may be unnecessary to deliberately recognize the LBB Po when it is difficult to detect LBB Po and meet the LBBP criterion.


Cerebrospinal fluid (CSF) lactate levels have been suggested to be associated with disease severity and progression in several neurological diseases as an indicator of impaired energy metabolism, neuronal death, or microglial activation. Few studies have examined CSF lactate levels in dementia due to Alzheimer’s disease (AD) and found higher values in AD patients compared to healthy controls (HC). However, these studies were mostly small in size, the inclusion criteria were not always well defined, and the diagnostic value and pathophysiological significance of CSF lactate in AD remain unclear.n=312), biologically and clinically well-defined sample of patients with AD at the stage of mild cognitive impairment (MCI-AD) and dementia (ADD), HC, and patients with frontotemporal lobar degeneration (FTLD).We examined CSF lactate levels and potentially associated factors in a large (Contrary to previous studies, patients with ADD and HC did not differ in CSF lactate levels. However, we found higher values for patients with MCI-AD compared to those with ADD and to HC in univariate analysis, as well as for MCI-AD compared to ADD when controlling for age and blood-brain barrier integrity. CSF lactate levels were associated with age and blood-brain barrier integrity but not with clinical severity or CSF biomarkers of AD.CSF lactate does not indicate biological or clinical disease severity in AD, nor does it differentiate between patients with AD and HC or patients with FTLD. However, higher CSF lactate levels were found in earlier stages of AD, which might be interpreted in the context of inflammatory processes. Metabolic dysfunction of the central nervous system (CNS) has been discussed as a pathophysiological key contributor in Alzheimer’s disease (AD). Evidence from animal and in vitro studies points to disturbances in glucose transport mechanisms, disrupted glycolysis, oxidative stress and impaired mitochondrial function , which iLactate in the CNS has traditionally been interpreted as a metabolic waste product, while in recent years it has been found to serve a wide range of physiological functions from modulating neuronal excitability to contributing to energy homeostasis . HoweverImpaired mitochondrial functioning and glycolysis pathways have been found in AD  and a smTaken together, previous work suggested elevated CSF lactate levels in ADD, but most of the studies were limited by small sample sizes. Furthermore, limited characterization of ADD patients and control subjects poses a major problem in these studies. Thus, in none of the studies was the inclusion of patients based on distinct cut-off values for core biomarkers of AD-like amyloid-beta 1–42 (Aβ42) or tau-proteins. In the meantime, this has become the gold standard for studies in the field of AD, which is reflected by recently published research criteria for AD . RegardiIn recent years, neuroinflammation and microglial activation have been discussed as key elements in the pathogenesis of AD . In thisIn summary, it is still unclear if CSF lactate concentrations differ between patients with AD and healthy controls. Moreover, data regarding associations of CSF lactate levels with clinical and biological proxies for AD disease severity are still missing.In this study, we compared CSF lactate levels in a large and well-defined sample of patients with AD dementia, MCI due to AD and healthy controls under consideration of potentially associated or even confounding factors like BBB integrity, age, and proxies of disease severity (CSF biomarkers of AD and cognitive measures). For comparison, we included a subset of patients suffering from dementia with frontotemporal lobar degeneration (FTLD).Data was extracted from the CSF biobank of the Department of Psychiatry and Psychotherapy, Klinikum rechts der Isar, Technical University of Munich. All study participants provided written informed consent.We analyzed CSF data from patients with ADD, MCI-AD, and FTLD. As part of clinical routine procedures, CSF lactate levels and Albumin CSF/serum ratios (Alb CSF/S) were assessed for differential diagnosis. As a control group, CSF samples from healthy volunteers were acquired in the context of spinal anesthesia prior to orthopedic or urologic surgery procedures. Inclusion criteria were the absence of any neurological or psychiatric diseases , absence of subjective cognitive impairment, and intact global cognitive functioning defined by Mini-Mental State examination scores (MMSE) ; of at lDiagnoses of ADD, MCI-AD and FTLD were made by board-certified psychiatrists and experts in the field of dementia and aging in accordance with internationally recognized consensus criteria. For ADD, the criteria by McKhann et al. (2011) were used. For being classified as MCI-AD, we used the general criteria by Albert et al. . In thisTo rule out loss of BBB integrity due to additional undiagnosed underlying nervous system pathology  we excluded patients with an Albumin CSF to serum ratio (Alb CSF/S) greater