Benefiting from the peculiar architectures, the fabricated NiSe2−1//Zn battery based on NiSe2 and the Zn plate exhibits a high specific capacity of 231.6 mAh g−1 at 1 A g−1, and excellent rate performance (162.8 mAh g−1 at 10 A g−1). In addition, the NiSe2//Zn battery also presents a satisfactory cycle life at the high current density of 8 A g−1 . Additionally, the battery device also exhibits a satisfactory energy density of 343.2 Wh kg−1 and a peak power density of 11.7 kW kg−1. This work provides a simple attempt to design a high-performance layered cathode material for aqueous Ni-Zn batteries.Transition metal organic framework materials and their selenides are considered to be one of the most promising cathode materials for nickel-zinc (denoted as Ni-Zn) batteries due to their low cost, environmental friendliness, and controllable microstructure. Yet, their low capacity and poor cycling performance severely restricts their further development. Herein, we developed a simple one-pot hydrothermal process to directly synthesize NiSe A large quantity of energy storage devices have been developed in the past few decades, such as supercapacitors, Li-ion batteries, and aqueous rechargeable batteries ,4,5,6,7.Zn anode ,14,15,16ode [−1) ,20,21,222-x-derived cobalt oxides nanoparticles synthesized by Tang et al. obtained a capacity of 7.42 mAh m−2 at 10 mA cm−2 with 0.02% cycle−1 capacity decay over 4200 cycles [2@NiOOH/CoOOH nanoflakes reported by Cui and his co-workers exhibited a capacity of 108.9 mAh g−1 at 1 A g−1 with 105.1% capacity retention after 4000 cycles [2 decorated Ni/Co selenide complex hollow arrays presented a capacity of 142 mAh g−1 at 2 A g−1 with ~36% capacity loss after 1000 cycles [Among current cathode materials, transition metal selenides (TMSs) own higher conductivity than transition metal oxides and hydroxides. For example, the binding energy of Ni-Se is lower than that of Ni-O and Ni-S with weaker electronegativity, reducing the energy consumption of conversion reaction . Additio0 cycles . Porous 0 cycles . MoSe2 d0 cycles . However2 stacked layered sheets without Ni-MOFs template and high temperature heat treatment. The NiSe2//Zn battery was assembled by using the synthesized NiSe2 nanosheet as the cathode and zinc as the anode of the aqueous Ni-Zn batteries, and the fabricated battery obtained a higher specific capacity of 231.6–162.8 mAh g−1 at various current densities of 1–10 A g−1, which indicated the good rate performance. Moreover, it shows excellent cycling performance with almost no decay compared to the initial specific capacity value after 1000 cycles at 8 A g−1. Impressively, the fabricated NiSe2//Zn battery shows outstanding performance with a maximum energy density of 343.2 Wh kg−1, and a maximum power density of 11.7 kW kg−1, substantially outstripping most of the recently reported Ni-Zn batteries.In this work, we propose a facile one-pot hydrothermal synthesis of NiSe2−X nanomaterials with different degrees of selenization can be obtained by adding different masses of SeO2 into the precursor solution of Ni-MOFs synthesis. 2 nanosheets under different SeO2 addition conditions. It can be seen that NiSe2−0.5 still retains part of the original nanosheets due to the lack of SeO2 during the synthesis process. Meanwhile, in 2−1 can be observed. Through the high magnification SEM images of the corresponding area  lattice plane of NiSe2 (JCPDS NO. 41-1495) [The Ni-MOFs nanomaterials were first synthesized by a simple one-pot hydrothermal process. As shown in ing area , it can ized TEM . Figure 41-1495) .2−X samples, which obviously demonstrates that the characteristic peaks of the four samples have changed significantly due to the different amount of SeO2 added during the synthesis process. The XRD diffraction patterns of NiSe2−1 sample, displaying six peaks at 2θ = 29.8°, 33.4°, 36.7°, 50.5°, 55.2° and 57.5° respectively, which are a right match (200), (210), (211), (311), (230), and (321) planes of NiSe2 (JCPDS NO. 41-1495), were also related to the lattice fringes of the NiSe2−1 material , OH- (533.6 eV) and adsorb water (532.5 eV), respectively . The corresponding GCD curves of NiSe2-1 at different current densities are depicted in −1 at 1 A g−1, and the discharge specific capacity was still maintained at 162.8 mAh g−1 with a high current density of 10 A g−1, proving its excellent rate performance. Meanwhile, the obvious charge/discharge voltage plateaus appear in the GCD curves at different current densities, and the voltage range of the plateau (at 1.85–1.95 V) matches the position of the redox peaks in the cyclic voltammetry curves correspondingly. This result further confirms that the NiSe2-1 electrode undergoes a redox reaction, which has the typical characteristics of a battery-type material. The rate performance of the original Ni-MOFs and NiSe2−1 electrodes applied to Ni-Zn batteries is shown in 2−1 exhibits an apparent increase in specific capacity during the initial cycles, which follows the same trend as that in −1 after 40 cycles, the average capacity retention of the NiSe2−1 electrode can even reach 117% compared to the initial average capacity, which is better than that of the Ni-MOFs electrode (96%) [In order to evaluate the electrochemical performance of the prepared electrode materials, we assembled Ni-MOFs, NiSectrolyte ,40. Figude (96%) .2+, we used different electrochemical methods to explore its kinetics. 2−1 electrode at different scan rates. It is obvious that with the increase of the scanning rate, the peak pattern of the redox peaks does not change, but the position of the redox shifts, which is mainly attributed to polarization. Theoretically, the peak current (i) and the scan rate (v) of the CV curves follow the equation: i = bav, where a and b are variable parameters. A b value of 0.5 indicates a diffusion-controlled behavior of the Zn-ion extract/insert electrode material, and a b value of 1 indicates a control by the capacitive process. The b values can be obtained by fitting a linear relationship between log i and log v [b values at each redox peak of the NiSe2−1 electrode are 0.74 and 0.76, respectively, between 0.5 and 1, suggesting the presence of both diffusion-controlled and capacity-based kinetic processes. The pseudocapacitive contributions of NiSe2−1 at 0.2, 0.4, 0.6, 0.8, 1 mV s−1, presented in i = 1vk + 2v1/2k, where 1k and 2k are constant values, and the calculated result of it is the theoretical value of the pseudocapacitive contribution [2-1 electrode, the 1vk value ranges from 67% to 98% at the scan range of 0.2–1 mV s−1. Typically, the original CV curve and fitted pseudocapacitive contribution (the shaded area) at 1 mV s−1 are shown in 2−1 tends to undergo redox reaction on the electrode surface to store charge, which can effectively protect the structure of the NiSe2−1 and improve the cyclic stability of the electrode material. More importantly, the near-surface pseudocapacitance effect can obtain a shorter ion transport path and a faster electron transport rate, which is conducive to enhancing the electrochemical performance of NiSe2−1 [2  are depicted in ct of NiSe2-1 electrode is 34.31 Ω, while the Rct of Ni-MOFs electrode is 40.6 Ω; in the low frequency region, NiSe2-1 electrode has a higher slope of the line than Ni-MOFs electrode, which means a faster electron diffusion. Therefore, the NiSe2−1 electrode exhibits better impedance performance than the pristine Ni-MOFs in both low and high frequency regions.In order to further understand the storage mechanism of Znnd log v ,43. As eribution ,45. For  NiSe2−1 . The ele NiSe2−1 ,47. It cSe2−1 [2 , the str2−1 as the cathode electrode of Ni-Zn batteries. It can be seen that the specific capacity of Ni-MOFs is slightly higher than that of NiSe2−1 during the very initial cycles, corresponding to the initial rate capability curve in 2−1 has a rather high percentage increase compared to the pristine Ni-MOFs, and the increase in capacity over about 100 cycles is attributed to the surface chemistry between NiSe2−1 and the KOH electrolyte, which activates the electrode [2-1 decayed by 50% of the peak capacity , as shown within 2−1//Zn battery were calculated to obtain the Ragone plots  [−1, 2.6 kW kg−1) [2@NiOOH/CoOOH//Zn battery  [2//Zn battery  [−1, 94.5 Wh kg−1) [−1, 85.8 Wh kg−1) [In order to further explore the practicability of the Ni-Zn batteries prepared, we investigated the cycle performance, energy density, and power density. lectrode ,40. Aftene plots b. NotewokW kg−1) , NiCoSe2Wh kg−1) , Co-dopeWh kg−1)  and flex3)2·6H2O and 1 mmol PTA were dissolved in a mixed solution consisting of 20 mL of DMF and 5 mL of 0.4 M NaOH, stirred well at room temperature and transferred into a 50 mL Teflon-lined stainless steel reactor, which was subjected to hydrothermal reaction at 160 °C for 12 h in an oven. After the reaction, the solution was cooled down to room temperature, centrifugally, and dried at 60 °C for 12 h to obtain Ni-MOFs.All chemicals in this work were utilized as received without any purification . 1 mmol Ni according to the molar amount of SeO2 added.The synthesis of NiSe2−X  electrode, Zn flakes, and a mixed aqueous consisting of 3 M KOH and 0.2 M Zn(CH3COO)2 as the anode, cathode, and electrolyte. The mass loading was about 2.4 mg cm−2. The electrochemical performances  of the fabricated batteries were characterized by an electrochemical workstation (CHI 660E) and Land 2001A.The aqueous Ni-Zn batteries were fabricated by a piece of Zn plate, the synthesized NiSeThe surface morphology, microstructure, crystalline structure, chemical composition, and elemental valence of the synthesized samples were investigated by the field-emission scanning electron microscope  equipped with an energy dispersive X-ray element analysis system (EDS), transmission electron microscope , X-ray diffraction  patterns, X-ray photoelectron spectroscopy , and Raman spectroscopy .All electrochemical characterization was carried out in a two-electrode system with a potential window of 1.6 to 2.0 V. Galvanostatic charge/discharge (GCD), Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements were performed using an electrochemical workstation (CHI 660E). The cycle life of the Ni-Zn batteries was tested by using a Land 2001A.2 with a layered structure. Benefiting from the unique nanosheet structure, the assembled aqueous NiSe2−1//Zn battery exhibits remarkable specific capacity (231.6 mAh g−1 at 1 A g−1), excellent rate performance (162.8 mAh g−1 at 10 A g−1), and prominent energy density (343.2 Wh kg−1) as well as excellent long-term cycling stability . Moreover, NiSe2 can be easily prepared by a one-pot hydrothermal synthesis method, which facilitates the widespread and commercial application of the energy storage devices and lays the foundation for future research and development of such materials.In summary, based on the synthesis of Ni-MOF, we have developed a simple one-pot synthesis method of NiSe


