The p38 mitogen-activated protein kinase  cascade is a central signaling pathway that transmits stress and other signals to various intracellular targets in the cytoplasm and nucleus. More than 150 substrates of p38α/β have been identified, and this number is likely to increase. The phosphorylation of these substrates initiates or regulates a large number of cellular processes including transcription, translation, RNA processing and cell cycle progression, as well as degradation and the nuclear translocation of various proteins. Being such a central signaling cascade, its dysregulation is associated with many pathologies, particularly inflammation and cancer. One of the hallmarks of p38α/β signaling is its stimulated nuclear translocation, which occurs shortly after extracellular stimulation. Although p38α/β do not contain nuclear localization or nuclear export signals, they rapidly and robustly translocate to the nucleus, and they are exported back to the cytoplasm within minutes to hours. Here, we describe the physiological and pathological roles of p38α/β phosphorylation, concentrating mainly on the ill-reviewed regulation of p38α/β substrate degradation and nuclear translocation. In addition, we provide information on the p38α/β ’s substrates, concentrating mainly on the nuclear targets and their role in p38α/β functions. Finally, we also provide information on the mechanisms of nuclear p38α/β translocation and its use as a therapeutic target for p38α/β-dependent diseases. The p38 mitogen-activated protein kinase  is a signaling protein kinase that operates within a signaling cascade to transmit extracellular signals to their intracellular targets. The p38 cascade is one of four similar cascades that are all key communication lines between the plasma membranes and the nucleus, and thereby, it is involved in fundamental cellular processes, including stress response, proliferation, differentiation and others ,2,3. TheBeing responsible for the various distinct and even opposing fundamental cellular processes, the p38 cascade needs to be tightly regulated. Indeed, several regulatory mechanisms that determine the specificity of the cascade have been identified, including the duration and strength of the signals ,14, whicThe p38α/β are best known for their involvement in stress signaling, and indeed, these kinases as well as JNKs were initially termed stress-activated protein kinases (SAPKs ). HoweveThe role of p38α/β in the regulation of protein degradation is widespread, mainly upon stress signals, and may involve several distinct mechanisms in both the cytoplasm and the nucleus. One such mechanism that mostly occurs in the nucleus involves phosphorylation of ubiquitin E3 ligases, such as Siah2, which is known to regulate PHD3 that further controls the stability of the transcription factor HIF1 α. p38α/β phosphorylate Siah2 on Ser24 and Thr29, thereby facilitating its activity towards degradation of PHD, and in turn destabilization of HIF1 α ,35. SimiAnother important process that is regulated by p38α/β is the dynamic change of protein localization upon stimulation. As described above regarding the regulation of protein degradation, the effect of p38α/β on nuclear translocation can be either global or specific to certain phosphorylated proteins. The global effects may be derived by p38α/β phosphorylation of either nuclear pore proteins or of karyopherins (importins/exportins). Indeed, it was shown that the nuclear pore proteins Nup62, Nup153, and Nup214 are phosphorylated by p38 (or ERK), and this phosphorylation inhibits the global nuclear protein shuttling initiated by viruses that affect the heart such as the encephalomyocarditis virus . A similAbnormal activity and dysregulation of the p38α/β cascade are associated with a variety of diseases. Indeed, p38α/β were implicated in the induction and maintenance of several pathologies such as inflammation , cancer In the past decade, substantial research has been devoted to studying the role of p38α/β in cancer, confirming that these kinases can act either as tumor promotors, or more frequently, as tumor suppressors ,30. The More than 150 direct substrates of p38α/β have been identified so far ,87,88,892-Xaa2-6-Φaa-Xaa-Φaa (where Φaa is a hydrophobic residue). The D-domains in the substrates bind to their counterpart docking site on p38α/β termed common docking motif (CD), which is composed of three negatively charged residues and at least two hydrophobic residues [As all other MAPKs, activated p38α/β execute their functions through the phosphorylation of downstream proteins. To the best of our knowledge, unlike ERK , no phosresidues . The CD residues . Bindingresidues , and tharesidues .However, the differences between the hydrophobic residues cannot fully explain the specificity of p38α/β phosphorylation. Rather, this is likely explained by a second docking site on p38α/β substrates termed DEF , which consists of two Phe residues separated by one residue and is often followed by a Pro residue . The DEFOne of the mechanisms that determines the ability of MAPKs to phosphorylate distinct substrates upon varying conditions or cell lines is the cellular localization of the substrates’ phosphorylation . Indeed,The ability of p38α/β to phosphorylate substrates in the cytoplasm as well as in the nucleus and the role of nuclear p38α/β in the regulation of cancer and inflammation diverted attention toward the subcellular localization of these kinases. Thus, it was initially shown that p38α/β may be localized in the cytoplasm of resting cells, and similarly to ERK , it may Although it is clear that p38α/β shuttle to the nucleus, either upon or without stimulation, the mechanism that regulates this is not fully understood. Similarly to ERK and JNK, p38α/β do not contain the canonical nuclear localization signals (NLS). In addition, they do not seem to interact with the classical Impα/β , or use In previous studies on the nuclear translocation of ERK, our group designed a myristoylated peptide (EPE peptide) based on the ERK interaction site with Imp7 ,121. WheThe response of cells to stress and other extracellular stimuli leads to the activation of several signaling pathways, including primarily those of p38α/β and JNK. The p38α/β signaling cascade is well-known for its ability to transmit stress signals to various targets within the cells. Thus, it regulates the activity of many transcription factors involved in stress response, and it was shown to also regulate cell cycle, RNA processes, and cytoskeletal elements. Here, we discussed in detail its involvement in the degradation and nuclear translocation of many proteins, which is an effect that may be in both cases either global, by affecting executing enzymes, or individual by acting specifically on degrading/translocating molecules. Regarding protein degradation, the global effects may be mediated by regulating proteasome or ubiquitin ligases, while the phosphorylation of individual substrates may either stabilize them or enhance their degradation. As for nuclear translocation, the global effect is possibly mediated by regulating importins or nuclear pore proteins (NUPs), while the individual effects can be mediated by either reduced or enhanced binding to the translocation machinery or to anchoring proteins. Being such central signaling pathways, the dysregulation of the p38α/β cascade results in pathologies, and indeed, almost all constantly nuclear targets were shown to play a role in the regulation of cancer and inflammation.One of the hallmarks of many stimulations is the rapid and robust nuclear translocation of p38α/β. This translocation is essential for the regulation of targets that are only localized in the nucleus, although it can enhance the phosphorylation of proteins that are phosphorylated in the cytoplasm and translocate to the nucleus upon stimulation . We found that the translocation is mediated by the binding of p38α/β with either Imp7 or Imp9, which further bind individually to Imp3. Then, the complex moves to the NUPs, where Imp3 stays, while Imp7 or Imp9 shuttles the p38α/β into the nucleus. In the nucleus, p38 is freed from the importins by Ran and then, it is able to execute its nuclear functions. The duration of p38α/β residence in the nucleus may vary between cells and conditions, after which the kinases are exported out of the nucleus by their NES-containing substrate, MK2. A few years ago, our group developed the PERY peptide that completely prevents the nuclear translocation of p38α/β and thereby prevents the growth of some cancer cells—particularly DSS-induced colon inflammation and inflammation-induced cancer. Thus, the nuclear translocation of p38α/β can serve as a good target for inflammation and cancer, and inhibitors of this kinase translocation should be further developed for clinical use.