than 15. The rationale behind that was that we examined the integrity of the BBB as a possible influencing factor on CSF lactate levels. Additionally, we excluded patients with CSF lactate concentrations > 3.5 mmol/l due to possible acute infectious central nervous processes.g and 4°C for 10 min, and aliquots were stored at – 80 °C. Peptide concentrations were analyzed with commercially available enzyme-linked immunosorbent assays  following standardized procedures in our round robin test-certified laboratory for neurochemical analyses.For CSF collection into polypropylene tubes, lumbar puncture was performed between segments L3/4 or L4/5. All samples were immediately stored on ice in upright position and processed within 1 hour. CSF lactate levels and albumin levels in CSF and serum were measured following clinical standard procedures. CSF for peptide concentrations  was centrifuged at 2000×R: A language and environment for statistical computing  = 10.78, p = 0.013). Dwass-Steel-Critchlow-Fligner pairwise comparisons revealed higher values for MCI-AD compared to ADD  and HC .Figure F  = 6.68, p < 0.001), each age  and Alb CSF/S  significantly predicted lactate concentrations. Group category was a significant predictor for ADD vs. MCI-AD , but failed to reach significance for HC vs. MCI-AD . After including the interaction of age and Alb CSF/S as a predictor in the regression model, only group category remained significant as a predictor . Further regression analyses with each ADD, FTLD and HC at the reference level did not provide additional information on categorial predictors. Multivariate regression under consideration of a more rigid exclusion criteria for BBB integrity as mentioned above (Alb-CSF/S > 10) showed comparable results with a significant model . Group category  and Alb CSF/S  significantly predicted lactate concentrations, while age did not (p = 0.107).In an additional analysis, we performed multivariate regression on lactate concentrations with diagnostic group  as a categorical predictor and—based on above mentioned correlational analyses—age and Alb CSF/S as continuous predictors. Group was dummy-coded . The overall model was significant  with HC and patients with FTLD, while taking into account BBB integrity, age, and proxies of disease severity. Across subgroups, we found correlations of CSF lactate with age and Alb CSF/S as a marker of BBB. Neither CSF biomarkers for AD nor MMSE scores were associated with CSF lactate levels for subgroups or in pooled analyses of all patients after adjusting alpha-values for multiple comparisons. Univariate between group comparisons revealed higher CSF lactate levels for MCI-AD compared to ADD and HC. In a multivariate analysis controlling for age and BBB integrity, group remained significant as a categorical predictor of lactate levels when comparing MCI-AD and ADD.These results are in contrast to the few existing studies on this topic, which predominantly found higher CSF lactate levels in patients with ADD compared to healthy controls –14. As oThe correlation of age and CSF lactate levels is in accordance with data from a large and clinically diverse sample . FurtherCSF lactate levels were lower in the ADD group compared to the MCI-AD group in our sample and we did not observe correlations of CSF lactate with CSF biomarkers of AD or cognitive scores. This speaks against a linear association of CSF lactate levels and disease severity in AD and stands in contrast to the previous finding of an inverse correlation of t-Tau and CSF lactate in patients with ADD described by Liguori and colleagues (2015).Several pre-analytical and physiological factors regarding CSF lactate levels should be taken into account that are of relevance when interpreting our results and previous studies. Lactate levels in body fluid samples should not be considered stable over time and are sensitive to temperature . While wTo apply uniform diagnostic criteria to the whole group of AD patients in this retrospective study, AD was diagnosed using the McKhann et al.  and not Furthermore, lactate not only originates in the CNS, but also in the periphery. As mentioned above, lactate crosses the BBB. Hence, CSF lactate levels should be interpreted cautiously when being understood as a proxy for metabolic processes in the CNS. Impaired regulatory mechanism of the BBB might account for variance. For this reason, peripheral blood lactate levels should be considered in future studies when examining CNS-related measurements of lactate. Also, associations of CSF lactate and additional CSF and peripheral markers of AD and neuroinflammation should be considered in future studies to further elucidate the relationship of AD and inflammatory processes in clinical samples.In summary, CSF lactate is not indicative of biological or clinical disease severity in AD, nor does it differentiate between patients with ADD and healthy controls or patients with FTLD. Interestingly, our results point to higher CSF lactate levels in earlier stages of AD, which might be interpreted in the context of microglial activation or the infection hypothesis of AD.