Whole-cell patch-clamp electrophysiology is an essential technique for understanding how single neurons translate their diverse inputs into a functional output. The relative inaccessibility of live human cortical neurons for experimental manipulation has made it difficult to determine the unique features of how human cortical neurons differ from their counterparts in other species.We present a curated repository of whole-cell patch-clamp recordings from surgically resected human cortical tissue, encompassing 118 neurons from 35 individuals . Recorded human cortical neurons derive from layers 2 and 3 (L2&3), deep layer 3 (L3c), or layer 5 (L5) and are annotated with a rich set of subject and experimental metadata. For comparison, we also provide a limited set of comparable recordings from 21-day-old mice (11 cells from 5 mice). All electrophysiological recordings are provided in the Neurodata Without Borders (NWB) format and are available for further analysis via the Distributed Archives for Neurophysiology Data Integration online repository. The associated data conversion code is made publicly available and can help others in converting electrophysiology datasets to the open NWB standard for general reuse.These data can be used for novel analyses of biophysical characteristics of human cortical neurons, including in cross-species or cross-lab comparisons or in building computational models of individual human neurons. Intracellular electrophysiology, as performed via the whole-cell patch-clamp technique, is a hallmark method for characterizing the biophysical features of neurons. While there have been numerous datasets characterizing these features from cortical neurons in the rodent brain , comparaRRID:SCR_014806) [However, collaborations between neurosurgeons and basic neuroscientists have recently made it possible to characterize living cortical neurons in brain slices immediately prepared from biopsies following routine neurosurgery . Still, _014806) .RRID:SCR_015242) electrophysiology data format via the Distributed Archives for Neurophysiology Data Integration (DANDI) data archive. We provide morphological reconstructions for N = 7 cells, made available at NeuroMorpho.org (RRID:SCR_002145). Each recording is made available with rich subject and experimental protocol metadata, enabling subsequent reuse and comparison with analogous datasets from other species and sources.Here, we describe an openly accessible dataset of electrophysiological recordings from human and mouse cortical neurons. The dataset encompasses 132 whole-cell patch-clamp recordings from surgically resected human tissue  or from 21-day-old mice (11 cells from 5 mice). These datasets are made available in the Neurodata Without Borders (NWB) (n = 34), frontal (n = 1), or parietal lobe (n = 1) epilepsy or brain tumors (n = 4) in 17 male and 18 female patients, age ranging from 21 to 59 years (mean age ± SD: 40.5 ± 12.0). Written informed consent was obtained from all study participants to use their tissue and to share the acquired data with anonymized demographic information—namely, subject age at time of surgery, sex, years of seizure, seizure frequency, secondarily generalized seizure frequency , antiepileptic drug treatment, and type of seizure.Resected human cortical tissues were obtained from Toronto Western Hospital . All procedures on human tissue were performed in accordance with the Declaration of Helsinki and approved by the University Health Network Research Ethics board . PatientThe resected cortical tissue from the temporal lobe–middle temporal gyrus exhibited no structural or functional abnormalities in preoperative magnetic resonance imaging and was considered “relatively healthy” by ourselves and others as it was located outside of the site of epileptogenesis , 12, 13.All experimental procedures involving mice were reviewed and approved by the animal care committees of the University Health Network in accordance with the guidelines of the Canadian Council on Animal Care. Mixed male and female wild-type C57Bl/6 J, age postnatal 21 days, were used for experiments. Mice were kept on a 12-hour light/dark cycle and had free access to food and water.2/5% CO2) artificial cerebrospinal fluid (aCSF) containing (in mM) the following: sucrose, 248; KCl, 2; MgSO4.7H2O, 3; CaCl2.2H2O, 1; NaHCO3, 26; NaH2PO4.H2O, 1.25; and D-glucose, 10. The osmolarity was adjusted to 300–305 mOsm. Transverse brain slices (400 μm) were sectioned using a vibratome (Leica 1200 V) Germany in cutting solution. Tissue slicing was performed perpendicular to the pial surface to help ensure that pyramidal cell dendrites were minimally truncated [2.2H2O, 1; MgSO4.7H2O, 1; NaHCO3, 26; NaH2PO4.H2O, 1.2; and D-glucose, 10, pH 7.40. All aCSF and cutting solutions were continuously bubbled with carbogen gas (95% O2–5% CO2) and had an osmolarity of 300–305 mOsm. Following this incubation, the slices were maintained in standard aCSF at 22–23°C for at least 1 hour, until they were individually transferred to a submerged recording chamber.Immediately following surgical human cortical resection, the cortical specimens were submerged in an ice-cold (∼4°C) cutting solution that was continuously bubbled with carbogenated  sucrose, 248; KCl, 2; MgSO4.7H2O, 3; CaCl2.2H2O, 1; NaHCO3, 26; NaH2PO4.H2O, 1.25; and D-glucose, 10. Mouse somatosensory cortical slices (350 μm) were prepared in the coronal plane similar to human slice preparation as described above.Brain slice preparation was done in a similar way for mice and human tissue. Mice were deeply anesthetized by isoflurane 1.5–3.0%. After decapitation, brains were submerged in (∼4°C) cutting solution that was continuously bubbled with 95% O2PO4, 1.25; NaHCO3, 30; HEPES, 20; glucose, 25; thiourea, 2; Na–L-ascorbate, 5; Na-pyruvate, 3; CaCl2.4H2O, 0.5; and MgSO4.7H2O 10 (mM). The pH of NMDG-HEPES aCSF solution was adjusted to 7.3–7.4 using hydrochloric acid, and the osmolarity was 300–305 mOsm. The cortical slices were prepared using a vibratome as described above. After slicing, slices were transferred to a recovery chamber filled with 32–34 °C NMDG-HEPES aCSF solution, which continuously bubbled with 95% O2–5% CO2. After 12 minutes, the slices were transferred to an incubation solution—HEPES aCSF—containing (in mM) NaCl, 92; KCl, 2.5; NaH2PO4.H2O, 1.25; NaHCO3, 30; HEPES, 20; glucose, 25; thiourea, 2; Na–L-ascorbate, 5; Na-pyruvate, 3; CaCl2.4H2O, 2; and MgSO4.7H2O, 2. After a 1-hour incubation at room temperature, slices were transferred to a recording chamber and continuously perfused with aCSF containing (in mM) NaCl, 126; KCl, 2.5; NaH2PO4.H2O, 1.25; NaHCO3, 26; glucose, 12.6; CaCl2.2H2O, 2; and MgSO4.7H2O 1 (mM) [A subset of cortical slices in both human and mouse was prepared using the N-methyl-D-glucamine (NMDG) protective recovery method . The cor2/5% CO2) aCSF containing (in mM) NaCl, 123; KCl, 4; CaCl2.2H2O, 1.5; MgSO4.7H2O, 1.3; NaHCO3, 26; NaH2PO4.H2O, 1.2; and D-glucose, 10, pH 7.40, at 32–34°C. Cortical neurons were visualized using an IR-CCD camera  Albany, NY with a 40× water immersion objective. Patch pipettes (3–6 MΩ) were pulled from standard borosilicate glass pipettes  using a vertical puller  Japan. For somatic recording of electrophysiological properties, patch pipettes were filled with intracellular solution containing (in mM) K-gluconate, 135; NaCl, 10; HEPES, 10; MgCl2, 1; Na2ATP, 2; and GTP 0.3, pH adjusted with KOH to 7.4 (290–309 mOsm). A subset of data was collected with excitatory  and inhibitory  synaptic activity blocked.For electrophysiological recordings, cortical slices were placed in a recording chamber mounted on a fixed-stage upright microscope  Oberkochen, Baden-Württemberg. Slices were continuously perfused with carbogenated . Subsequently, electrical signals were digitized at 20 kHz using a 1320X digitizer or a 1440A digitizer . The access resistance was monitored throughout the recording , and neurons were discarded if the access resistance was >25 MΩ. Recordings were not corrected for bridge balancing due to the short duration of recording time. We note that stimulus parameters for each recording are not identical across recorded cells, in part due to technical considerations by the experimentalist, for example, to prevent losing the cell recording.RRID:SCR_011323) 9.0, which output ABFv1 files, and pClamp >10.0, which output ABFv2 files, to ensure valid conversions while incorporating the essential metadata. The key aspects of our usage of these data conversion computer scripts relate to defining which ABF channels correspond to stimulus and response traces and ensuring that appropriate scale and offset factors are applied properly upon conversion. We incorporated the ndx-dandi-icephys metadata extensions to allow for inclusion of user-defined “Subject” and “Lab” metadata fields to be able to include specific metadata, including “subject_id,” “age,” “species,” “cell_id,” and “tissue_sample_id.”The x-to-nwb repository was used to convert current clamp recordings in axon binary format (ABF) to NWB format. Separate converters were used for files recorded using pClamp  toolbox was used to extract features from converted NWB files , 20. AllWe performed both automated and manual quality control checks of converted recordings to ensure dataset quality and maximize reuse potential. Using features automatically extracted via IPFX, we checked whether the baseline voltage of a sweep  deviated by more than 10 mV from the initial measure in the first current injection step. Any cell recordings that had any sweep deviate beyond the 10-mV threshold were not included in the final contributed dataset. We also included the measures for maximum drift of baseline Vm in each recording's metadata under the field max_drift_Vm.Also, individual recordings were manually inspected at 3 injected current steps . In addition, we further manually inspected each neuron recording's frequency/input curve to identify any abnormal responses and also to identify putative recordings from interneurons.Following this manual inspection process, we note that in some instances, we observed some evidence for spike saturation at higher steps of current injection. We also noted some instances of cells spiking spontaneously , but we chose not to reject these sweeps or cells according to our quality control criteria.t-test, Wilcoxon rank-sum test, Kruskal–Wallis rank-sum test, or Pearson correlation using the statistical functions in base R.To detect statistical differences across experimental groupings, we report results using the 2-sample All statistical tests were performed using R version 4.1.2 .In Table In current clamp mode, hyperpolarizing and depolarizing current injections  were used to characterize biophysical features of cortical neurons, with examples from 3 recorded cells shown in Fig. n = 18) and regular aCSF ; t(19) = 4.94, P = 9.37e-05. However, in Fig. t(28) = 1.14, P = 0.265, between neurons recorded following application of synaptics blockers and regular aCSF. Similarly, in Fig. t(40) = 0.259, P = 0.797, between the same groups.In Fig. t(11) = 1.32, P = 0.210, or average firing rate, t(25) = 0.0709, P = 0.944, observed between the recordings from the 2 species. However, when comparing the width of APs from recordings in human neurons  and mouse neurons , there was a significant difference detected, t(23) = 2.23, P = 0.0355.To illustrate comparisons across species, in Figs. t(16) = 2.52, P = 0.0224, of higher measured input resistance in the recordings made following preparation in the NMDG  recovery solution compared to the sucrose solution . In Fig. t(13) = 0.317, p = 0.756. The statistical comparisons made in Fig. To compare the effect of solution used for the brain slice preparation on intrinsic properties, we compared the input resistance and sag ratio recorded following preparation in either solution. In Fig. 2 = 