The chemical structure and performances of the NS–NR2 were characterized through a series of experiments, which revealed that NS–NR2 can react with CO2 in water and nanofluid with low viscosity revealed better CO2 capture. The CO2 capture mechanism of NS–NR2 was studied by kinetic models. From the correlation coefficient, the pseudo second order model was found to fit well with the experiment data. The influencing factors were investigated, including temperature, dispersants, and cycling numbers. Results has shown the additional surfactant to greatly promote the CO2 adsorption performance of NS–NR2 because of the better dispersity of nanoparticles. This work proved that NS–NR2 yields low viscosity, high capacity for CO2 capture, and good regenerability in water. NS–NR2 with high CO2 capture will play a role in storing CO2 to enhanced oil recovery in CO2 flooding.To improve CO Moreover, because of the high energy costs involved, cryogenic distillation is not optimally suitable. Solvents and solid sorbents have been reported for CO2 capture, such as basic solvents, supported amine, and ammonium materials, as the primary classes of chemical sorbents  solid sorbents have been investigated for CO2 capture given their high pore volume, large surface, and ease of functionality  was investigated further in terms of temperature, dispersants, and cycling numbers.Therefore, this study aimed to develop a sorbent to avert the limits of aqueous amine solutions and take advantage of tertiary amines for CO7H8), 3–(trimethoxysilyl)−1–propanamine (KH−540), ethanol (C2H5OH), formic acid (HCOOH), formaldehyde (HCHO), hydrochloric acid (HCl), N, N–dimethylformamide (DMF), and sodium hydroxide (NaOH) were purchased from Chengdu Kelong Chemical Reagent Factory . Nano–SiO2 (10–20 nm) was obtained from Aladdin Chemistry Co. . All chemical reagents were analytical–grade. CO2 (g) and N2 (g) were purchased from Jingli Gas Company . Water was double deionized with a Millipore Milli Q system to produce the 18 MM deionized water.Methylbenzene(C2 loaded with primary amines (NS–NH2) was prepared first using 3–Aminopropyltrimethoxysilane (KH540) as modifiers, and then it was used as the matrix material to synthesize branched nanomaterials with a tertiary amine group on its surface (NS–NR2) via methylation of primary amines based on formic acid and formaldehyde. The mechanism is shown in The nano–SiO2, NS–NR2, and the preparation methods of the nanofluid are shown in 2 are displayed in The specific reaction conditions of NS–NH2 was characterized using an electron microscope (ZEISS Libra 200 FE). The carbon and nitrogen contents were detected by elemental analysis using a Var10EL III instrument. The hydrodynamic diameter and proportion of the nanoparticles were received by a BI 200SM wide-angle dynamic light scattering (DLS) instrument. The rheological property of NS–NR2 dispersion was measured with a HAAKE MARS III rheometer at 25°C to assess the viscosity.Fourier transform–Infrared (FT–IR) spectra were acquired by the KBr pellet method using a WQF520 spectrometer. Thermogravimetric analysis (TGA) was conducted on a synchronous comprehensive thermal analyzer (Netzsch Scientific Instruments). The microtopography of NS–NR2 dispersion was introduced into a gas adsorption bottle. The gas adsorption bottle was put in a constant temperature water bath. The gas flow of CO2 was controlled at 1 L/min by a gas flow controller, and CO2 was bubbling into the high concentrated sulfuric acid to adsorb microscale water in CO2 gas in a hermetic wild-mouth bottle. After that, the dry CO2 was flowed through the gas adsorption bottle to reacted with absorbent in the water. The mass change of dispersion was confirmed by an accurate analytical balance (±0.1 mg) until the weight has no change. The amount of adsorption CO2 on nanoparticles could be calculated under a control test (no nanoparticles). As shown in 2 adsorption capacity of NS–NR2 was measured using a gas adsorption system, as shown in 2 desorption experiment was simply carried out by bubbling N2 around 1 L/min instead of injecting CO2 and keeping the system temperature at 25°C to avoid the huge energy depletion.NS–NR2 adsorption into NS–NR2 are often executed using kinetic models including pseudo first order, pseudo second order, and intraparticle diffusion model. The parameter R2 and relative error (ε) were applied to evaluate the reliability of kinetic models in predicting adsorption capacity, as defined in Equation (1). Based on Equation (1), qe, cal is the predicted date acquired from the model analysis, and qe, exp is the experiment date.The mechanism studies of COThe pseudo first order model that introduced by Langergren in 1898 year :where qt is the adsorption capacity at a special time (mmol/g), k is the rate constant of intraparticle diffusion (mmol/g min1/2) and C (mmol/g) is as the thickness of the boundary layer; the intercept is positively correlated with the boundary layer. 3NH2 reacted to –(CH2)3N(CH3)2 on the surface of nano–SiO2.The FT–IR spectrum of the nanoparticles is shown in 2 was shown to be approximately 15 nm. The nanoparticles aggregated slightly because of the particle size being in the nanometer scale  and NS–NR2 (SiO2-C5H10N), respectively. Here, the molar ratio of carbon to nitrogen was adopted to further confirm the grafting of tertiary amines on nano–SiO2.The element contents of raw SiO2 could also be proved by thermos gravimetric analysis (TGA). Based on the TGA curves in 2, NS–NH2 and NS–NR2 at 900°C under the air atmosphere were 96.86, 89.38, and 87.06%, respectively. For raw nano-SiO2, the mass depletion is attributed to the surface dihydroxylation. In terms of the structure of NS–NH2 and NS–NR2, when the temperature up to 900°C, the primary amine and tertiary amine chains were grafted on the nano-SiO2 has decomposed, respectively. Therefore, compared with the TGA curve of raw nano-SiO2, the surface of nano-SiO2 was modified to primary amine. The different weight loss between in the NS–NH2 and NS–NR2 indicated that tertiary amine was synthesized from primary amine successfully.The successful synthesis of NS–NR2 in the presence of water . The second derivative of the VHCl-pH curve was obtained  completely saturated CO2 at 1 L/min CO2 gas velocity in 20 min.Any amine with a pKa value >5 can react with COobtained , with th2 capture is significant influenced by the viscosity of absorbent  at 25°C, as shown in 2 adsorption capacity of 0.1–21.45 mmol/g)  theory on interparticle interaction potential (Ilyas et al., 2 capture. Therefore, adding the surfactant improved CO2 adsorption by enhancing the dispersibility of nanoparticles.CO 56.7 nm . The ter2 adsorption capacity (Ma et al., 2 absorbent was carried out at 25°C. After CO2 adsorption in the nanofluid for 20 min, the nanofluid was shifted to CO2 desorption for regeneration. The CO2 desorption test was executed for another 20 min by bubbling N2 around 1 L/min before the absorbent was used for the next round of adsorption. As illustrated in 2 adsorption of absorbent was set as the 100 % baseline. After five cycles, NS–NR2 adsorbent shown favorable regeneration capacity with a slight decrease of 14.3% in water for NS–NR2 compared to the initial capacity. The results shown regeneration and efficient CO2 adsorption capacity of nanoparticles adsorbent. Considering cycling capacity, NS–NR2 material shows intriguing regeneration ability for CO2 adsorption.It is crucial that an absorbent is reusable and retains an efficient CO2 successfully. The measurements of pKa value and nanofluid viscosity change proved that NS-NR2 can react with CO2 in water and nanofluid has a low viscosity. NS–NR2 shown better CO2 adsorption capacity, and adsorption kinetics revealed the pseudo second order model was found to fit well with the experiment data. The influence of factors such as temperature, dispersants, and cycling numbers on CO2 adsorption was investigated. Results indicated higher temperature to work against CO2 adsorption of NS–NR2. The CO2 adsorption performance of NS–NR2 was greatly promoted because of a better dispersity of nanoparticles with added surfactant. After recycling of absorbent, the NS–NR2 maintained an efficient CO2 capture and shown favorable regeneration capacity. The measurements of NS–NR2 properties on the bases of viscosity, kinetic models, CO2 capture, and regeneration manifests that NS–NR2 exhibits satisfying performance to capture CO2. NS–NR2 with high CO2 capture will play a role in storing CO2 to enhanced oil recovery in CO2 flooding.This work synthesized tertiary amino functionalized nano–SiOAll datasets generated for this study are included in the article/2. QZ wrote the manuscript. DQ and KC came up with ideas for the manuscript. QZ, LT, and DW discussed and analyzed the experiment data. LT and DW revised the manuscript. NL supervised the whole research work.NL, QZ, and LT conceived the idea. QZ, WH, TY, and YC implemented the preparation, characterization, and measurement of NS–NRDQ was employed by the Engineer Technology Research Institute, CNPC Xibu Drilling Engineering Company Limited company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


According to the “Macrophage phagocytosis” part in the Materials and Methods of the manuscripts, N0 stood for the number of colonies formed by phagocytized bacteria by macrophage after 1 h infection, and N1 stood for the number of colonies formed by survival bacteria inside macrophage 1 h after elimination of extracellular bacteria. The rate of phagocytosis was calculated as N0/ (%), and the rate of bacterial killing was calculated as (N0-N1)/N0 (%).2 × 10The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