Hybrid fixation techniques including the both modified cortical bone trajectory (MCBT) and traditional trajectory (TT) at the L4 and L5 lumbar segment are firstly proposed by our team. Therefore, the purpose of this study is to evaluate and provide specific biomechanical data of the hybrid fixation techniques including the MCBT and TT.Four human cadaveric specimens were from the anatomy laboratory of Xinjiang Medical University. Four finite-element (FE) models of the L4–L5 lumbar spine were generated. For each of them, four implanted models with the following fixations were established: TT-TT , MCBT-MCBT , hybrid MCBT-TT , and TT-MCBT . A 400-N compressive load with 7.5 N/m moments was applied to simulate flexion, extension, lateral bending, and rotation, respectively. The range of motion (ROM) of the L4–L5 segment and the posterior fixation, the von Mises stress of the intervertebral disc, and the posterior fixation were compared.p ≤ 0.009), lower ROM of the posterior fixation (p < 0.001), lower intervertebral disc stress (p < 0.001), and lower posterior fixation stress (p ≤ 0.041). TT-MCBT groups showed a significant lower ROM of the L4–L5 segment (p ≤ 0.012), lower ROM of the posterior fixation (p < 0.001), lower intervertebral disc stress (p < 0.001), and lower posterior fixation stress (p ≤ 0.038).Compared to the TT-TT group, the MCBT-TT showed a significant lower ROM of the L4–L5 segment (The biomechanical properties of the hybrid MCBT-TT and TT-MCBT techniques at the L4–L5 segment are superior to that of stability MCBT-MCBT and TT-TT techniques, and feasibility needs further cadaveric study to verify. For decades, the pedicle screw fixation technique has been the mainstay in the lumbar spine surgery , but it In 2009, Santoni et al.  proposedWe firstly proposed the hybrid MCBT-TT and TT-MCBT techniques to make up for the defects of CBT and MCBT techniques, especially nerve decompression effect, and aim to provide the fixation technique with superior stability and strength. The ROM of the fixation segment and posterior fixation are inversely proportional to the stability of the vertebral body and the fixation. The equivalent stress of the intervertebral disc and the fixation are related to stability of the cage after lumbar fusion and the strength limit of fixation . In thisWe have completed the construction and validation of the FE models in L4–L5 lumbar spine and analyzed the TT-TT group in the earlier study . In thisFour different posterior fixations were established: (1) TT-TT group, pedicle screws at the cranial, and caudal levels  19); ; All models were fixed at the inferior surface of the L5 vertebral body. A 400 N vertical axial preload with 7.5 Nm moment was applied on the superior surface of the L4 vertebral body to simulate flexion, extension, left-right lateral bending, and left-right rotation. The relationship between the L4 and L5 vertebral body and the intervertebral disc was defined as mutual contact, using face-to-face correspondence. The ROM of the L4–L5 segment and posterior fixation, von Mises stress of the intervertebral disc, and posterior fixation were investigated.post hoc test was performed using the least significant difference (LSD) method. For all results, p < 0.05 was considered statistically significant.SPSS 26.0 software was used for data analysis and processing. Mean values of quantitative data were presented as mean ± standard deviation. One-way analysis of variance was used for the analysis of differences. When the differences were statistically significant, p ≤ 0.012), and compared with the MCBT-MCBT group, the TT-MCBT group showed significantly lower ROM only in flexion and right lateral bending (p ≤ 0.045). There was no significant difference between the MCBT-TT group and the TT-MCBT group (p > 0.05). Among the four posterior fixation models, the TT-MCBT group showed the lowest ROM . Compared to the MCBT-MCBT group, the MCBT-TT group showed significantly lower ROM only in flexion and right rotation conditions (p ≤ 0.021). There was no significant difference between the MCBT-TT and TT-MCBT groups in all working conditions (p > 0.05). Among the four posterior fixation models, the TT-TT group showed the largest ROM (2.38° ± 0.22°) and the TT-MCBT group showed the lowest ROM (0.46° ± 0.07°)  . Comparep < 