2.7968, df = 2, P = 0.247) or sag ratio  were found across the 3 resected locations. In Figs. t(70) = 1.38, P = 0.172, or sag ratio, t(70) = 0.0644, P = 0.949. Note that all cells from frontal and parietal cortices were recorded from tissue resected near the site of the epileptogenic focus, whereas all cells from the temporal cortex were recorded distal from the epileptogenic focus (with the exception of 1 subject).To illustrate the rich diversity of the metadata for each of the human recordings, in Fig. Additionally, we illustrate the relationship of the input resistance and sag ratio against both patient age at time of surgical resection Figs. , B and yRRID:SCR_014806).The recordings in this database permit the quantification of biophysical properties from a diverse set of neurons, including human and mouse neurons with a well-described set of metadata. Independent variables collected include age, sex, seizure history, and cortical layer from which the tissue was resected. Additionally, experiments on the human neurons were performed with the use of synaptic blockers and without, allowing for comparisons and integration with other intrinsic electrophysiological databases comprising patch-clamp recordings, including from the Allen Cell Types Database (in silicoapproaches. Usage of these models in neuronal or circuit simulations can thus help to further predict and unveil the potential effects of differences in neuronal physiology across demographic groupings [These data from current-clamp experiments are particularly beneficial for the development of conductance-based models of human neurons , 22, 23.roupings , 28.The repository provided is focused mainly on cortical neurons derived from human tissue. There are comparatively fewer recordings for analyses of mouse neuron function provided, and all of these were performed using synaptic blockers that were shown to have a baseline effect on input resistance.The recordings from human specimens derived from tissue during the surgical resection of diseased tissue for patients with intractable epilepsy or brain tumors. Along with having suffered seizures for an extended period of time, the patients may have concurrently taken 1 or a variety of antiepileptic drugs that could have affected baseline neuronal excitability characteristics.While these data were collected for the purpose of characterizing intrinsic properties of human neocortical neurons, we note that they were collected using different sets of experimental conditions, including those related to different recording solutions as well as cutting solutions. Our analyses suggest such experimental condition differences likely contribute to differences in downstream electrophysiological properties and are consistent with prior analyses by ourselves and others , 29. ForIn contrast, the comparisons of electrophysiological measures following different cutting solutions highlight potential effects on neuronal excitability. We observe variability in the measured input resistance but consider that these effects may be due to changes in conductivity across the membrane or also experimental biases in selection of healthy neurons for patch-clamp protocol due to differential response to solutions of different osmolarity. Furthermore, there exists contrasting results in the literature on the effects of NMDG on neuronal excitability and synaptic transmission, which shows the context dependence of the many experimental variables , 34. TakABF: axon binary format; aCSF: artificial cerebrospinal fluid; DANDI: Distributed Archives for Neurophysiology Data Integration; IPFX: Intrinsic Physiology Feature Extractor; NMDG: N-methyl-D-glucamine; NWB: Neurodata Without Borders.Both mouse and human data are available on the DANDI platform , 36.GigaScience GigaDB database [Conversion, analysis scripts, and other associated metadata for recordings are available at GitHub . All othdatabase Derek Howard Conceptualization, Project Administration, Formal Analysis, Software, Investigation, Validation, Data Curation, Writing—Original Draft Preparation, Writing—Review & Editing, Visualization Homeira Moradi Chameh Project Administration, Investigation, Data Curation, Writing—Review & Editing Alexandre Guet-McCreight Investigation, Visualization Huan Allen Hsiao Software, Investigation Maggie Vuong Software, Investigation Young Seok Seo Software, Investigation Prajay Shah Investigation Anukrati Nigam Investigation Yuxiao Chen Investigation Melanie Davie Investigation Etay Hay Writing—Review & Editing  Taufik A Valiante Supervision, Resources, Funding Acquisition, Writing—Review & Editing Shreejoy Tripathy Conceptualization, Supervision, Methodology, Validation, Resources, Funding Acquisition, Writing—Original Draft Preparation , Writing—Review & Editing , Visualization.giac108_GIGA-D-22-00068_Original_SubmissionClick here for additional data file.giac108_GIGA-D-22-00068_Revision_1Click here for additional data file.giac108_GIGA-D-22-00068_Revision_2Click here for additional data file.giac108_Response_to_Reviewer_Comments_Original_SubmissionClick here for additional data file.giac108_Response_to_Reviewer_Comments_Revision_1Click here for additional data file.giac108_Reviewer_1_Report_Original_SubmissionKoen Kole -- 5/11/2022 ReviewedClick here for additional data file.giac108_Reviewer_2_Report_Original_SubmissionNathan Gouwens -- 5/13/2022 ReviewedClick here for additional data file.giac108_Reviewer_2_Report_Revision_1Koen Kole -- 8/23/2022 ReviewedClick here for additional data file.


N,N-dimethylacrylamide)gel networks enhancesswelling and allows reversible chemically driven actuation. The volumeswelling ratio of the gels is shown to depend on both the type ofanion and its concentration. 1H NMR and UV–vis titrationswith the squaramide crosslinkers reveal a relationship between anionbinding affinity and the concentration-dependent swelling behavior.Gel swelling is shown to be reversible, and by embedding a solid supportinto rod-shaped gels, soft actuators are fabricated that undergo forwardand backward bending motion in response to changing anion concentration.The swelling and bending process, which is accompanied by intensegreen coloration of the gel, is achieved by using only low amountsof crosslinker. This macroscopic actuation achieved by anion bindingto specific molecular entities in the polymer network will open newopportunities in the field of chemically responsive materials.Supramolecular anion binding to squaramide crosslinkersin poly( Indeed, soft actuatorsmade from stimuli-responsive polymer gels have emerged as a thrivingresearch field, with promising applications in electronics, healthcare,and robotics.15 Actuation of these gels is generally induced by stimuli such asheat, light, or an electrical or magnetic field.20 In particular for medical applications, however,chemically responsive actuators would be beneficial, because theycould act upon internal stimuli in the body where external stimulican be hard to apply. Yet, successful examples of mechanical actuationdriven by changes in the chemical environment  are limited and typically based on changes in pH value,humidity,15 or—more rarely—site-specific interaction with metalcations.24The ability of living organisms to adapttheir shape and colorto the surrounding environment has been a constant source of inspirationin the design of smart materials.27 which have proven useful in applications such as analyte sensing,29 wastewater extraction,30 and transmembranetransport.33 Additionally, anions have been used to alter theproperties of low-molecular-weight supramolecular gels.36 Their binding to certain gelators can result in disruption of anintermolecular hydrogen bonding network, leading to an irreversiblegel–sol transition. Yet, while complex and programmable shapedeformation of polymer gels has been achieved by various physicaland chemical stimuli,14 to our best knowledge, reversibleactuation of (polymeric) soft materials through binding of anionicspecies has not been demonstrated.Anions and anionic substances are omnipresent in nature andfulfillimportant roles in various chemical and biological processes. A largenumber of artificial anion receptors have therefore been developed,37 Forexample, thiourea-functionalized polymers have been used as colorimetricsensors for acetate and bicarbonate,38 whileurea-containing polymers have shown enhanced binding of phenylphosphonate.39 Interestingly, Flood and co-workers found thatincorporation of aryl-triazole units into poly(methyl methacrylate)(PMMA) did not only enhance the chloride extraction capability butthat formation of crosslinks by the anion also affected the polymer’shydrodynamic radius.40 In addition, thegroup of Sessler observed that anion extraction using PMMA copolymersbearing calix[4]pyrrole or tetracationic macrocycles altered the polymer’sphysical properties because of receptor–anion interactions.42 Despite these observations, efforts to gain control over macroscopicproperties of polymer materials using anionic stimuli are scarce.So far, to the best of our knowledge, only the groups of Sada43 and Song44 reportedanion-dependent swelling  ofthiourea-containing polymer gels. However, no reversibility was demonstrated.We envisioned that the use of a stronger anion-binding motif wouldenhance swelling at low anion concentration, which, in addition tothe previously described swelling decrease in the presence of excessamount of anion salt,44 could give rise to reversiblebehavior.Some anion receptors havebeen attached to polymeric supports inorder to improve sensing and extraction properties.SQ1 and SQ2 . While electron-withdrawingaromatic substituents increase squaramide proton acidity and henceanion binding affinity,46 they will add to therigidity of the crosslinker. For comparison, we therefore also synthesizedthe aliphatic derivative SQ2, which comprises two methacrylategroups, providing a higher degree of molecular flexibility. Becauseof increased electron density, this derivative should have lower anionbinding affinity with respect to SQ1. The synthesis of SQ2 was carried out in similar way as for SQ1, now using 2-aminoethyl methacrylate instead of vinyl aniline inthe substitution reaction. Furthermore, as opposed to zinc triflateas catalyst, N,N-diisopropylethylaminewas used in this case as the acid scavenger to give the desired compoundin 48% yield .The design ofcrosslinkers tors see 1.45,46 TN,N-dimethylacrylamide (DMAAm)as main monomer was chosen. In contrast to, for example, acrylamideor dimethylaminoethyl acrylate, DMAAm does not have NH protons thatcould engage in interactions with anions, thus allowing us to solelyinvestigate the effect of anion binding to the squaramide crosslinkers.Polymerizations were carried out in small volumes (0.25 mL) with a1.0 M concentration of the main DMAAm monomer (10 wt % in DMSO), 5mol % of either the SQ1 or SQ2 crosslinker,and 1 mol % of thermoinitiator AIBN. The samples were placed in anoven to cure overnight at 60 °C resulting in small pellet-likegel specimens.To obtain the anion-responsive gel networks, free radical polymerizationusing Fmix), which is counteracted by the networks’contractile elastic energy (Fel).50 An influx of solvent molecules into the network will then lead toa volume expansion until the equilibrium swollen state (Fmix = −Fel) is reached.It should be noted that in nonionic networks this volume expansionis normally independent