SMN1). SMN1 is located in a duplicated region on chromosome 5q13 that contains Alu elements and is predisposed to genomic rearrangements. Due to the genomic complexity of the SMN region and genetic heterogeneity, approximately 50% of SMA patients remain without genetic diagnosis that is a prerequisite for genetic treatments. In this work we describe the diagnostic odyssey of one SMA patient in whom routine diagnostics identified only a maternal heterozygous SMN1Δ(7–8) deletion.Spinal muscular atrophy (SMA) is an inherited neuromuscular disease affecting 1 in 8,000 newborns. The majority of patients carry bi‐allelic variants in the survival of motor neuron 1 gene , estimated SMN genes dosage, and mapped genomic rearrangement in the SMN region.We characterized Alu‐mediated deletion encompassing exons 2a‐5 of SMN1 on the paternal allele and a complete deletion of SMN1 on the maternal allele as the cause of SMA in this patient.We identified an Alu‐mediated rearrangements in SMN1 can escape routine diagnostic testing. Parallel analysis of SMN gene dosage, SMN transcripts, and total SMN protein levels in PBMC can identify genomic rearrangements and should be considered in genetically undefined SMA cases. Spinal muscular atrophy (SMA) is devastating inherited neuromuscular disease resulting from variants of SMN1 and deficiency of the survival motor neuron protein (SMN). In this work, we describe the diagnostic odyssey for one SMA patient in whom routine diagnostic procedures identified only a heterozygous, maternally inherited deletion of exons 7 and 8 in SMN1. Using an individual approach we found in this case that SMA was caused by a novel Alu‐mediated deletion. SMA is the most prevalent monogenic cause of death in infancy  is a homologue of SMN1. Genetic investigations have revealed zero to six copies of SMN2 that are located next to SMN1 on 5q13  encode for the same SMN protein. However, expression of the SMN protein from SMN2 is substantially lower than from SMN1 due to a single nucleotide sequence difference at the sixth position of exon 7 in SMN2 . This sequence difference alters splicing and results in the predominant production of an SMN2 transcript that skips exon 7 (SMN2Δ7) and encodes for an unstable and less functional protein. SMN2 is therefore unable to fully compensate the deficit of SMN1. However, SMN expression from SMN2 may be increased in a gene dose‐dependent manner ‐repetitive DNA sequences called Alu elements. The Alu elements are about 280 base pairs long and are formed by two diverged dimers ) that appear to be caused by Alu‐mediated rearrangements  assays, which are the current gold standard of SMA diagnostics  at Genexpress. The coding regions of SMN genes were analyzed using paired‐end sequencing of PCR amplicons on Illumina MiSeq (Illumina) at Alpha Medical.Genomic DNA (gDNA) was isolated from peripheral venous blood in the patient and her parents using standard protocol. The patient’s karyotype was assessed by G‐banding and comparative genomic hybridization (aCGH) was performed using Agilent SurePrint HD 4x44 platform at the Medirex Group, Slovakia. Genes associated with a set of neuromuscular disorders were analyzed using a custom SeqCap EZ kit (Roche) and Illumina sequencing at CMBGT in Brno, Czech Republic. The TruSight One Sequencing Panel (Illumina) was used for analysis of more than 4,800 genes associated with human diseases at Medirex. The presence of the deletion of exons 7 and 8 in 2.4SMN1 and SMN2 cDNAs were PCR amplified using primers SMN575_F . PCR products were analyzed using the agarose gel electrophoresis, and Sanger sequenced using the version 3.1 Dye Terminator cycle sequencing kit (ThermoFisher Scientific) with electrophoresis on an ABI 3500XL Avant Genetic Analyzer (ThermoFisher Scientific).Total RNA and cDNA were isolated and prepared from peripheral blood mononuclear cells (PBMC) using ProtoScript® II Reverse Transcriptase (NEB). Full‐length 2.5The quality and amount of the SMN protein were assessed in lysates of PBMC using Western blot analysis. PBMC were isolated using the Histopaque‐1077 reagent (Sigma‐Aldrich). The cell pellet was resuspended in 50 mM Tris pH 6,8, 50 mM DTT, 2% SDS, and Complete Protease Inhibitor Cocktail (Roche), sonicated using the Covaris S2 Ultrasonicator (Covaris), and denaturated at 100°C for 10 min. The protein content in the supernatant was determined using an infrared spectrometer Direct Detect (Millipore) according to the manufacturer's protocol. Protein lysates equivalent to 22 μg of protein were reduced and denatured at 100°C for 10 min in a sample buffer with 1% beta‐mercaptoethanol before SDS‐PAGE electrophoresis. After protein transfer to the polyvinylidene fluoride (PVDF) membrane, the membrane was blocked by 5% skimmed milk and 0.1% Tween 20 in PBS for 1 hr at room temperature (RT). The SMN protein was visualized by incubation with mouse monoclonal SMN antibody  at 1:5,000 in 5% BSA and 0.1% Tween 20 in PBS over night at 4°C, followed by incubation with goat anti‐mouse HRP (Pierce) at 1:10,000 in 0.1% Tween 20 in PBS for 60 min, and detection was performed by Clarity Western ECL Substrate (Bio‐Rad). The actin protein was visualized by incubation with rabbit Actin antibody  at 1:1,000 in 0.1% BSA and 0.1% Tween 20 in PBS for 1 hr at RT, followed by incubation with goat anti‐rabbit HRP (Pierce) under conditions and using detection as described above. Relative quantification of the SMN protein was performed using GeneTools software . SMN protein levels were normalized to actin; the experiment was performed in three technical replicates. The statistical significance was determined using one‐way ANOVA test.2.6SMN genes  in four overlapping PCR products  analysis of four retrotransposon‐free SMN genomic regions: (a) in intron 1 (I1), (b) in the exon 3–intron 3 junction (E3I3), (c) in the intron 5–exon 6 junction (I5E6), and (d) ~1 kb downstream from exon 8 (+1 kb)  of b) Table  was perf2.8Alu PCR was performed as described previously by Majer et al.    Table , and (b)SMN1  and exon 6 of 33.1SMN1 exons 7 and 8 and two copies of SMN2 exons 7 and 8. Heterozygous deletion of SMN1 exons 7 and 8 was also found in the patient’s mother, who also carried only one copy of SMN2 exons 7 and 8. The father carried two copies of exons 7 and 8 of both SMN genes  were performed but no gross chromosomal abnormalities were detected. The panel sequencing of genes associated with a set of neuromuscular disorders and the TruSight One Sequencing Panel did not reveal any definitive or likely pathogenic variants related to the phenotype. Due to inconclusive results of genetic testing, the patient was referred to the Research Unit for Rare Diseases of the First Faculty of Medicine, Charles University in Prague, and included in the “Undiagnosed Disease Program” that aims to identify genetic diagnosis in cases of rare genetic diseases with negative results of genetic and genomic analyses.Subsequent targeted sequencing of the coding regions of SMN genomic region in this patient whose clinical phenotype was highly suggestive of SMA.Before looking for other genetic causes, the primary focus was to further analyze the 3.2SMN1 transcript we performed reverse transcription polymerase chain reaction (RT‐PCR) and amplified the full‐length SMN cDNAs from PBMC of the patient, both parents and controls. Using the agarose gel electrophoresis, we observed in the patient and her father an abnormal PCR product of ~600 bp that was not present in mother and controls   Figure .3.3To analyze the effect of identified mRNA changes on the protein quality and abundance, we immunodetected and quantified SMN protein in PBMC lysates from the patient, her parents, and a control. In all samples we detected only one immune‐reactive protein at ~40 kDa, corresponding to the predicted molecular weight of full‐length SMN Figure . The pro3.4SMN gene dosage, we performed qPCR analysis of four retrotransposon‐free SMN genomic regions: (a) in intron 1 (I1), (b) in the exon 3–intron 3 junction (E3I3), (c) in the intron 5–exon 6 junction (I5E6), and (d) ~1 kb downstream from exon 8 (+1 kb). Compared with four copies of SMN genes that are normally present in each of four tested loci in controls, we found that the patient has three copies at the I1, I5E6, and +1 kb loci and two copies at the E3I3 loci. The mother has three copies through the I1 to I5E6 loci and two copies at the +1 kb loci. The father has three copies only at the I5E6 loci _(70249850_?)del), where the genomic coordinates denote the central position of the qPCR probes targeting the I1 and +1 kb genomic loci, and a deletion encompassing Δ2a‐5 exons of SMN1 on the paternal allele.To assess i Figure . Togethei Figure , this an3.5SMN1 exons 2a‐5 in the patient's and father's cDNA, we PCR amplified the genomic DNA and Illumina sequenced four overlapping long‐range amplicons (PCR1–PCR4) spanning exons 1–8 of SMN1 and SMN2. Using specific single nucleotide sequence differences that distinguish the SMN genes, we found that in the patient amplicon PCR4, spanning intron 6, exon 7, intron 7, and exon 8  AluSq sequence of intron 5 and the sequence originating from a new chimeric Alu resulting from the rearrangement with AluSp element originating from the intron 1  SMN content in PBMC that must be expressed exclusively from SMN2  SMN content in PBMC compared with the father (41% of controls), who has only the ~9 kbp deletion on one of the SMN1 alleles and both SMN2 alleles intact.In summary, the genetic analysis Figure  establis2 Figure ,c. In ad4SMN1 and deficiency of the survival motor neuron protein (SMN). Several clinical or experimental therapies for SMA augmenting levels of SMN are currently available or in development  of SMN1 on the paternal allele and by deletion of whole SMN1 on the maternal allele.In this work, we describe the diagnostic odyssey for one SMA patient in whom routine diagnostic procedures identified only a heterozygous, maternally inherited deletion of exons 7 and 8 in SMN1 escaped detection by the routine MLPA assay that targets only exons 7 and 8. To the best of our knowledge, this is only the second reported Alu‐mediated deletion in SMN1 that does not encompass the exons 7 and 8  variant of Alu‐mediated rearrangements in the SMN region that escape detection with routine genetic testing may be more common and should be considered in SMA cases who remain without a genetic diagnosis after standard genetic testing.Our work suggests that other SMN gene dosage, full characterization of SMN transcripts, and precise characterization of the eventual genomic rearrangements. All these parameters are critical for targeted genotyping and eventually for prenatal or preconception analysis in affected families and therapeutic eligibility for affected individuals. We are interested to study other similar cases and can provide the genetic testing and biochemical analyses described here. Please contact ivana.jedlickova@lf1.cuni.cz.We demonstrate that in these cases measurement of SMA protein in PBMC may successfully identify patients with SMA in whom a genetic diagnosis cannot be made. These individuals can then undergo further testing, including All authors have no conflict of interest to declare.IJ, AP, LN, FM, GM, MG, and SK were involved in writing of the manuscript and revised the manuscript. IJ, LN, FM, HH, KHo, HT, KHý, PS, and MG performed the laboratory experiments and interpreted the data. AP, VS, and GM performed bioinformatic studies. MG ascertained the patient and provided the clinical evaluation of the patient. IJ, MG, and SK coordinated the study.Fig S1‐S2Click here for additional data file.