0.001). Compared to the MCBT-MCBT group, the MCBT-TT group was significantly reduced in flexion, extension, and rotation (p < 0.006), and there was no significant difference in lateral bending (p > 0.05). There was no significant difference between the MCBT-TT and TT-MCBT groups (p > 0.05). Compared to the TT-TT group, MCBT-MCBT group was reduced by 47%, 42%, 34%, 37%, 37%, and 33% in flexion, extension, left-right lateral bending, and left-right rotation, respectively. These were 64%, 54%, 42%, 46%, 53%, and 55% in the MCBT-TT group, and 64%, 53%, 43%, 45%, 52%, and 50% in the TT-MCBT group. Compared to the MCBT-MCBT group, MCBT-TT group was reduced by 32%, 21%, 26%, and 32% in flexion, extension, left rotation, and right rotation, respectively. These were 32%, 19%, 24%, and 25%, respectively in the same working conditions, in the TT-MCBT group , and significantly lower in the MCBT-TT and TT-MCBT groups in all conditions (p < 0.041). There was no significant difference between the MCBT-TT and MCBT-MCBT groups (p > 0.05). Compared to the TT-TT group, MCBT-MCBT group was reduced by 45%, 37%, 36%, and 44% in flexion, extension, left lateral bending, and left rotation, respectively. In the MCBT-TT group, the posterior fixation stress was reduced by 56%, 48%, 62%, 47%, 60%, and 48% in flexion, extension, left-right lateral bending, and left-right rotation, and 56%, 49%, 64%, 48%, 58%, and 47% in the TT-MCBT group, respectively . This difference may be due to the length of the MCBT screw (L = 40 mm), the head and hub of the MCBT screw were in the cortical bone area while the CBT screw hub was in the cortical bone, but the head is not. McLachlin et al. . In patients with osteoporosis, the purchase of the screw was not strong because of the reduced bone mineral density in cancellous bone , while the pedicle screw diameter (D = 6.0 mm) was thicker than the MCBT screw (D = 4.5 mm). Although the length of the two screws were similar, but the ROM of the posterior fixations were different because the bony environment at the head and hub of the two screws were different. These results indicated that screw thickness or length was not an independent factor determining the stability of the fixation, which was consistent with the results of McLachlin et al. (p < 0.05), both of them were reduced by 56%. This result was consistent with the results of Ren et al.  in the study of Wang et al.  used in this study, but the length was shorter than the MCBT screw. The possible reason for this difference was that the contact of the screw head with the anterior lateral edge of the upper endplate of the vertebral body, the increase in screw length improved the stability of the vertebral body, which was consistent with the results of Matsukawa et al. , MCBT screw with a larger diameter may further increase the stability of hybrid fixation. Pedicle screw damages the facet joints inevitably, resulting in reduced vertebral stability and adjacent segment degeneration (ASD)  , MCBT scon (ASD)  and Rodron (ASD)  demonstre effect , 25, whie effect , 25.As with any FE analysis, certain limitations were inherent in the present study. First, there was a lack of an adjacent segment that provides an indispensable role in evaluating the relationship between hybrid techniques and ASD. Second, the limited number of specimens was insufficient to allow more convincing conclusions. Third, there was only one size of TT and MCBT screws, thus the biomechanical effects of various screw diameters and lengths in the hybrid techniques have not been considered extensively in the present study. The present study did not standardize the sizes of the MCBT and the TT screws in the hybrid techniques.This study reflects the biomechanical properties of the hybrid MCBT-TT and TT-MCBT techniques. The stability of the hybrid MCBT-TT and the TT-MCBT techniques were superior to that of TT-TT and MCBT-MCBT techniques, providing a preliminary biomechanical theoretical basis for the application of hybrid lumbar fixation techniques in patients with osteoporosis. However, the present study did not analyzed the effects of the hybrid techniques on the adjacent segment. The authors concluded that the hybrid MCBT-TT and the TT-MCBT techniques can be the optimal choices for lumbar posterior fixation in patients with osteoporosis.