of the concentration of salts being present.We hypothesized that, in our case, the squaramide crosslinkers wouldinfuse the nonionic network with anion-specific swelling behavior,introducing a third energy contribution (Fion) to account for (Fmix + Fion = −Fel).52To characterize themacroscopic behavior of the polymer networks, the obtained gel specimenswere subjected to swelling experiments. In general, if a gel sampleis immersed in excess solvent, the surrounding solution may exhibitan osmotic pressure on the network to balance the free energy of mixing for 24 h or 7days. After this time, the volume swelling ratios (Qv) were calculated to assess the expansionof the gels . Interestingly, thegels showed a distinct swelling pattern dependent on the concentrationfor each anion. For example, when SQ1-crosslinked gelswere immersed in solutions of [Bu4N]+[F]−, a peak-shaped profile was obtained for the volumeswelling ratio as a function of the concentration , which is mostlikely due to a weaker interaction of the anion with the crosslinkeras compared to the experiments with [Bu4N]+[F]− and [Bu4N]+[AcO]− (vide infra). In this case, also no color changewas observed, highlighting that the gels could be used in the colorimetricsensing and detection of the more basic F– and AcO– anions. Experiments carried out with gels containingcrosslinker SQ2, exhibiting a higher degree of molecularflexibility, showed similar peak-shaped swelling profiles. Now, after24 h, maxima of QV =41.7 and QV = 63.0 wereobtained around 0.01 M for [Bu4N]+[F]− and [Bu4N]+[AcO]−, respectively.In [Bu4N]+[Cl]− solution,as was also observed for the gels containing SQ1, these SQ2-crosslinked gels displayed a flat swelling profile after24 h and very minor concentration dependency after 7 days  was used as the crosslinker, which does not bind anions, noconcentration-dependent swelling was observed . This control experiment confirms our expectationthat anion binding is crucial to enhance swelling of the squaramide-crosslinkedgels.In this case, the maxima increased to r 7 days 2F, again54 behavinglike ordinary nonionic networks. However, in the [Bu4N]+[F]− and [Bu4N]+[AcO]− solutions, the polymer network becomes ionically “charged”owing to the presence of anion-binding sites. The enhanced swellingin this case, beyond the volume swelling ratio obtained using thesalt free solution, can be ascribed to charge repulsion between polymerchains. At higher salt concentrations, however, the amounts of anionsbound to the polymer (note that only 5 mol % of crosslinker is incorporated)becomes negligible as compared to the high quantities of salt beingpresent inside and outside of the network. As a result, the chargesclose to the polymer chains become increasingly shielded by the surplusof salt, and the gels do not show enhanced swelling anymore, in linewith the behavior of polyelectrolytes.55The anion-specific and concentration-dependent swellingis thusexplained by the adsorption of anions, which is facilitated by thesquaramide crosslinkers 2. In bri4N]+[F]− gave large responses, samples of SQ1- and SQ2-crosslinked gels were each immersed for 24h in 0.01 and 0.8 M solutions of this salt, as well as a salt freesolution, after which oscillatory frequency sweeps were measured.For SQ1-crosslinked gels, the storage modulus (a measurefor the stiffness and energy that can be stored within the network)for a gel immersed in 0.8 M [Bu4N]+[F]− was determined as 390 Pa .This value is similar to that measured in salt free solution (380Pa), which is in agreement with the similar volume swelling ratios. Here, the storage moduluswas 136 Pa for the gel that was immersed in the 0.8 M salt solutionand 93 Pa for the one in the salt free solution, while for the 0.01M salt solution the value decreased to 63 Pa. These rheological measurementsare thus in agreement with the volume swelling experiments describedabove. Furthermore, all measurements showed frequency-independentbehavior, which is expected for covalently crosslinked polymer networks.Oscillatoryrheological measurements were conducted to confirmthe anion-induced swelling via appropriate changes in the stiffnessof the gels. It is well-known that the degree of swelling directlycorrelates with the viscoelastic properties and, thus, storage andloss moduli of the gels. As the use of +[I]− did not produce any noticeable chemical shift changes,suggesting that neither of them bound iodide . Where also the titration of SQ2 with [Bu4N]+[Br]− did notlead to noteworthy changes in chemical shifts, addition of this saltto SQ1 produced a downfield shift of the squaramide NHsignal, indicative of bromide binding . Addition of [Bu4N]+[Cl]− to SQ1 and SQ2 showedsimilar downfield shifting of the NH signal, and analysis of the titrationdata revealed weak to moderate binding . The largest downfield shift was observedfor the titration of SQ2 with [Bu4N]+[AcO]−, and fitting of the data revealed a muchhigher binding constant for acetate than for chloride .59 Unfortunately,addition of [Bu4N]+[AcO]− to SQ1 as well as [Bu4N]+[F]− to both SQ1 and SQ2 resulted in severepeak broadening of the squaramide NH signals into the baseline . Such an observationhas previously been ascribed to either strong hydrogen bonding47 or fast proton exchange/deprotonation.58To elucidate the influence ofcrosslinker–anion binding on the swelling behavior, 2O. When [Bu4N]+[AcO]− was added to SQ1, the absorption maximumat λ = 365 nm decreased and bathochromically shifted to λ= 410 nm, in line with what has been previously reported for anionbinding to squaramides.60 The titrationdata was successfully fitted to a 1:1 binding model using BindFit62 to afford an association constant that is nearly 2 orders of magnitudelarger than the one determined for SQ2 .59 Addition of [Bu4N]+[F]− to SQ1 resulted in similar UV–vis spectral changes, whilefor SQ2 mainly a decrease in absorption was observed. Fitting of the datato a 1:1 binding model gave affinities in the same range as determinedfor AcO– binding to SQ1 and SQ2 .63The stability constant of these monomer/anion combinationswastherefore determined by UV–vis spectroscopic titrations inDMSO/0.5% H64SQ1 was treated with excess [Bu4N]+[OH]−, revealing a much larger bathochromic shift (λmax = 480 nm) than when [Bu4N]+[AcO]− or [Bu4N]+[F]− were added. In multiple examples reportedin the literature, the absorption maximum of squaramide derivativeswith aromatic substituents was found to red-shift to 460 nm,47 485 nm,65 and 540nm upon single deprotonation,64 while double deprotonationled to absorption above 600 nm.65 The much smaller bathochromicshifts observed upon addition of [Bu4N]+[AcO]− or [Bu4N]+[F]− to SQ1 and SQ2, similar to what was reportedearlier for anion binding to squaramide,60 thus suggest that neither of these crosslinkers experiences significantdeprotonation during the UV–vis titration experiments.To confirm that the observed spectral changes stem from bindingrather than deprotonation,– < Br– <Cl– < AcO– ∼ F–, covering 5 orders of magnitude. Most importantly, they correlatewith the volume swelling ratios of our gels (vide supra). That is, samples immersed in [Bu4N]+[I]−, [Bu4N]+[Br]−, or [Bu4N]+[Cl]− displayedconcentration-independent swelling, reflecting the weak binding ofthe anions to SQ1 and SQ2. On the otherhand, [Bu4N]+[F]− or [Bu4N]+[AcO]− solutions containinganions with Ka > 1 × 103 M–1 gave enhanced swelling behavior, resultingin the peak-shaped profiles shown in Overall, the binding constants show an increase according to theanion series ISQ1 were usedin these experiments as they showed moderate stiffness and a largeanion response. A gel sample that was first immersed in a 0.01 M solutionof [Bu4N]+[F]− for 24 h reacheda volume swelling ratio of QV = 34.4. Subsequent immersion in a 0.8 M [Bu4N]+[F]− solution induced shrinking to givea value of QV = 19.3,which is in agreement with what was observed at the higher concentrationsin the gel swelling experiments (vide supra). Furtheralternation between 0.8 M [Bu4N]+[F]− and salt free solutions led to repeated swelling and shrinking exhibiting QV values of approximately 21.5and 26.5, respectively  with solutionsof either [Bu4N]+[F]− or [Bu4N]+[AcO]−, showing similar expansionand shrinking behavior .To assess whetherthe anion-induced swelling process was reversible, the gel specimenswere sequentially immersed in solutions with different salt concentrations.The polymeric networks crosslinked with ectively 3. These SQ1), with a thin stripof cellulose filter paper embedded into the gel . Once the gel sample was fully cured, it was removedfrom the mold and cut into dimensions of 20 × 2.5 × 5.0mm (L × H × W). Then, the gel-embedded filter strip was fixed by using a clampedpair of tweezers, and the gel sample was immersed in a [Bu4N]+[F]− solution . Initially, at a concentration of 0.01 mM, thesample was nearly straightened (5°), yet as time progressed thebending angle increased to a maximum of 72° after 24 h , the degree of bending reached a plateaualready at 55° . Moreover,bending was less reversible upon replacement with the highly concentrated0.8 M [Bu4N]+[F]− solution,resulting in an angle of 39°. This observation is in line withthe same volume swelling ratios determined for the salt free solutionand the highly concentrated salt solutions.66The process of bending was accompanied by an intensegreen colorationof the gel similar to what was observed in the swelling experiments.When the original immersion solution was replaced by a highly concentratedone of 0.8 M [Bu4N]+[F]− solution,which was partially reversed by 12° after replacing it for the0.8 M solution . In comparison,a control sample that was first immersed in a salt free solution (asopposed to 0.01 mM [Bu4N]+[F]−) did not show any reversibility . It should be noted that, despite the low quantities of anion-bindingcrosslinker (2.5–5.0 mol %) in these polymeric gels, the effectof bending was readily perceivable. Overall, the higher crosslinkerconcentration narrowed the bending angle boundaries , improving the reversibility of the anion-inducedbending process.By reducing the crosslinker concentration to 2.5 mol %, afinalbending angle of 82° was achieved after 24 h of immersion inthe 0.01 M +[F]− gave rise to reversible bending, whichwas monitored over time. Additionally, the anion-induced swellingand bending process was accompanied by an intense green colorationof the gel, which may be applicable for sensing and detection purposes.Our results demonstrate that squaramide crosslinkers enable very effectiveanion-responsive swelling as well as actuation of soft polymer gels,which opens the path toward materials and robots that autonomouslyreact with motion and a change in physical appearance to the anionicspecies that surround them.In summary, we have demonstrated the capacityof two differentsquaramide crosslinkers to infuse polymeric gel networks with anion-responsiveproperties. These networks were successfully prepared by using smallamounts (2.5–5.0 mol %) of the crosslinker together with DMAAmas the main monomer in a free radical polymerization process. Thevolume swelling ratio of the hence obtained gels (determined after24 h and 7 days) showed peak-shaped dependency on the concentrationof the most strongly binding anions , N,N-dimethylacrylamide, ethylene glycol dimethacrylate , 2,2′-azobis(2-methylpropionitrile) , tetrabutylammonium fluoride hydrate , tetrabutylammoniumchloride , tetrabutylammonium bromide , tetrabutylammoniumiodide , and tetrabutylammonium acetate  were used as received.AIBN was recrystallized from methanol prior to use and stored below5 °C. To remove the residual inhibitor, the main monomer DMAAmwas destabilized via a short column of basic alumina prior use.Unless otherwise indicated, all solventswere purchased from commercial sources and were used without furtherpurification. 