C‐phosphonates (M6Po). The M6Po's are stable M6P mimics that are resistant to cleavage of the phosphate group by endogenous phosphatases. Two different strategies for the incorporation of the M6Po clusters in the conjugate have been developed: the first relies on a “post‐assembly” click approach employing an M6Po bearing an alkyne functionality; the second hinges on an M6Po C‐glycoside amino acid building block that can be used in solid‐phase peptide synthesis. The generated conjugates were further equipped with a TLR7 ligand to stimulate dendritic cell (DC) maturation. While antigen presentation is hindered by the presence of the M6Po clusters, the incorporation of the M6Po clusters leads to increased activation of DCs, thus demonstrating their potential in improving vaccine adjuvanticity by intraendosomally active TLR ligands.Mannose‐6‐phosphate (M6P) is recognized by the mannose‐6‐phosphate receptor and plays an important role in the transport of cargo to the endosomes, making it an attractive tool to improve endosomal trafficking of vaccines. We describe herein the assembly of peptide antigen conjugates carrying clusters of mannose‐6‐ Enhancing the immune response: Two mannose‐6‐C‐phosphonate (M6Po) building blocks have been synthesized that can be incorporated into a conjugate by a “post‐assembly” click or an online SPPS approach to improve endosomal trafficking of vaccines. The generated M6Po conjugates, which were also equipped with a TLR7 ligand, were able to enhance the immune response by activation and maturation of dendritic cells. The mannose‐6‐phosphate receptor (MPR), a P‐type lectin, plays an important role in this transportation through binding to M6P.[N‐hexanoyl‐d‐erythro‐sphingosine with M6P‐functionalized liposomes.[Carbohydrates play an important role in many biological processes, such as cell‐cell communication, pathogen recognition and protein folding. Mannose‐6‐phosphate (M6P), a g to M6P.1, 2 TC‐phosphonate ester.[C‐phosphonate proved to be a stable and effective replacement for the phosphate monoester.The activity of peptide‐based anti‐cancer vaccines may be enhanced by the targeted delivery of these antigens to antigen presenting cells. In this context, mannosylated antigens, destined for the mannose receptor or DC‐SIGN present on dendritic cells (DCs), have been widely explored.15, 161–8, Figure 9 and 10, respectively, are used to construct M6Po‐clusters and conjugate them to either the N‐ or the C‐terminus of a synthetic long peptide (SLP). In this study, two different ovalbumin derived SLPs are used: DEVA5K (DEVSGLEQLESIINFEKLAAAAAK), which harbours the MHC‐I presented epitope SIINFEKL, and HAAHA (ISQAVHAAHAEINEAGRK), which contains an MHC‐II epitope. Our goal is to enhance the immune response by increasing endosomal trafficking of the peptide vaccine via the MPR which led to the design of bis‐conjugates  in which the Toll‐like receptor 7 ligand (TLR7L) 2‐alkoxy‐8‐oxo‐adenine is added at either the N‐ or the C‐terminus of the M6Po‐SLP.[O‐M6Po building block 9 to allow for the copper mediated 1,3‐dipolar cycloaddition to azide functions incorporated in the SLP, while C‐M6Po building block 10 was used to generate the HAAHA‐conjugates via solid‐phase peptide synthesis (SPPS). This building block is a C‐analogueO‐M6Po, in which the anomeric oxygen is replaced with a CH2, preventing hydrolysis under the acidic conditions used in SPPS. An additional advantage of this SPPS‐compatible building block is the possibility to prepare conjugates of peptides that are not suitable for copper mediated 1,3‐dipolar cycloaddition. It also allows one to incorporate azide or alkyne click handles in conjugate vaccine constructs that can be exploited for labelling or visualization purposes.We here describe the incorporation of a cluster of mannose‐6‐phosphonates (M6Po) in two types of peptide antigen‐conjugates  building block (9) to azide containing peptides. Synthesis of the required building block 9 started from propargyl‐d‐mannose 11 , we placed an isopropylidene group over the 2,3‐cis‐diol system to prevent this intramolecular side reaction.[11 and subsequent installation of the isopropylidene gave 13, of which the remaining alcohol was masked as a p‐methoxybenzyl ether to give the fully protected mannose 14. The alkyne in 14 was protected with a TMS group using TMSCl and nBuLi at −78 °C. Removal of the trityl in the thus obtained 15 with a catalytic amount of p‐toluenesulfonic acid in CH2Cl2/MeOH was accompanied by partial removal of the isopropylidene ketal. Reinstallation of the isopropylidene and subsequent deprotection of the mixed ketal simultaneously formed on the primary alcohol gave 16 in 98 % over three steps. Alcohol 16 was treated with Tf2O and pyridine at −40 °CnBuLi in THF at −70 °C, yielding compound 17 in 72 % over two steps. Removal of the TMS protecting group gave 18, which was transformed into key building block 9 by a two‐step deprotection sequence. The deprotection of the phosphonate using TMSBr was followed by the removal of the p‐methoxybenzyl and isopropylidene groups by treatment with AcOH/H2O at 90 °C to deliver key building block 9 in 27 % yield over 15 steps starting from d‐mannose.The first type of reaction.24, 2510 6‐SIINFEKL conjugates was undertaken. Immobilized peptides 25 and 28 were prepared through standard SPPS HCTU/Fmoc chemistry using Tentagel S Ram as solid support  treatment removed all protecting groups and cleaved the peptides from the resin to give peptides 26 and 29 in 1 and 6 % yield, respectively, after purification. Alternatively, the MMT protecting group at the C‐terminal lysine of 25 was selectively deprotected using a cocktail of TFA/TIS/CH2Cl2 . The released amine was subsequently coupled with the spacer 33 and the Boc‐protected TLR7 ligand building block 34.27 and 30 were obtained in a 2 % yield. Coupling of O‐M6Po building block 9 to peptides 26, 27, 29 and 30 was performed using a cocktail of CuSO4, sodium ascorbate and tris(benzyltriazolylmethyl)amine in DMSO/H2O,1–4 were obtained in 5 % (0.3 mg), 18 % (0.9 mg), 18 % (1.0 mg) and 31 % (3.3 mg) yield respectively.Next, the assembly of the  at the C terminus will be used for elongation at a later stage of the synthesis. Six consecutive coupling and Fmoc removal cycles with building block 10 gave immobilized peptide 31. Peptide 32, bearing the C‐M6Po cluster at the C‐terminal end, was generated by assembling the hexa‐C‐M6Po peptide through manual couplings of building block 10, followed by automated SPPS to assemble the rest of the peptide. Immobilized and protected peptides 31 and 32 were deprotected and simultaneously cleaved from the resin with the TFA/TIS cocktail to provide conjugates 5 (13.3 mg) and 7 (3.1 mg) after purification by RP‐HPLC in 10 and 8 % yield, respectively, demonstrating the suitability of 10 for SPPS. To obtain conjugate 6, bearing the TLR7 ligand, the MMT group in 31 was selectively removed with a cocktail of AcOH/TFE/CH2Cl2 . The obtained free amine was elongated with spacer 33 and Boc‐protected TLR7 ligand building block 34 to give conjugate 6 (11.0 mg) in 2 % yield, after removal of all the protecting groups, cleavage from the resin and RP‐HPLC purification. The same sequence of events was applied to the N‐terminal amine in immobilized peptide 32 to afford conjugate 8 (17.0 mg) in 8 % yield.The HAAHA peptide contains two histidines, which can coordinate to copper and thereby inhibit the reduction of Cus Scheme B using C1–8 to induce maturation of dendritic cells (DCs) and stimulate antigen presentation . The activation of DCs can be measured by the detection of the production of interleukin‐12 (IL‐12).We assessed the capacity of the conjugates n Figure . In thesO‐M6Po conjugates 1–4 were evaluated  does not induce DC maturation. In contrast, the TLR7 ligand SLP conjugates (36 and 37) induce IL12 production due to stimulation of the TLR7 receptor.First, the d Figure A. To thi2 and 4) induced a stronger activation of the DCs than their counterparts solely bearing a TLR7 ligand . The position of the M6Po‐clusters and TLR7 ligand in these conjugates did not seem to influence the activity of the conjugates. Similar effects were observed for the conjugates 6 and 8 with the C‐M6Po‐clusters, indicating that the C‐mannosyl‐6‐C‐phosphonate is an adequate mimic of its O‐mannosyl counterpart and that the enhanced stimulatory effect is independent of the peptide sequence  to CD4+ T cells. For the former assay, the hybridoma (B3Z) CD8+ T cell line was used, while the latter employed the OTIIZ hybridoma CD4+ T cell line. As can be seen in Figure 35 vs. 36/37 and 38 vs. 40), however the inclusion of the M6Po‐clusters hampered presentation through both the MHC class I and MHC class II pathways . This indicates that the conjugation of M6Po‐clusters affects intracellular trafficking or processing of the peptides.Processing of the two peptides was investigated by assessing presentation of the SIINFEKL epitope on MHC‐I the DCs to CD8C‐phosphonate (M6Po) building blocks which allow for stabilized M6P‐analogues to be incorporated in peptide sequences to target the M6P receptor. To prevent dephosphorylation by endogenous phosphatases, the O‐phosphate in the naturally occurring mannose‐6‐phosphate was replaced by a C‐phosphonate moiety. The first building block carries an O‐propargyl group at the anomeric center of the mannose‐6‐C‐phosphonate, which allows for the incorporation of the M6Po in peptide sequences through an azide‐alkyne click reaction. The second building block, a C‐mannoside, was designed and synthesized for application in solid‐phase peptide synthesis. The acid‐stable anomeric linkage and protecting groups used enabled the streamlined in‐line incorporation of the building block during SPPS. With the building blocks, various peptide conjugates were assembled, containing either an MHC‐I or an MHC‐II epitope, an M6Po‐cluster presenting six mannose‐6‐phosphonates, and a TLR7 ligand. Although immunological evaluation has shown that conjugation of the M6Po‐clusters inhibits antigen presentation, the ability of the TLR7 ligand conjugates to induce DC maturation was significantly improved. While the M6Po clusters effectively trafficked the conjugates to the endosome, where the conjugates interacted with TLR7 receptor, the processing of the conjugates was impeded by the M6Po‐clusters. Future conjugates will be designed featuring cleavable linkers that allow for the release of the clusters during endolysosomal processing, effectively liberating the incorporated peptide antigens for presentation.In conclusion, we have described the development of two mannose‐6‐The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