3,4-Diethoxycyclobut-3-ene-1,2-dione , zinctrifluoromethanesulfonate , 4-vinylaniline , 2-aminoethyl methacrylate hydrochloride , 1H and 13C NMRspectra were recorded on a Bruker Avance 500 NMR spectrometer  and are reported as follows: chemical shiftδ (ppm) ,number of protons, assignment). The residual protiated solvent signals were used as reference. Chemical shifts are reported in ppm tothe nearest 0.01 ppm for 1H and the nearest 0.1 ppm for 13C. Infrared spectra were recorded on a PerkinElmer FT-IRSpectrum Two spectrometer using an ATR unit. Absorbance maxima arereported in wavenumbers (cm–1), and only selectedintensities are reported. High-resolution mass spectrometry (HRMS)was performed on a Thermo Scientific Q Exactive HF spectrometer withelectron spray ionization (ESI). Rheological data were recorded ata Discovery HR-2 hybrid rheometer (TA Instruments). For all measurementsa sand-blasted plate stainless steel 8 mm SMART-SWAP plate (TA Instruments)was applied at a gap size between 700 and 1100 μm at 298 K.Samples were sliced into thin pieces using a razor blade and quicklyloaded onto the rheometer as they slowly experienced syneresis overtime. Oscillatory frequency sweeps were recorded at 1% strain in afrequency range between 0.1 and 100 Hz. Results were analyzed usingTA Instruments TRIOS software. UV–vis spectra were recordedon an Agilent Cary 8454 spectrometer in a 1 cm quartz cuvette. High-resolutionmass spectrometry (ESI-MS) was performed on a Thermo Scientific QExactive HF spectrometer with ESI ionization.47 Vinylaniline  was added to a stirred solutionof 3,4-diethoxycyclobut-3-ene-1,2-dione  and zinctrifluoromethanesulfonate  in ethanol  at rt. The reaction mixture was stirred at rt for 24 h. Next,the light brown precipitate was filtered off, washed with a smallamount of cold ethanol, and dried in vacuo to giveproduct SQ1 as a light brown powder . Mp> 400 °C (decomp.).1H NMR  δ 9.93 ,7.55–7.39, 6.70 , 5.77 , 5.20 . 13C NMR δ 182.2 (C=O), 166.0 (=CNH), 138.7(NHC), 136.5 (HC=CH2), 132.9 (C7), 127.8 (p-Carom), 119.1 (o-Carom),113.7 (HC=CH2). IR (ATR): ν3145 (m), 3092 (m), 3065 (m), 3007 (m), 1800 (m), 1795 (w), 1712 (s),1676 (s), 1598 (s sh), 1561 (s), 1539 (s sh), 1518 (s sh), 1461 (s),1439 (s sh), 1424 (w sh), 1409 (s), 1355 (m), 1328 (m), 1234 (m),1188 (m), 1163 (w), 1130 (m), 1099 (w), 1080 (w), 1036 (w), 1017 (w),995 (m), 968 (w), 907 (s), 863 (w), 838 (s), 823 (w sh), 809 (w),784 (m), 774 (m), 752 (s), 726 (s), 661 (w), 640 (w), 622 (w), 592(m), 559 (w), 491 (s). HRMS (ESI) m/z: 317.1284 (M + H)+, calculated for C20H17N2O2+: 317.1285.The procedure was adaptedfrom the one reported by Manesiotis et al.2Cl2 (50mL), and washed with 1 M HCl(aq) (2 × 50 mL). Theorganic layer was evaporated under reduced pressure, and the residualcrude solid was redissolved in CH2Cl2 (10 mL).The resulting white suspension was filtered off to give product SQ2 as a white powder . Mp 163.1–166.2°C. 1H NMR  δ 7.50 , 6.01 , 5.67 , 4.20 , 3.87–3.69 , 1.85 . 13C NMR  δ182.7 (C=O), 166.4 (OC=O),135.7 (C=CH2), 126.0 (C=CH2), 64.2 (NHCH2), 42.3 (OCH2), 17.9 (CH3). IR (ATR): ν = 3174 (m br), 3089 (m br), 3047(m br), 2971 (m br), 1807 (w), 1724 (s), 1641 (m), 1575 (s), 1489(m), 1455 (m), 1442 (m), 1398 (m sh), 1362 (m), 1323 (w), 1297 (m),1276 (m), 1202 (w sh), 1159 (s), 1036 (m), 1018 (m), 941 (m), 917(w), 866 (w), 832 (m), 815 (m), 777 (br), 723 (m), 657 (w), 634 (w),610 (w) cm–1. HRMS (ESI) m/z: 337.1391 (M + H)+, calculated for C16H21N2O6+: 337.1394; 359.1213(M + Na)+, calculated for C16H20N2NaO6+: 359.1214.DIPEA  was added dropwise over 1 h to a stirred suspension of 3,4-diethoxycyclobut-3-ene-1,2-dione and 2-aminoethyl methacrylate hydrochloride  in ethanol  at 0 °C. The reactionmixture was allowed to warm to rt and stirred for 24 h. Next, thereaction mixture was filtered, and the filtrate was concentrated underreduced pressure, redissolved in CHSQ1  and AIBN  in DMSO  at room temperature. A volumeof 250 μL of this gel precursor solution was then dispensedinto 1 mL glass vials (40×) capped with a rubber seal. The solutionsin these vials were degassed by purging with argon for 10 min. Finally,these solutions were heated to 60 °C overnight in an oven togive the cured anion-responsive networks as transparent slightly yellowishgels. Reactions with SQ2 or ethylene glycol dimethacrylate(EGDMA) as crosslinkers were carried out analogously. For the gelsthat were used in actuation experiments, the gel precursor solutioncontained either 2.5 mol % or 5 mol % crosslinker.DMAAm was added to solution of


Purpose: Heart failure (HF) is a leading cause of morbidity and mortality worldwide, and it is characterized by cardiac hypertrophy and fibrosis. However, effective treatments are not available to block cardiac fibrosis after cardiac hypertrophy. The QiShenYiQi pill (QSYQ) is an effective treatment for chronic HF. However, the underlying mechanism remains unclear.Methods: In the present study, a pressure overload-induced cardiac hypertrophy model was established in rats by inducing ascending aortic stenosis for 4 weeks. QSYQ was administered for 6 weeks, and its effects on cardiac fibrosis, myocardial apoptosis, RP S19 release, macrophage polarization, TGF-β1 production, and TGF-β1/Smad signaling were analyzed. In vitro studies using H9C2, Raw264.7, and RDF cell models were performed to confirm the in vivo study findings and evaluate the contribution to the observed effects of the main ingredients of QSYQ, namely, astragaloside IV, notoginsenoside R1, 3,4-dihydroxyl-phenyl lactic acid, and Dalbergia odorifera T. C. Chen oil. The role of four-and-a-half LIM domains protein 2 (FHL2) in cardiac fibrosis and QSYQ’s effects were assessed by small interfering RNAs (siRNAs).Results: QSYQ ameliorated cardiac fibrosis after pressure overload-induced cardiac hypertrophy and attenuated cardiomyocyte apoptosis, low FHL2 expression, and TGF-β1 release by the injured myocardium. QSYQ also inhibited the following: release of RP S19 from the injured myocardium, activation of C5a receptors in monocytes, polarization of macrophages, and release of TGF-β1. Moreover, QSYQ downregulated TGF-βR-II expression induced by TGF-β1 in fibroblasts and inhibited Smad protein activation and collagen release and deposition.Conclusion: The results showed that QSYQ inhibited myocardial fibrosis after pressure overload, which was mediated by RP S19-TGF-β1 signaling and decreased FHL2, thus providing support for QSYQ as a promising therapy for blocking myocardial fibrosis. Heart failure (HF) is a common outcome of various cardiovascular diseases and has poor prognosis . DespiteMounting evidence has established the role of cardiac myocyte apoptosis in the regulation of the progression of cardiovascular disease to heart failure . HoweverThe four-and-a-half LIM-domain protein 2 (FHL2) is the second member of the four-and-a-half LIM domain protein family, and it is most abundant in the heart tissue . Evidenc® (QSYQ) is a compound Chinese medicine approved by the China State Food and Drug Administration in 2003 for the treatment of cardiac dysfunction , Panax notoginseng (Burkill) F. H. Chen , Salvia miltiorrhiza Bunge , and Dalbergia odorifera T. C. Chen  . Clinical data show that QSYQ partially reverses left ventricular hypertrophy in hypertensive patients  . The majpatients , improvepatients , and incpatients . In addipatients  and acutpatients . Our prepatients . Howeverin vitro study using cardiomyocytes, monocytes, and fibroblasts provide further insights into the mechanisms underlying the effects of QSYQ.In the present study, an ascending aortic stenosis (AAS) rat model was used to investigate the effects and mechanisms of QSYQ and its main ingredients ASIV, R1, DLA, and DO on cardiac fibrosis caused by pressure overload. The results of the in vivo study. Both QSYQ and TMZ were dissolved in saline  to generate solutions with concentrations of 0.16 g/ml .QSYQ (batch number: 20030139) was obtained from Tasly Pharmaceutical Co. Ltd. . Trimetazidine hydrochloride tablets  were obtained from Servier Pharmaceutical Co., Ltd.  for the .16 g/ml  and 2 mg.16 g/ml , respect® 594 conjugate) and rhodamine-phalloidin were purchased from Invitrogen . Hematoxylin and Eosin (HE) was purchased from Zhongshan Goldenbridge Biotechnology Co., Ltd. . Antibodies against TGF-β1, p-Smad3, Smad3, Smad4, Smad6, Smad7, matrix metalloproteinases 9 (MMP9), MMP2, tissue inhibitor of metalloproteinases 1 (TIMP1), TIMP2, RP S19, and FHL2 were purchased from Abcam ; antibodies against CD68, CD163, and CD80 were purchased from Santa Cruz Biotechnology ; antibodies against TGFβR II, Bax, Bcl-2, cleaved caspase-3, caspase-3, cleaved caspase-3, caspase-3, and GAPDH were obtained from Cell Signaling Technology ; and antibodies against C5aR, collagen I, and collagen III were obtained from Proteintech . Enzyme-linked immunosorbent assay (ELISA) kits for Adenosine triphosphate (ATP), Adenosine diphosphate (ADP), Adenosine monophosphate (AMP), lactate dehydrogenase (LDH), malondialdehyde (MDA), TGF-β1, monocyte chemoattractant protein-1 (MCP-1), RP S19, and N-terminal pro-brain natriuretic peptide (NT-ProBNP) were purchased from Beijing Andihuatai Technology Co. Ltd. .Wheat germ agglutinin  were purchased from the Animal Center of the Peking University Health Science Center . Animals were housed at a temperature of 20 ± 2°C and a humidity of 40 ± 5% with 12/12-h light/dark cycles and fed standard rat chow and water. Animal care complied with the institutional guidelines of the Peking University Animal Research Committee, and the experimental protocols were approved by the Committee on the Ethics of Animal Experiments of Peking University Health Science Center (LA2020308) and consistent with the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health (NIH). All efforts were made to minimize animal suffering and the number of sacrificed animals based on the rules of replacement, refinement, or reduction.Rats were fasted for 12 h before the experiment while allowed free access to water. The animals were anesthetized with 2% pentobarbital sodium (60 mg/kg) via intraperitoneal injection . After tAfter 4 weeks of AAS challenge, the animals were examined by echocardiography, and the rats with a left ventricular wall 20% thicker than that in the sham group were identified as successful in modeling  and randThe harvested hearts were cut horizontally, and the upper one-third was fixed in 4% paraformaldehyde solution for 48 h and processed for paraffin sections (7 μm) using an Automatic Paraffin slicer . The sections were stained with HE, Masson’s trichrome, or Sirius red. The sections were observed under a microscope , and four fields were randomly selected for evaluation. Collagen accumulation in the interstitial and perivascular areas was analyzed using ImageJ 1.53c software  to assess the degree of cardiac fibrosis.Following dewaxing in xylene and rehydration in ethanol, antigen retrieval was performed in 0.01 mol/L sodium citrate buffer (pH 6.0) in a microwave. Endogenous