N‐alkyl‐(2‐pyridinemethanimine) ligands and new complexes: [Fe(L1)2](ClO4)2 (1); [Fe(L2)2](ClO4)2 (2); [Co2(L3)2](ClO4)4 (3); [Fe2(L3)2](ClO4)4 (4), which were crystallised and their structure confirmed by single‐crystal X‐ray diffraction determination, as well as a range of new supramolecular clusters discovered in solution using high‐resolution mass spectrometry.We present a chemical discovery robot for the efficient and reliable discovery of supramolecular architectures through the exploration of a huge reaction space exceeding ten billion combinations. The system was designed to search for areas of reactivity found through autonomous selection of the reagent types, amounts, and reaction conditions aiming for combinations that are reactive. The process consists of two parts where reagents are mixed together, choosing from one type of aldehyde, one amine and one azide  with different volumes, ratios, reaction times, and temperatures, whereby the reagents are passed through a copper coil reactor. Next, either cobalt or iron is added, again from a large number of possible quantities. The reactivity was determined by evaluating differences in pH, UV‐Vis, and mass spectra before and after the search was started. The algorithm was focused on the exploration of interesting regions, as defined by the outputs from the sensors, and this led to the discovery of a range of 1‐benzyl‐‐ A chemical robot was designed, assembled and utilised for the discovery of supramolecular architectures through the exploration of a chemical space exceeding 109 possible reactions. By searching for reactivity differences, a range of new 1‐benzyl‐‐N‐alkyl‐(2‐pyridinemethanimine) ligands were found and four new complexes of Fe and Co were discovered, autonomously discovering the rules of self‐assembly for these systems. Exploring supramolecular chemical space for new assemblies is difficult not only because it is vast and sparse, but because we are looking for novel outcomes not easily predictable from a given set of input reagents and conditions. In addition, most reactions are done with specific possible outcomes in mind or rely on established heuristics. This means that the discovery of new and unpredicted coordination complexes is challenging and often occurs serendipitously.Not only are the reaction operations automated,The autonomous decision makingII(ClO4)2] or [CoII(ClO4)2]. Including the second coordination step, the number of possible experiments in the chemical system increases to 4×1014. The cleaning, reaction activation, and decision‐making operations are all fully automated, allowing the continuous operation of the system without human involvement. The system is controlled using bespoke code written in Python such that the chemical robot can perform all the liquid‐handling operations, including cleaning procedures between one experiment and the next, as well as control of analytical instruments and data analysis. The design and the connectivity of the system is shown in Figure The reactor for the synthesis step consists of a catalytically active 10 mL capacity copper coil,The routing of chemicals and solutions through the system is conducted in a non‐deterministic fashion .The calculation of α is based on the ratio of differences between the analytical measurements of the ligand with its starting materials; and between the ligand and its resulting complex. Thus, this value constrains the exploration range for the next randomly chosen experiment. The calculation of the differences for each analytical measurement is detailed in the Supporting Information. In general, the differences are all based on the comparison of the two data sets—the ligand mixture and starting materials; or complex combination and ligand mixture. Specifically, MS changes are calculated using the most intense and highest N‐alkyl‐(2‐pyridinemethanimine) ligands which were applied in the discovery of new complexes: [Fe(L1)2](ClO4)2 (1); [Fe(L2)2](ClO4)2 (2); [Co2(L3)2](ClO4)4 (3); [Fe2(L3)2](ClO4)4 (4). Exact details for the conditions used in the syntheses of any isolated compounds can be found in the Supporting Information. Complex 1 is formed through tridentate coordination of each ligand to FeII, with one N‐donor atom from each of the pyridine, imine, and triazole groups. Despite L1 being more flexible than L2 , the favored coordination mode is still tridentate with all nitrogen containing moieties bound. In the UV‐Vis spectrum of the complex mixture , three new absorbance bands are detected at 356, 476, and 544 nm. This evidence lets us conclude that the intermediate [Cu(L1)2]2+ formed in the flow reactor undergoes a metal‐exchange process in the presence of the FeII salt to give [Fe(L1)2](ClO4)2. Also, the pH values and the ESI‐MS spectra of the two analysed mixtures are in accordance with this observation. In fact, a significant change in the acidity of the new complex from 6.29 to 4.34 is detected. The ESI‐MS shows the presence of peaks with m/z of 390.23 in the ligand mixture and m/z of 387.25 in the complex mixture. These could be assigned to [Cu(L1)2]2+ and 1 respectively.Using this system we have been able to autonomously synthesise a range of 1‐benzyl‐‐2)2](ClO4)2 (2) was discovered from an alternate set of input reagents yielding the general ligand framework 1‐benzyl‐‐N‐alkyl‐(2‐pyridinemethanimine). Solid‐state structures of these complexes are shown in Figure 2(L3)2](ClO4)4 (3) and [Fe2(L3)2](ClO4)4 (4) forming an M2L2 complex using a ligand based on a structure 3 type motif.Using this same methodology [Fe(L2)2]ClO2 (2) was4 through crystallisation from the reaction solution but the ESI‐MS measurements of the reaction mixture reveals more species to be present in solution  or a cage‐like arrangement (E). However, other peaks have m/z values that can be associated with the mass of specific cages, such as circular triangular (C) or square (D) arrangements  must change to a bidentate motif. This makes it possible that three bidentate motifs are coordinated to one metal center to form the proposed structures illustrated in Figure II under one reaction condition.All the assumed adducts are also summarised in Figure II‐ and FeII‐ coordination architectures. The screening of a huge chemical space with the described ligand system resulted in four new coordination structures, which could be isolated, and their molecular structures could be ascertained by X‐ray diffraction.In summary we have achieved the exploration of a vast range of potential experiments without any heuristics or other chemical knowledge. Using this approach, the system actively searches through the available space based on the information obtained about the system as it is running. The ligand system targeted has not been previously reported in the literature, although it is simple and can lead to many outcomes. Furthermore, to the best of our knowledge, no coordination architectures are known to be self‐assembled through the presented coordination moiety. Our studies show the complexity of this ligand system in the outcome of several Co1529980, 1529981, 1529982 and 1529983 (1–4) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre. Code is also available from the Croninlab git pages: https://github.com/croningp/InorganicFinder.A volume explaining the platform, software, search data, analytical data and crystallographic data is available. CCDC The work in this publication has been filed as a patent by the University of Glasgow.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Medicago sativa L.) is an important forage legume in farming and animal husbandry systems. This study assessed the effects of arbuscular mycorrhizal fungi (AMF) and phosphate-solubilizing bacteria (PSB) on alfalfa growth under different phosphorus application levels. In this experiment, a complete randomized block design was used. The following four bacterial applications were used: inoculation of Funneliformis mosseae (Fm), inoculation of Bacillus megaterium (Bm), inoculation of mixed species (Fm × Bm) and noninoculation treatment (CK). Phosphorus (P) treatment was applied at the following four levels: 0 mg kg−1 (P0), 50 mg kg−1 (P1), 100 mg kg−1 (P2) and 150 mg P kg−1 (P3). The results showed that with the increase in phosphorus application, each index increased first and then decreased. The J2 treatment was significantly greater than the J0 treatment (P < 0.05) under the same bacterial treatment. In each cropping period the difference in each index to alfalfa was extremely significant under J, P treatment and J × P interactive treatment (P < 0.01). The indexes were compared by membership function. The priority order was as follows: J3P2 > J1P2 > J3P1 treatment. Therefore, when phosphorus was applied at 100 mg kg−1, the mixed inoculation of Fm × Bm was optimal, benefitting mycorrhiza growth and the production performance of alfalfa.Alfalfa ( Medicago sativa L.), as a perennial legume forage, is a high-quality protein feed for livestock, and it is an essential cultivated forage in the arid and semiarid areas of western China  are widely distributed and are one of the microorganisms most closely related to plants in the soil. AMF plays a critical role in the uptake of plant nutrients in the natural environment, and they promote the absorption of phosphorus nutrients and enhance the resistance of plants to biological and abiotic stresses  are important microorganisms in the soil for phosphorus uptake by plants. PSB secrete organic acids and enzymes, and they convert organic phosphorus and insoluble phosphorus into useful phosphorus that can be absorbed and utilized by plants, which improves the utilization efficiency of phosphorus in the soil, thereby promoting the growth of crops  was selected. The inoculant was rhizosphere soil, including host plant root, mycorrhizal fungal spore and ectomycorrhizal mycelium. The density of the spore was 25–35 g, which was provided by the Qingdao Agricultural Mycorrhizae Research Institute of China.For the phosphate-solubilizing bacteria, Bacillus megaterium (Bm) were purchased from the Agricultural Culture Collection of China . The host plant alfalfa variety tested was WL354HQ.Funneliformis mosseae (Fm), single inoculation of Bacillus megaterium (Bm), inoculation of mixed strains (Fm × Bm) and noninoculated bacteria (CK), which were labeled as J1, J2, J3, and J0, respectively. In the single inoculation treatment group, 10 g of bacteria was used in each pot. In the mixed inoculation treatment, 5 g of each bacterial strain was applied in each pot . The noninoculated (J0) treatment strains were subjected to sterilization. The noninoculated (J0) treatment strains were subjected to sterilization. The following four levels of phosphorus treatment were used, and each treatment was repeated 6 times: 0 mg kg−1 (P0), 50 mg kg−1 (P1), 100 mg kg−1 (P2) and 150 mg kg−1 (P3).The experiment adopted a two-factor random block design with two factors of bacterial application and phosphorus application. The following four bacterial treatments were used: single inoculation of Funneliformis mosseae (Fm) 5 cm below the soil surface in the pot. Full and uniformly sized alfalfa seeds were selected and disinfected with 10% H2O2 for 10 min, repeatedly washed with distilled water and seeded on May 1, 2019 with ten seeds per pot. Equivalent water supply was provided for every pot after sowing, and seedlings were interplanted when seedlings grew to the three-leaf stage. Each cup retained five alfalfa seedlings with the same growth trend. Each treatment was replicated six times, and the pots were randomly placed. The phosphate fertilizer used was pandemonium phosphate (containing 52% of P and 11% of N). Because pandemonium phosphate contains N, urea (containing 46% of N) was added to maintain the same N content in each treatment. All fertilizers were implemented in two batches on June 18, 2019 and September 19, 2019, and the method of fertilization was to supply by water drops. Alfalfa was harvested in two crops throughout the year at the initial flowering stage (flowering 5–10%), and the remaining stubble height was 2 cm. The specific cutting time was August 2, 2019 and October 12, 2019.The experiment was performed at the Shihezi University Experimental Base  from May to October 2019. The pot experiment was conducted with a nutrient bowl with a top diameter of 23 cm, a bottom diameter of 15 cm and a height of 16 cm. The test soil was derived from the test field of Shihezi University . The soil was sterilized in a high-pressure steam sterilizer at 121 °C for 2 h and then air-dried for use, and each pot was filled with 3 kg to destroy the fungus in the dried soil. To promote root colonization of alfalfa, we inoculated −1) and underground biomass (g pot−1) using the following formulas:−1); FY is fresh yield of alfalfa (g pot−1); MC is the moisture content; U is underground biomass of alfalfa (g pot−1); and FR is fresh root of alfalfa (g pot−1).Each pot was considered as a unit, and three pots of alfalfa plants with consistent growth were selected. The alfalfa plants in the sample plot (cut to 2 cm) were cut with scissors and weighed, and the yield of fresh alfalfa forage was recorded three times for every treatment. A soil shaking method was used to remove the underground part of the alfalfa plant. The root system was collected, rinsed and weighed, and the fresh weight was recorded. The samples were first oven-dried at 105 °C for 30 min and then at 65 °C to a constant mass. The moisture content was measured and converted into alfalfa aboveground biomass  of the absolute height was reported. A Vernier caliper with an accuracy of 0.02 mm was used to measure the alfalfa at 5 cm from the ground stem diameter (mm).The crude protein content (CP) was determined using the Kjeldahl method  was obtained.After the aboveground biomass of alfalfa was measured, fresh leaves of alfalfa plants were harvested, and chlorophyll content was determined by the acetone method  and DPS 8.0 software  were used for statistical analyses. Two-way ANOVA was used to analyze the interaction of J, P and J × P, and Duncan’s method was used for multiple comparisons. Data were plotted with Origin 8.0 software  and expressed as the mean ± standard error.Pearson’s correlation coefficient measures the degree of correlation between two variables, and it is a value between 1 and − 1 as follows: 1 detects an entirely positive relationship of variables; 0 indicates no correlation; and − 1 indicates an entirely negative correlation. The correlation among mycorrhizae infection rate, growth traits and soluble sugar content of alfalfa was analyzed by Pearson’s correlation analysis.The subordinate function evaluation method was used to comprehensively evaluate the optimal treatment using the following formulas:1 treatment, and the P1 treatment had a significantly higher infection rate than that under the P0 and P2 treatments (P < 0.05). The second crop reached the highest infection rate under the P2 treatment, except for the J0 treatment, and the P2 treatment had a significantly higher infection rate than that under the P0, P1 and P3 treatments (P < 0.05). Phosphorus application treatment resulted in a higher infection rate than without phosphorus application under the J2 treatment, and the P1 treatment resulted in a higher infection rate than the P2 and P3 treatments (P < 0.05). Both single inoculation (J1 or J2) and mixed inoculation (J3) treatments had significantly higher infection rates than those without vaccination (J0) (P < 0.05) under the same phosphorus application treatment.The mycorrhizae infection rate of alfalfa increased first and then decreased with the increase of phosphorus application rate under the same treatment conditions Fig. . The firThe degree of alfalfa mycorrhizae infection varied under different treatments Fig. . Compare2 treatment than in P0 treatment, except that the aboveground biomass of the first crop reached the highest value under the P1 treatment (P < 0.05). Compared to the noninoculated treatment, all treatments, except the P1 treatment under the J3 condition , reached the maximum value under the P2 treatment under the J3 condition . The aboveground biomass, plant height and stem diameter of alfalfa under different inoculation treatments were greater in the first crop than in the second crop. The aboveground biomass, plant height and stem diameter of alfalfa under the J, P and J × P treatments were significantly different in each cultivation (P < 0.01).The aboveground biomass, plant height and stem diameter of alfalfa increased first and then decreased with the increase of phosphorus application Table . The aer2 treatment with the J0 condition, except for the second crop P3 treatment, and the P2 treatment was significantly greater than the P0 treatment (P < 0.05). The difference in CP content between the P1 treatment and the P2 treatment was not significant (P > 0.05). Under the J1 and J2 conditions, the P2 treatment resulted in significantly higher CP content than that of the P0, P1 and P3 treatments (P < 0.05). The CP content reached the highest value under the P2 treatment with the J3 condition, except for the second crop P1 treatment, and there was no significant difference between the P2 and P3 treatments (P > 0.05). The CP content after treatment with J2 in the first crop was markedly higher than that of alfalfa treated with J0 and J3 under the same phosphorus application conditions (P < 0.05). The CP content of alfalfa treated with J2 in the second crop was considerably higher than that after treatment with J0, J2 and J3 (P < 0.05). Under the P1 condition, the CP content of alfalfa treated with J2 in the first crop was significantly greater than that after treatment with J0 (P < 0.05). The CP content of alfalfa treated with J3 in the second crop was substantially higher than that of alfalfa treated with J0 and J2 (P < 0.05). Under the conditions of P0 and P2, the CP contents of alfalfa treated with J1 and J3 were significantly greater than that of alfalfa treated with J0 (P < 0.05), and there was no significant difference in CP contents among the J1, J2 and J3 treatments (P > 0.05). The alfalfa CP content after the J1 treatment was significantly greater than that after the J0 treatment under the P3 condition (P < 0.05), and there was no significant difference between the J2 and J3 treatments (P > 0.05).The CP content of alfalfa increased first and then decreased with the increase of phosphorus application under the same bacterial application conditions Fig. . The CP 3 treatment (P < 0.05). The NDF content after treatment with P1 was significantly greater than that after treatment with P0 with the J0 and J3 conditions (P < 0.05), and the NDF content after treatment with P2 was the lowest with the J2 condition. The NDF and ADF contents of alfalfa after all treatments were lower than that of the J0 treatment under the constant phosphorus application condition, except for the NDF content after the J3 treatment in the first crop, which was markedly greater than that of J0 treatment under the constant phosphorus application condition (P < 0.05). The NDF and ADF contents in alfalfa were higher in the first crop than in the second crop under different inoculation treatment conditions. The NDF and ADF contents of alfalfa were significantly different under the J, P and J × P treatments in each plant (P < 0.01).The NDF and ADF of alfalfa first increased and then decreased with increasing phosphorus application under the same bacterial application Table . The NDF2 and J3 conditions (Table 1 treatment than after the other treatments under the J0 condition (P < 0.05). In the second crop, the chlorophyll content and soluble sugar content reached the highest values under the P2 treatment. The chlorophyll content after the P2 treatment was significantly greater than that after the other treatments under the J1 condition (P < 0.05). The soluble sugar content in the first crop was significantly higher in the P3 treatment that after the other treatments (P < 0.05), and the soluble sugar content in the second crop was higher after the P1 treatment than after the P0 treatment with the J1 condition (P < 0.05). Both indexes of the first crop were significantly higher after the P2 treatment compared to the other treatments with the J2 condition (P < 0.05), and the chlorophyll content of the second crop was higher after P1 treatment than after the other treatments (P < 0.05). The chlorophyll content in the first crop was higher after the P1 treatment than after the other treatments (P < 0.05), and the chlorophyll content in the second crop and the soluble sugar content in the first crop were significantly higher after the P2 treatment than after the other treatments with the J3 condition (P < 0.05). The soluble sugar content in the second crop was considerably higher after the P3 treatment than after the P0 treatment with the J3 condition (P < 0.05). The inoculation treatment was markedly greater than the noninoculation treatment under the same phosphorus application conditions (P < 0.05). In each crop, the chlorophyll content and soluble sugar content were significantly different under the J, P and J × P treatments (P < 0.01).The chlorophyll content and leaf soluble sugar content of alfalfa first increased and then decreased with increasing phosphorus application under the same phosphorus application, except for the second crop under the JP < 0.01), and the aboveground biomass was positively correlated with mycorrhizae infection rate, stem diameter and chlorophyll content (P < 0.05) (Table P < 0.01). The mycorrhizae infection rate was positively correlated with crude protein content (P < 0.05). Alfalfa plant height was positively correlated with stem diameter and crude protein content (P < 0.05). Stem diameter was positively correlated with crude protein content, NDF and ADF (P < 0.05). The crude protein content was positively correlated with chlorophyll content and soluble sugar content (P < 0.05). There was a significant positive correlation between NDF and ADF contents (P < 0.01).The aboveground biomass of alfalfa was positively correlated with plant height and crude protein content (5) Table . The myc3P2 > J1P2 > J3P1.Because each treatment had different effects on different indicators, we could not comprehensively assess the optimal inoculation treatment with any single indicator. Thus, we used the following nine indicators of alfalfa for a comprehensive evaluation: aboveground biomass, infection rate, plant height, stem diameter, crude protein, NDF, ADF, chlorophyll content and soluble sugar content. We comprehensively evaluated the growth characteristics, nutritional quality, chlorophyll content and soluble sugar content of alfalfa under different inoculation treatments Table . The resArbuscular mycorrhizal fungi (AMF) are ubiquitous plant-fungal symbionts in nature  decreases the activity of enzymes in organic acid metabolism and the secretion of organic acids, thus reducing the activation and diffusion of phosphorus (Wang et al. The essential nutrient element for plant growth is phosphorus, which plays a critical role in plant metabolism and is also one of the leading nutrient elements affecting the improvement of crop yield. Phosphorus participates in various metabolic processes in plants in a variety of ways, thereby affecting plant physiology and morphology (Shen et al. The important indexes for evaluating the nutritional quality of alfalfa include the CP, ADF and NDF contents (Larson and Mayland The chlorophyll content and leaf soluble sugar content can be used as essential indicators of nutrients for efficient growth of alfalfa (Elfanssi et al. −1 phosphorus application and coinoculation of Fm and Bm, which promoted the growth of mycorrhiza, promoted the growth of alfalfa shoots and improved the nutritional quality. Further investigation on the interaction between AMF and PSB will provide a theoretical basis for practical production and application.In conclusion, the present study showed that the appropriate phosphorus application and inoculation with AMF and PSB significantly increased the mycorrhizae infection rate, aboveground biomass, chlorophyll content in leaves and soluble sugar content of alfalfa. In contrast, excessive phosphorus application inhibited the increase in the above indicators. The CP, NDF and ADF contents of alfalfa showed a tendency to increase first and then decrease with increasing phosphorus application under the same bacterial treatments. Considering all factors, the best combination treatment was 100 mg kg


Halobacterium strain 63‐R2 was recently reported and provides the opportunity to resolve long‐standing issues regarding the source of two widely used model strains of Halobacterium salinarum, NRC‐1 and R1. Strain 63‐R2 was isolated in 1934 from a salted buffalo hide (epithet “cutirubra”), along with another strain from a salted cow hide . Both strains belong to the same species according to genome‐based taxonomy analysis (TYGS), with chromosome sequences showing 99.64% identity over 1.85 Mb. The chromosome of strain 63‐R2 is 99.99% identical to the two laboratory strains NRC‐1 and R1, with only five indels, excluding the mobilome. The two reported plasmids of strain 63‐R2 share their architecture with plasmids of strain R1 . We detected and assembled additional plasmids using PacBio reads deposited at the SRA database, further corroborating that strain differences are minimal. One plasmid, pHcu190  corresponds to pHS1 (strain R1) but is even more similar in architecture to pNRC100 (strain NRC‐1). Another plasmid, pHcu229, assembled partially and completed in silico , shares most of its architecture with pHS2 (strain R1). In deviating regions, it corresponds to pNRC200 (strain NRC‐1). Further architectural differences between the laboratory strain plasmids are not unique, but are present in strain 63‐R2, which contains characteristics from both of them. Based on these observations, it is proposed that the early twentieth‐century isolate 63‐R2 is the immediate ancestor of the twin laboratory strains NRC‐1 and R1.The genome of  T and 63‐R2) were isolated in 1934 by Lochhead from cow and buffalo hides. From the results of these comparisons, we conclude that strain 63‐R2 is the immediate ancestor of the two, widely used laboratory strains NRC‐1 and R1.The complete genomes of four Halobacterium salinarum strains were compared in detail. Two strains (91‐R6 This was the original isolate and designated the type strain of Halobacterium salinarum (according to the currently approved taxonomy). Later, this strain was lost.The use of salt in the preservation of food (curing) and the tanning of leather are traditional processes dating back hundreds of years. In 1922, searching for the cause of “red discolorations” on salted codfish, which was seen as a threat to the Canadian fishery industry, Harrison and Kennedy isolated a red pigmented microorganism  of Hbt. salinarum. The other Lochhead isolate (63‐R2), obtained from a buffalo hide, was given the species epithet “cutirubra.” It was considered a distinct species by Lochhead, but this was later revised  of salted hides, which were causing losses for Canadian leather manufacturers   according to the international code of nomenclature of prokaryotes  were only rarely used for experimental analyses, the twin pair of laboratory strains  was extensively studied. Both were assumed to be derived from Hbt. salinarum  have now been determined, allowing detailed comparison and analysis. The genome sequence of Hbt. salinarum strain NRC‐1 was the first haloarchaeal genome sequence that became publicly available in 2000   , and this has been a focus of study from the 1980s onwards  was found to be exceedingly similar to the laboratory twins NRC‐1 and R1, and the types of changes seen are consistent with strain 63‐R2 being the ancestral strain from which the two laboratory strains were derived. We believe that the origin of these laboratory strains has now been resolved.2https://doi.org/10.5281/zenodo.7780801. For convenience, summaries of these methods are given below.Detailed methods are provided in the supplementary material, deposited at Zenodo: 2.1In‐house tagged versions of the genome sequences of strains 63‐R2, 91‐R6, R1, and NRC‐1 were generated in which all unique sequences between MGEs were identified, as well as each MGE and associated target sequence duplication (TSD).After the removal of  comments, a “total” sequence was available for each strain. The concatenation of these sequences resulted in a “total” database for subsequent analyses, especially the determination of positions in the original genome sequences. In this “total” database file, line breaks around MGEs are preserved so that their visual identification is simple, especially when the MGE is enclosed by a TSD. A copy of that file served as the initial version of the “core” database, open for subsequent manual modification, most importantly the removal of strain‐specific copies of MGEs.2.2T, and because of this, the initial analyses were restricted to these three strains. Applying an iterative comparison procedure, “core” chromosome sequences devoid of strain‐specific MGEs were generated. The build‐up of this “core” database is described in Supplementary Methods, deposited at Zenodo: https://doi.org/10.5281/zenodo.7780801. All eliminated MGEs, including their position in the original genome sequence, are documented in Table Preliminary genomic comparisons  had indicated that the genome sequence of strain 63‐R2 was much more closely related to those of the twin laboratory strains NRC‐1 and R1 than to that of strain 91‐R6BLASTn analyses of the “core” sequences resulted in a complete set of HSPs (high‐scoring pairs) that correlated the complete “core” sequence of the chromosome from strain 63‐R2 against the core chromosomes from the twin laboratory strains NRC‐1 and R1.https://doi.org/10.5281/zenodo.7780801).HSP positions of the interstrain comparison are reported for the “core” database, but to allow easy correlation with biological features, all “core” database positions have been correlated with the corresponding positions in the original sequences of the “total” database .2.4Preliminary comparisons (BLASTn) indicated that the sequence of plasmid pHcu235 from strain 63‐R2 is most closely related to plasmid pHS3 from strain R1, so these plasmids were compared in detail using the same procedure as described for chromosomal comparison  indicated that the unique, 2.3 kb sequence of plasmid pHS4 from strain R1 is closely related to a region on plasmid pHcu43 from strain 63‐R2. Thus, the sequences of these two plasmids were compared. A plasmid corresponding to pHS4 has not been reported for strain NRC‐1, and thus a plasmid from this strain was not included in the analysis.The position of strain‐specific MGEs and their associated TSD which were removed upon generation of core plasmid sequences, are listed in Table 2.5https://doi.org/10.5281/zenodo.7780801.This is based on an analysis of Illumina sequence reads obtained upon resequencing of strain NRC‐1  unique sequences from other strains which were not covered in the set of contigs from strain 63‐R2, (b) sets of PacBio reads selected according to a yet unexplored junction between a unique sequence and a duplication; this enabled the minimum length of the duplicated sequence which is connected to that junction to be determined, and (c) optional MGE's, where some reads contained the MGE‐free sequence version, while others exemplified the junction between the MGE and the adjacent unique sequence.2.8Some sequences in strains R1 and NRC‐1 are strain‐specific and are not represented in the other strain   as well as to Hbt. salinarum DSM 669 . The most relevant data for taxonomic analyses generated by the TYGS server  are given in Table 4) values were above 90% in comparison to the type strain 91‐R6T, confirming they are all strains