peroxidase inhibition and blocking were performed. The paraffin sections were then incubated overnight with antibodies against CD68 (1:100), CD80 (1:100), CD163 (1:50), TGF-β1 (1:100), or RP S19 (1:100) after blocking with bovine serum albumin. Specific binding was detected by incubation with an HRP-conjugated secondary antibody, and positive staining was visualized with a DAB substrate kit. PBS was used instead of the primary antibody as a control. Images were captured using a digital camera connected to a microscope . The number of CD68/CD80/CD163-positive cells and the mean optical density of TGF-β1 and RP S19 were determined using ImageJ 1.53c software .in situ Cell Death Detection kit, Fluorescein  according to the manufacturer’s protocol. Paraffin sections were dewaxed, hydrated, and permeabilized with protease K before incubation with 50 μL TUNEL reaction mixture (enzyme solution: label solution = 1:9) for 2 h in a 37°C incubator. H9C2 cells were seeded on 8-well glass slides , and after the corresponding treatment, the cells were fixed with methanol and incubated with the TUNEL reaction mixture for 2 h in a 37°C incubator. Thereafter, paraffin sections and cells were counterstained with Hoechst 33342 for nuclear staining. Samples that were added only to the label solution served as negative controls. Images were acquired using a laser scanning confocal microscope .Cardiomyocyte and H9C2 apoptosis were determined using an 3) were excised from the left ventricle of rats 4 or 10 weeks after AAS. The tissues were fixed with 3% glutaraldehyde and post-fixed with 1% osmium tetroxide. The specimens were processed into ultrathin sections and stained with uranium acetate and lead citrate, and the ultrastructure of the myocardial tissue was examined using a transmission electron microscope .Fresh myocardial tissues  containing 4 mM L-glutamine, 4.5 g/L glucose, and 10% fetal bovine serum  at 37°C in a humidified incubator with 95% air and 5% CO2. All cells were used in passages 5–10. The cells were seeded, grown to 70–80% confluence, and starved in a serum-free medium for 24 h before treatment. H9C2 cells were treated with 1 μM Ang II  for 24 h for the in vitro studies , RDFs, a rat fibroblast cell line , and Raw264.7 cells, a mouse monocyte/macrophage cell line , were used for the  studies . QSYQ an studies . The dos studies . The dos studies . TMZ  and observed with a laser scanning confocal microscope . The average cross-sectional areas of 7–10 cells in each section were calculated using ImageJ 1.53c software . F-actin was labeled with rhodamine-phalloidin (1:50).Cells were fixed using 4% paraformaldehyde in PBS for 20 min at room temperature and permeabilized with 0.3% Triton X-100 at 37°C for 30 min. After blocking with 1% BSA and 22.52 mg/ml glycine in PBST (PBS+ 0.1% Tween 20) at room temperature for 30 min, monocytes and fibroblasts were incubated overnight at 4°C with anti-C5aR (1:50) plus anti-RP S19 (1:100) and anti-TGFβR I (1:100) plus anti-TGFβR II (1:100) antibodies, respectively, in 1% BSA in PBST. H9C2 cells were incubated overnight at 4°C with anti-FHL2 (1:200) and anti-TGF-β1 (1:100). After three washes in PBS, cells were incubated for 2 h at 37°C with DyLight-488/549-labeled secondary antibodies. The cells were counterstained with Hoechst 33342 for nuclei staining. Immunostaining without primary antibodies served as the control. Images were acquired using a laser scanning confocal microscope .ELISA was performed using a specific kit to determine the levels of ATP, ADP, AMP, LDH, MDA, TGF-β1, RP S19, and MCP-1 in the myocardium and myocyte supernatants  and collagen I, collagen III, and TGF-β1 in fibroblast supernatants  using a microplate reader . Each test was performed according to the manufacturer’s instructions.Approximately 50 mg of myocardium tissue was harvested from each animal (n = 4), quickly frozen in liquid nitrogen, and stored at −80°C for a maximum of 1 week before use. The total protein content was extracted using a protein extraction kit  and mixed with a 5× electrophoresis sample buffer. After electrophoresis, the separated proteins were transferred onto polyvinylidene difluoride membranes. After blocking with 5% nonfat dry milk, the membrane with target proteins was incubated overnight at 4°C with antibodies against RP S19, TGF-β1, TGFβR II, Smad3, p-Smad3, Smad4, Smad6, Smad7, MMP2, MMP9, TIMP1, TIMP2, collagen 1, collagen 3, cleaved caspase-3, caspase-3, cleaved caspase-9, caspase-9, cathepsin B, or GAPDH. After washing, the membrane was incubated with the secondary antibody for 1 hour at room temperature, and immunoreactive bands were detected using an enhanced chemiluminescence system. The protein signal was quantified by scanning densitometry and further evaluated using a bioimage analysis system .5 cells/well in 12 well plates. After incubating for 24 h at 37°C, the cells were transfected with 20 nm FHL siRNA or NC siRNA. Transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Western blotting was used to validate the transfection efficiency of FHL2 siRNA 48 h after transfection, and whole-cell extracts were collected to test the FHL2 and TGF-β1 contents.FHL2 small interfering RNA (siRNA) and negative control siRNA (NC siRNA) were provided by HANBIO . The sequences of the siRNAs and the negative control used in the present study were as follows: FHL2 siRNA, 5′-GGACUUGUCCUACAAGGAUTT-3′ and 3′-AUCCUUGUAGGACAAGUCCTT-1′; and negative control (NC siRNA), 5′-UUCUCCGAACGUGUCACGUTT-3′ and 3′-ACGUGACACGUUCGGAGAATT-1′. H9C2 cardiac myocytes were cultured to 40–50% confluence and seeded at 1–3×10t-test. The correlation between two variables was determined using Pearson’s correlation coefficient. The level of statistical significance was set at p < 0.05. All measurements were performed at least three times.All results are presented as the mean ± standard error of the mean (SEM). Statistical tests were conducted using the GraphPad Prism 9 software . Differences between groups were compared using one-way ANOVA or unpaired Student’s p < 0.05). Interestingly, what was observed in vivo could be simulated in vitro because H9C2 cells exposed to Ang II responded similarly to rat cardiomyocytes after AAS in terms of changes in cell size and ANF/18S, BNP/18S, and Myh7/18S mRNA levels. QSYQ and its main ingredients inhibited Ang II-induced H9C2 cell hypertrophy (p < 0.05).Heart failure presents as myocardial dysfunction, which was observed in the present study at 10 weeks after AAS surgery, as evidenced by the lower ejection fraction, fraction shortening, and reduced MBF in rats. Notably, treatment with QSYQ significantly restored heart function after AAS . Myocardertrophy  (p < 0.0p < 0.05).Next, we explored whether the QSYQ treatment inhibited cardiac injury and fibrosis after AAS. The transmission electron microscopy results showed that, compared with the sham groups, the AAS10W surgery resulted in prominent damage to cardiac myocytes, which manifested as ruptured myofibrils, swollen mitochondria, and edematous intermyofibril apace; however, this damage was improved by QSYQ treatment . The prop < 0.05). QSYQ post-treatment elicited a significant decrease in the average collagen volume after the AAS challenge (p < 0.05). Overall, QSYQ reduced cardiac fibrosis secondary to cardiac hypertrophy induced by pressure overload.We then investigated whether the QSYQ treatment could reduce cardiac fibrosis. Masson’s trichrome staining  and Sirivia apoptosis (p < 0.05). The effect of TMZ on apoptosis was similar to that of QSYQ. The serum LDH and MDA levels were also measured to assess the effects of QSYQ on myocardial injury, thus demonstrating that QSYQ significantly reduced both the serum LDH and heart MDA levels compared with that of AAS10W (p < 0.05). Collectively, the above results indicate that post-treatment with QSYQ ameliorated myocardial apoptotic injury induced by chronic pressure overload.Sustained pressure overload ultimately triggers maladaptive cardiac hypertrophy and induces cardiomyocyte loss poptosis . In the f AAS10W  (p < 0.0p < 0.05). The QSYQ treatment significantly decreased the cleaved caspase-3 content but not the cleaved caspase-9 content after AAS10W treatment (p < 0.05). TMZ had no effect on the elevated cleaved caspase-3 and caspase-9 levels. We also measured the levels of Bax and Bcl-2, which are two proteins related to apoptosis. As shown in p < 0.05). Collectively, the above results indicate that the post-treatment with QSYQ inhibited cardiac myocyte apoptosis induced by chronic pressure overload.Apoptosis involves various proteolytic enzymes, including caspases . Hence, p < 0.05). Moreover, TMZ exhibited an effect similar to that of QSYQ. The LDH levels were significantly upregulated after Ang II stimulation and significantly decreased after treatment with QSYQ, TMZ, and R1 but not with ASIV, DLA, and DO (p < 0.05). The MDA levels were decreased compared with the Ang II group after treatment with QSYQ, TMZ, ASIV, and DLA but not with R1 and DO (p < 0.05). The protein levels of apoptosis-related proteins were assessed by Western blotting, which showed that the QSYQ, DLA, and DO treatments reduced the ratio of cleaved caspase-3 to caspase-3 induced by Ang II; however, the other ingredients did not have a significant effect (p < 0.05). Compared with the Ang II group, QSYQ and DLA inhibited the ratio of cleaved caspase-9 to caspase-9 (p < 0.05), and TMZ and QSYQ decreased the ratio of Bax to Bcl-2 (p < 0.05). The effects of QSYQ on the Ang II-induced impairment of ATP production in H9C2 cells were also observed (p < 0.05). Mitochondrial respirometry of H9C2 cells was performed using 2-channel high-resolution Oxyraph-2k, and the results showed that QSYQ significantly improved the function of mitochondrial complex II and upregulated ATP production, which was impaired by Ang II (p < 0.05). In summary, consistent with the in vivo experiment, QSYQ alleviated cardiomyocyte injury and cell apoptosis, and DLA was a major player.H9C2 cells exposed to Ang II were used to further verify the effect of QSYQ on cardiac myocyte apoptosis and identify its main ingredients. As shown in , and DO  . Consistent with the ELISA results, immunohistochemical staining of RP S19 in AAS10W rat tissue also showed upregulated RP S19 expression (p < 0.05). QSYQ post-treatment had a pronounced inhibitory effect on RP S19 expression and inhibited the expression of MCP-1 both in vivo and in vitro (p < 0.05). The RP S19 (p < 0.05). Interestingly, component R1 was found to inhibit the expression of RP S19  using immunofluorescence and Western blotting. Raw264.7 monocytes were stimulated with RP S19 and treated with QSYQ and the main ingredients. p < 0.05). Nevertheless, QSYQ significantly inhibited monocyte infiltration and macrophage polarization after the AAS10W surgery, and TMZ had a similar effect (p < 0.05).Macrophages are extensively implicated in cardiac remodeling and fibrosis in HF and are associated with left ventricular pressure overload . Therefoin vivo and in vitro. The in vivo results showed that TGF-β1 was dramatically upregulated in the rat hearts subjected to AAS challenge, as revealed by the immunohistochemical staining (p < 0.05). Nevertheless, the QSYQ treatment distinctly suppressed the augmentation of TGF-β1 in the heart (p < 0.05). p < 0.05).TGF-β1 plays a critical role in the pathogenesis of cardiac remodeling and fibrosis . Therefostaining  and ELISstaining  (p < 0.0p < 0.05). In addition, QSYQ inhibited the nuclear translocation of p-Smad3, and the main components exhibited similar effects, with R1 being the least effective (TGF-β1 binds to its type I (TGFβR I) and type II (TGFβR II) receptors, thereby triggering phosphorylation and nuclear translocation of Smad2 and Smad3 and inducing profibrotic gene transcription . Thus, wffective .in vivo study showed that the protein levels of TGFβR II and p-Smad3 were significantly upregulated after the AAS10W treatment (p < 0.05). The QSYQ treatment significantly inhibited p-Smad3 expression and tended to lower the expression of TGFβR II.Consistent with the above results, the reatment  (p < 0.0p < 0.05). In summary, QSYQ inhibited the expression of TGFβR II and p-Smad3 and upregulated the protein levels of Smad7.Smad6 and Smad7 are well-known inhibitory Smads that inhibit TGF-β signaling by interfering with Smad4 activation. Intriguingly, we found that QSYQ markedly upregulated the protein levels of Smad7 after the AAS challenge compared with AAS10W  (p < 0.0in vivo. As shown in p < 0.05). The AAS challenge also upregulated the expression of cathepsin B (p < 0.05). In line with the in vivo results, QSYQ markedly suppressed the expression of MMP9 (p < 0.05). All the effective ingredients downregulated the expression of MMP2, while only DO showed an inhibitory effect on MMP9 after TGF-β1 stimulation (p < 0.05). These results suggest that post-treatment with QSYQ restored the balance of the collagen degradation system impaired by pressure overload stress.In addition to TGF-β1 signaling, which mediates collagen synthesis, matrix metalloproteinases (MMP2 and MMP9), tissue inhibitor of metalloproteinases (TIMP) , and cathepsin B , which w of MMP9  and MMP2 of MMP9  in RDF cmulation  . The ELISA results showed that QSYQ significantly decreased the serum collagen III levels (p < 0.05). The QSYQ, DLA, and R1 treatment markedly decreased the collagen I concentration in vitro in the supernatants of TGF-β1 stimulated RDF cells, whereas QSYQ, R1, and DO significantly decreased the collagen III concentration (p < 0.05). These results suggested that QSYQ and its main ingredients inhibited collagen production.These results suggest that QSYQ post-treatment may repress collagen production and deposition, even after cardiac hypertrophy is induced by pressure overload stress. To test this hypothesis, we examined the collagen expression in rats under various conditions. Western blotting showed that the QSYQ post-treatment significantly restored the f AAS10W  (p < 0.0I levels  (p < 0.0ntration  (p < 0.0p < 0.05). Therefore, we explored the mechanism by which cardiac myocytes express TGF-β1. Our previous proteomic results (in vitro study showed that QSYQ and its main ingredients except for DO upregulated FHL2 in Ang II-stimulated H9C2 cells (p < 0.05). To further clarify the role of FHL2 in the effects of QSYQ, FHL2 was knocked down using siRNA. p < 0.05). Next, H9C2 cells were stimulated with Ang II. Both the Western blotting (p < 0.05). However, after FHL2 knockdown, QSYQ failed to inhibit TGF-β1 production. These results demonstrated that FHL2 mediated the protective effect of QSYQ on TGF-β1 in cardiac myocytes.In a study on the production of TGF-β1 after chronic pressure overload stress, large numbers of phalloidin-labeled cardiac myocytes were found to colocalize with TGF-β1 . In addi results  suggeste results  and West results  results C2 cells   inhibited cardiac fibrosis after pressure overload-induced cardiac hypertrophy. This effect was related to the inhibition of myocardial apoptosis, cardiomyocyte RP S19 release, monocyte recruitment, TGF-β1 release, and the fibroblast TGF-β1/Smad signaling pathway. In addition, this study showed for the first time that QSYQ inhibited cardiomyocyte TGF-β1 production through the restoration of FHL2 expression. The results of the present study provided novel insights for a better understanding of the mechanism underlying the beneficial role of QSYQ in protecting myocardial fibrosis. However, dose-dependent effect of QSYQ on inhibition of cardiac fibrosis needs to be examined, and clinical studies are needed to verify whether QSYQ inhibits cardiac fibrosis after cardiac hypertrophy.


Of the 13 HE strains, 10 belong to “Candidatus Nanosynbacter sp.” strain HMT-352  (6), enabling us to explore both the phenotypic and genomic strain variations within a single TM7 species. We show that TM7 HMT-352 strains exhibit a diverse host range and varied growth dynamics during the establishment of their episymbiotic relationship with host bacteria. Furthermore, despite HMT-352 strains sharing a majority of their genes, we identified several gene clusters that may play a pivotal role in host affinity. More importantly, our comparative analyses also provide TM7 gene candidates associated with strain-level phenotypic variation that may be important for episymbiotic interactions with host bacteria.Saccharibacteria (TM7), which are obligate episymbionts growing on the surface of host bacteria, may play an important role in oral disease, such as periodontitis . As TM7 is a newly cultured lineage of bacteria, its research is limited by the small number of isolated representatives relative to the number of TM7 genomes assembled from culture-independent studies (3–5). A comprehensive view of both TM7 taxa and TM7 strain-level variations remains opaque. In this study, we expanded our previously developed TM7 baiting method into using many host bacteria in parallel, which allowed us to obtain 37 TM7 strains from the human oral cavity. These strains were further classified into low-enrichment  bacteria comprise a poorly understood phylum that is estimated to encompass ∼26% of all diversity of domain bacteria. Among CPR bacteria, the Saccharibacteria lineage (TM7) is of particular interest, as it is found in high abundance in the mammal microbiome and has been associated with oral disease. While many CPR genomes, TM7 included, have been acquired through culture-independent methods, only a small number of representatives have been isolated. Such isolated representatives, however, shed light on the physiology, pathogenesis, and episymbiotic interactions of TM7. Combined with genomic analyses, experiments involving isolated representatives can distinguish phylogenetic to phenotypic discrepancies and better identify genes of importance. In this study, we utilized multiple host bacteria in parallel to isolate TM7 bacteria and examined strain-level variation in TM7 to reveal key genes that may drive TM7-host interactions. Our findings accentuate that broad phylogenetic characterization of CPR is the next step in understanding these bacteria. Candidatus Nanosynbacter lyticus” TM7x, from the human oral cavity and is associated with the human microbiome (3The candidate phylum radiation (CPR) is a large monophyletic radiation of bacteria >73 phyla) with reduced genomes, currently referred to as the superphylum Patescibacteria 3 phyla w. Among tcrobiome . In recerobiome 35, 10, 11robiome 3, 13. TM7robiome 3.Schaalia odontolytica strain XH001 (previously known as Actinomyces odontolyticus)  . When TMlyticus) . When TMlyticus) . While cbacteria . Studyinbacteria .One of the major impediments in researching TM7 remains the limited number of isolated TM7 representatives, both in phylogenetic breadth and the number of strains per species , 17. To Porphyromonas gingivalis strains with distinct immune-stimulatory capacities, Coats et al. revealed that the fimB allele was the key virulence gene. Further, a single polymorphism between the genomes resulted in the reduced immune-stimulatory capacity of one of the strains   . AdditioActinobacteria members. To expand on this previously short list of basibionts, we cultured high-value host bacteria that may bait TM7 from the oral cavity  were targeted based on their taxonomic relationship with previously known basibionts. Many new Actinomyces and Schaalia strains (N = 41) were cultivated from the healthy and periodontal human oral cavity using three independent blood agar selection plates   . We name10.1128/msystems.01488-21.5TABLE S1Table S1, XLSX file, 0.01 MB.List of basibionts and their growth conditions. Detailed taxonomy, growth, and source information on basibionts that were tested. Download Copyright © 2022 Nie et al.2022Nie et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Using this basibiont plate as a screening platform, we exposed 16 periodontal filtered patient saliva samples to each unique basibiont, creating ∼1,200 unique coculture combinations  . From th10.1128/msystems.01488-21.6TABLE S2Table S2, XLSX file, 0.02 MB.Summary of all the TM7 strains that were isolated using high-throughput host bait method. Identity determined by 16S sequences. High abundance, HA; low abundance, LA. Download Copyright © 2022 Nie et al.2022Nie et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in situ hybridization (FISH) imaging. TM7 were visually abundant in the HE group , and cocultures with small amounts of TM7  . The enrHE group  to C butHE group . DespiteHE group . This la10.1128/msystems.01488-21.1FIG S1FIG S1, PDF file, 0.3 MB.LE and HE TM7 and their basibionts. Phase-contrast (top row) and FISH (bottom row) images of LE and HE groups. FISH images of TM7 (red) and host bacteria (green) visualized by species-specific 16S rRNA probes. The LE cocultures were PCR positive for TM7 but displayed a weaker signal (see insets for the phase-contrast image). Download Copyright © 2022 Nie et al.2022Nie et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Ca. Nanosynbacter spp.” [Ca. Nanosynbacter featherlites” [Ca. Nanosynbacter spp.”) strains  352 (“er spp.” ). A few erlites” ) and HMT strains  to C. HM strains . Isolatisibionts . Althougsibionts  to C.Schaalia and Actinomyces spp. with each isolated TM7 strain , similar to the previously described HMT-952 strain TM7x, while HMT-957 strains primarily grew on clade 2 (Actinomyces spp.), similar to the previously described BB001 strain, which also belongs to HMT-957 . Sur. SurScha HMT-957 . TM7-079Remarkably, although only one strain of HMT-352, TM7-072, was originally baited with basibiont ICM47, in our infection experiment, almost all HMT-352 strains, with the exception of TM7-001 and HMT-076, were able to grow with this basibiont, suggesting the broad ability of this basibiont to host HMT-352 strains . Alterna600) and TM7 score (amount of TM7) during subsequent passages . TM7-001 was the only strain that failed to infect either JN023 or ICM47  and TM7 scores (squares). See Materials and Methods for detailed experimental setup. Download FIG S2, PDF file, 1.9 MB.Growth-crash phenotype in the JN023 basibiont that was infected with 10 different HMT-352 strains from the HE group. Expanded from Copyright © 2022 Nie et al.2022Nie et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/msystems.01488-21.3FIG S3x axis represents passage numbers across time, while the y axis represents both cell density measurements for host bacteria  and TM7 scores (squares). See Materials and Methods for detailed experimental setup. Download FIG S3, PDF file, 1.6 MB.Growth-crash phenotype in the ICM47 basibiont that was infected with 10 different HMT-352 strains from the HE group. Expanded from Copyright © 2022 Nie et al.2022Nie et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Ca. Nanosynbacter lyticus” TM7x). Thus, we infer that these new genome assemblies are of similar quality to complete TM7 genomes. We excluded the TM7-033 genome from our study, since it was 100% identical to TM7-037 and was isolated from the same patient.We sequenced the 10 HMT-352 strains  using Illumina short-read technology to assemble high-quality draft genomes . GenomesPhylogenetic analysis using GToTree  based onN = 534, 98%). These core genes encoded a number of conserved housekeeping functions, like ribosomal proteins, as well as other functions, like the arginine deiminase system and type IV secretion machinery  over alignable genome fractions supported this view of substantial intraspecies genetic diversity among HMT-352 strains –33. Thus–To further investigate this intraspecies genetic diversity, we compared the amino acid identity among the 545 gene clusters shared by all nine HMT-352 strains . The majN = 17 single-copy core gene clusters. Of these, 6 could be annotated, encoding a protein secreted by the type IV secretion system, the tyrosine tRNA charger tyrS, an adenylate cyclase, a protein predicted to be involved in chromosome condensation, a vitamin K epoxide reductase, and a cpoA glycosyl transferase . Download Table S6, XLSX file, 0.01 MB.Six single-copy core gene clusters that can be annotated with a mean of less than 90% amino acid identity across the HMT-352 genomes   . This obtiaceae) .in vitro conditions, and they may change according to the environmental niche in the oral cavity. For example, external forces such as nutrient availability, surrounding bacteria in the biofilm, and the immune system all can affect the phenotypic observations that we made. However, we believe that fundamental features such as selecting specific host bacteria do not change.Our previous study demonstrated that, in relation to TM7, basibionts could either be completely resistant to TM7 infection or susceptible to TM7 infection . Here, hCa. Nanosynbacter sp.” strain HMT-352 presents a surprising contrast between their apparent similarity based on shared gene content yet dissimilarity based on ANI. Each of the HMT-352 strains shared a majority of its genome with all other studied HMT-352 strains, with approximately 70% of the genes in each genome being core to all HMT-352 strains. This proportion is similar to that of other studied bacterial species, although the proportion can vary substantially based on both the number of genome representatives and the degree of gene flow . For example, HMT-352 and HMT-952 mostly infect clade 1 Schaalia, while HMT-957 mostly prefers clade 2 Actinomyces. However, little congruence could be found between the strain-level phylogeny of HMT-352 and their host range patterns. For example, within HMT-352 strains, the host range pattern is poorly correlated with Schaalia phylogeny. This result indicates that host range is not a predominantly vertically transmitted trait or perhaps that host range is a fast-evolving trait relative to the evolution of phylogenomic marker genes. Exploring these possibilities requires additional improvements to our current infection model, the model’s subsequent readout, and our TM7 strain-level collection.Host range is a fundamental feature of parasitic interactions. Compared to phages  and predSchaalia or Actinomyces hosts in the mouth, which led to the decoupling of genes related to host association from the rest of the TM7 genome. From this perspective, the predominant selective forces that shape oral TM7 speciation may be factors such as defense against phage. An alternative explanation involves horizontal gene transfer among different TM7, producing the observed result whereby host JN023 was sensitive to TM7-076 while ICM47 was not.Furthermore, using the most susceptible hosts found in this study, JN023 and ICM47, we tested host sensitivity to TM7 infection by assessing the TM7 ability to infect and induce the growth-crash phenomenon. The permissive hosts demonstrated the growth-crash phenotype, while the nonpermissive hosts did not display the growth-crash phenotype relative to specific TM7 strains . InteresHere, we establish a high-throughput method for TM7 isolation. We effectively isolated many TM7 strains from the oral cavity. By exploring the phenotypic characteristics of these strains, we found that they could be divided into low-enrichment and high-enrichment groups, adding a third phenotype to the permissive and nonpermissive characterizations of TM7 infection. In this study, we examined strain variations of TM7 for the first time both phenotypically and genetically. TM7 strains from the same species showed diverse relationships with hosts. Most importantly, we demonstrated that strains with nearly identical genetic structure can have divergent ANI and AAI characteristics and display different host affinities. Future studies focused on investigating the potential gene functions that drive host bacterial responses in oral TM7 may eventually reveal the mechanisms governing the episymbiotic interaction between TM7 and their host bacteria.Actinobacteria strains used in this study were cultured in brain heart infusion medium (BHI) at 37°C under microaerophilic  or anaerobic  conditions . The study was done under the Forsyth IRB protocol number 21-02. The collected samples were centrifuged at 3,000 × r plates  and inocterature  45, 46), 46g fornd 1492R  primers 10.1128/msystems.01488-21.7TABLE S3Table S3, XLSX file, 0.01 MB.Actinobacteria selective medium composition. Download Copyright © 2022 Nie et al.2022Nie et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/msystems.01488-21.8TABLE S4Table S4, XLSX file, 0.01 MB.PCR primers used in this study with specific conditions. Download Copyright © 2022 Nie et al.2022Nie et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the g for 5 min. The supernatant was filtered through a 0.45-μm filter, and the flowthrough was diluted to 5 mL with BRF medium composed of 50% BHI, 45% RPMI, and 5% fetal bovine serum (FBS) broth; 50 μL of dilutions was added to each well of a 96-well plate containing 200 μL of the basibiont culture, bringing the final volume of each well to 250 μL. To prepare the basibionts, we added 76 different basibionts  from 16 patients with periodontitis were collected, vortexed vigorously for 5 min, and centrifuged at 3,000 × sibionts  into 76 Campylobacteria as a contaminant. However, by controlling the flow of the saliva through a 0.45-μm filter , we generally avoided contamination. If, however, contamination occurred and persisted after a few passages, we conducted the following procedures to clean the culture.The cocultures were passaged at a dilution of 1:10 every 2 days into fresh BRF medium. The presence of TM7 was monitored by phase-contrast microscopy (Nikon Eclipse E400) and PCR  at the 43-, 104-, and 105-fold with BHI, and then 100 μL was plated on BHI blood agar and incubated for 5 to 7 days in an anaerobic or microaerophilic chamber  and incubated for 7 to 9 min at 37°C. Cells were pelleted and washed by resuspending each pellet in 0.5 mL of 50%, 80%, and 90% ethanol, with pelleting between each wash. For each pelleting step, centrifugation of 13,000 × g for 5 min was used. Finally, the cell pellets were dried in a speed vacuum dryer for 10 min.FISH was carried out according to our previously described protocol . BrieflyFor staining, cells were resuspended in 0.5 mL of hybridization solution  and incubated at 37°C for 15 min. Fluorescently labeled oligonucleotide probes  were added to a final concentration of 5 ng/μL. The solution was incubated for 3 h at 42°C in the dark and centrifuged to collect the cell pellet. The pellet was washed twice by resuspension in 0.1 mL of 0.1× saline sodium citrate buffer  and incubated for 10 min at 37°C in the dark. For the last wash step, 5 μM  Syto9 dye  was added to stain DNA of the basibiont bacteria. Cells were mounted on a PolyLys -coated coverslip with 4 μL of SlowFade gold antifade reagent (Invitrogen). To coat the coverslip, we dipped the coverslip in PolyLys solution for 2 min, washed it with water, and dried it. The slides were visualized using a Zeiss 780 confocal microscope equipped with a 63×/1.4 Plan-Apochromat oil immersion objective . Images GTGACTGGGCGTAAAGAGTT-3′; AJ14-R, 5′-CTACGGATTTCACTCCTAC-3′). For TM7, the reactions were processed at an initial denaturation temperature of 95°C for 1 min, followed by a 50-cycle amplification step, which consisted of denaturation at 95°C for 30 s, annealing at 54°C for 30 s, and extension at 72°C for 30 s. A standard curve was generated using a plasmid containing the 16S sequence of the TM7 bacteria. Using the generated standard curve, the coculture gDNA TM7 16S copy number was determined.The genomic DNA (gDNA) of each coculture was isolated using the MasterPure Complete DNA purification kit  by following the manufacturer’s guidelines, and its concentration was determined using Qubit (Invitrogen). The gDNA was diluted to 0.1, 0.01, 0.001, 0.0001, and 0.00001 ng/μL for both the DNA template standard and the bacterial coculture. A volume of 4.5 μL of gDNA from each dilution was mixed with 5 μL PowerUp SYBR green master mix and 0.25 μL of the appropriate forward and reverse quantitative reverse transcription-PCR primers . The flowthrough was ultracentrifuged at 80,000 × g for 80 min. The pellets were resuspended in 1 mL of fresh BHI. The purity of the isolated TM7 cells was confirmed by phase-contrast microscopy, and, using the EpiCenter Gram-positive DNA isolation kit, the TM7 gDNA was extracted from both the filtration-isolated TM7 and the original coculture to produce two genomic libraries per TM7 strain.The TM7 coculture was reinoculated as described above and expanded into 200 mL of BHI culture. The coculture was then centrifuged at 3,500 × gDNA was randomly fragmented by sonication, and then the gDNA fragments were end polished, A-tailed, and ligated with the full-length adapters of Illumina sequencing. Further PCR amplification was then performed with P5 and indexed P7 oligonucleotides. The PCR products, as the final construction of the libraries, were purified with the AMPure XP system. Libraries were checked for size distribution by an Agilent 2100 Bioanalyzer  and quantified by real-time PCR to pool libraries at a final concentration of 3 nM DNA. The libraries were then sequenced on an Illumina Novaseq .https://www.borlab.org/resources. For each new TM7 strain, genomes were assembled using anvi’o  was downloaded from NCBI and used as an outgroup. The resultant tree was visualized with FigTree.A phylogenomic tree was constructed with GToTree  using a Average nucleotide identity (ANI) was computed between all genome pairs in the anvi’o pangenome using BLASTN  via pyanAmino acid similarities among gene sequences for core gene clusters were calculated by exporting sequence alignments for all core genes using the anvi-get-sequences-for-gene-clusters command . The resultant fasta was then parsed using a custom R script (narrative methods) to obtain percent identities between all pairwise combinations of gene sequences for each gene cluster. Percent identities were calculated in R using the seqinr package.PRJNA784561.The genomes were deposited in GenBank with the accession numbers JAJQJX000000000, CP089290, JAJQJW000000000, JAJQJV000000000, CP089289, JAJQJU000000000, JAJQJT000000000, JAJQJS000000000000, CP089288, and JAJQJR000000000 under BioProject no.