of Hbt. salinarum because they exceed the 70% species delineation threshold  and are slightly less related (93%–94% dDDH[d4]) to strain 91‐R6.Complete genome sequences consisting of both chromosomes and plasmids of the Lochhead strains 91‐R6 and 63‐R2, and the laboratory strains NRC‐1 and R1 were submitted to the TYGS server for taxonomy assignment based on comparison of complete genomes  even though this name is flagged as a “younger heterotypic synonym” so that this strain is nowadays assigned to the species Hbt. salinarum (see above).At the time of analysis (January 2022) and within TYGS, strains 91‐R6 (NCBI WGS project VRYN01) and 63‐R2 (NCBI WGS project JACHGX01) were represented by draft genomes. Strain 63‐R2 is represented in the results from TYGS by the name under which it has been validly published  and then used for comparison.For transparency, all strain‐specific MGEs which were removed during this procedure are documented in Table https://doi.org/10.5281/zenodo.7780801.Due to the in silico removal of MGEs, the nucleotide positions listed in Tables https://doi.org/10.5281/zenodo.7780801). A duplication of only 38 bp is sufficient to be recorded as an event, which shows that the applied method is highly sensitive. Two breakpoints are novel and exemplify differences between strain 63‐R2 to both laboratory strains. The other three events are known from the comparison of the twin laboratory strains NRC‐1 and R1  and five (R1) HSPs, all showing 99.99% nucleotide sequence identity, needed to completely describe their relation to strain 63‐R2 (Tables 3.3https://doi.org/10.5281/zenodo.7780801). Also, due to the extreme similarity of strain 63‐R2 with strains NRC‐1 and R1, no additional relevant information can be gained by extending the comparison beyond the Lochhead strains. Thus, comparative data are only reported for strains 63‐R2 and 91‐R6; but in descriptions of the corresponding regions from strain 63‐R2, the near‐identical regions from the thoroughly analyzed laboratory strains are also referenced.The chromosomal sequences of strains 63‐R2, 91‐R6, NRC‐1, and R1 were used for detailed analysis, but not all pairwise comparisons need to be performed because strain 91‐R6 had previously been compared to the twin laboratory strains NRC‐1 and R1 . Within two of the long breaks are a small number of homologous sequences which are short  and which show reduced sequence similarity . Details about each of them are provided in Supplementary Text S2: https://doi.org/10.5281/zenodo.7780801. Here, we only summarize key observations for the most relevant unique regions.While only four or five HSPs from BLASTn comparisons are required to completely represent the relationship between Lochhead strain 63‐R2 and the twin laboratory strains NRC‐1 and R1, a much larger number of HSPs (38) is required to describe the relationship between the two Lochhead strains. These 38 HSPs sum up to 1,850,787 bp of which 1,844,079 are identical, giving an overall sequence similarity of 99.64%. The HSPs are separated by unique sequence breaks that are typically short (below 2 kb). Only a few of these are longer than 3 kb (11 of the unique regions). There are eight long breaks (3.2 kb to 9.4 kb), two very long pairs of unique sequences (47.0 to 78.2 kb), and one extremely long unique sequence (164.2 kb). Of the eight long breaks, six have features that are characteristic for proviruses . One long unique sequence codes for a type I restriction enzyme (break 2), and one long break is due to deletion of five genes, including https://doi.org/10.5281/zenodo.7780801). A 78.2 kb sequence in strain 91‐R6 is replaced by a 44.1 kb sequence in strains 63‐R2, NRC‐1, and R1 . In both cases, several encoded proteins are closely related (up to 85% protein sequence identity) and even gene synteny is partially retained. However, there is little similarity at the nucleotide sequence level except for short patches of restricted sequence homology (short HSPs) (Supplementary Tables S2.3 and S2.7 in Supplementary Text S2: https://doi.org/10.5281/zenodo.7780801). Overrepresented among encoded proteins are glycosyltransferases and other enzymes acting on carbohydrates. These may be associated with the N‐glycosylation pathways or may be required for the production of extracellular polysaccharides (EPS).All breaks >10 kb are chromosomal replacements. The term replacement is used to refer to the result of a deletion‐coupled insertion  which has characteristics of an integrated plasmid and which replaced 2.3 kb from strain 63‐R2, NRC‐1, and R1. A subregion, totaling 42.5 kb, shows a close but complex relationship to plasmid pHS3  have an equivalent in strain 63‐R2. The PacBio reads from genome sequencing of strain 63‐R2 were successfully scrutinized for matches to unique regions from R1 plasmid pHS2. Using a supervised approach within Geneious, pHS2 as a reference, and subassembly walking (see methods and Supplementary Methods), a contig was assembled that is longer than 170 kb. Despite considerable efforts, it was not possible to further extend this contig which runs at both termini into the long inverted duplication known from pHcu190, pNRC100, and pNRC200 Figure . Extensihttps://doi.org/10.5281/zenodo.7780801.After the removal of strain‐specific MGEs  are common when compared to strain 63‐R2. To confirm that the residual R1‐specific sequences were truly absent from strain 63‐R2, the complete set of PacBio reads was searched for these sequences.Unexpectedly, two R1 specific sequence regions were represented in the 63‐R2 PacBio reads even though they occurred neither in the chromosome of strain 63‐R2 nor in any of the four plasmids of this strain . Contigs were iteratively extended by subassembly walking resulting in two contigs  which proved resistant to further extension. Both represent plasmid sequences that have integrated into the chromosome at position 1.190 Mb. Extensive duplications were encountered. ContigDRAFT1 and contigDRAFT2 have a common sequence of 24.2 kb and they duplicate regions of the plasmids from strain 63‐R2.https://doi.org/10.5281/zenodo.7780801.The duplication between pHcu190 and contigDRAFT1 in strain 63‐R2 is caused by the heterogenous PacBio reads and thus must reflect strain‐internal variability. It is undecided if ATCC 33170 (63‐R2) contains a mixed cell population or if the heterogenous sequences occur within the same cell. However, it is remarkable that both sequence versions known from the twin laboratory strains NRC‐1 and R1 occur in strain 63‐R2. Given that the previously designated “R1 specific” sequences were subsequently detected in contigDRAFT1 and contigDRAFT2 of strain 63‐R2, they must have been ancestral to strains 63‐R2, NRC‐1, and R1. Consequently, the insertion of a 1.9 kb sequence in pHS1 at the expense of a deletion of 58.4 kb was not an event that occurred in strain R1 but had occurred earlier in the lineage leading to this strain . The most parsimonious interpretation is that these three strains originate from a single cultivation event, the isolation of strain 63‐R2 by Lochhead in 1934. Later, at unknown time points, samples were taken, probably independently, and further cultivated as either strain NRC‐1 or as the immediate parent (DSM 670) of the spontaneous gas‐vesicle‐free mutant strain R1 (DSM 671). At the same time, the originally cultivated cells have been further expanded as strain 63‐R2, and additional events likely altered the genome before the cells were deposited as ATCC 33170 and subsequently sequenced. This would explain the two novel breakpoints in strain 63‐R2, while strains R1 and NRC‐1 are identical in these regions. Details about the contigDRAFT1, contigDRAFT2, and the various duplicated regions in strains 63‐R2, NRC‐1, and R1 are provided in Supplementary File S3: 4Hbt. salinarum, R1, and NRC‐1 was previously unclear, although genome comparisons had shown their chromosomes and plasmids were extremely similar, confirming that they both came from the same parental strain  excluded strain 91‐R6T as being the parent of the two laboratory strains  which is absent from strain R1. This element has integrated into the The evolutionary signals conveyed by the plasmids of strains 63‐R2, NRC‐1, and R1 were also intriguing. While they are extremely well conserved in sequence, the architecture of the plasmids carried by these strains varied greatly. In a previous report  while only one copy is found in strain R1 on the pHcu235‐related plasmid pHS3, and only one copy is found in strain NRC‐1 on the pHcu190‐related plasmid pNRC100.In a surprising discovery, while attempting to confirm the absence of the pHS1‐specific 1.9 kb sequence from strain 63‐R2, PacBio reads were found that carry this sequence (lacking an ISH2 element and thus being a 1.3 kb sequence). Expansion of that sequence uncovered remnants of a plasmid that is found integrated into the chromosome and which occurs in two variants. Thus, both architectures, that of plasmids pHcu190/pNRC100 and that of plasmid pHS1 must already have occurred in the common ancestor of strains 63‐R2, NRC‐1, and R1. Similarly, one variant of the integrated plasmid contains a junction that is specific to pNRC200 and joins sequences from pHS3 and pHS2 . Again, both architectures, that of plasmids pHS3 and pHS2 and that of pNRC200 must already have occurred in the common ancestor, which probably is the original isolate 63‐R2.This suggests the following hypothetical scenario for plasmid evolution from the common ancestor to 63‐R2, the parent of the twin laboratory strains NRC‐1 and R1 Figure . The preHbt. halobium” ; formal analysis ; investigation ; project administration (lead); validation ; writing—original draft ; writing—review and editing . Mike Dyall‐Smith: Formal analysis ; investigation ; visualization (lead); writing—original draft ; writing—review and editing .None declared.None required.


Furthermore, combining accurate structural information and theoretical calculations reveals that the inserted hydrogen‐bonded inorganic layers provide the proton source and a networks of hydrogen−bonds leading to efficient proton transport, meanwhile reducing the bandgap of hybrid architecture and increasing the band electron delocalization of the metal–organic layer to greatly elevate the electron transport of intrinsic 2D metal–organic frameworks.The key to designing and fabricating highly efficient mixed protonic–electronic conductors materials (MPECs) is to integrate the mixed conductive active sites into a single structure, to break through the shortcomings of traditional physical blending. Herein, based on the host–guest interaction, an MPEC is consisted of 2D metal–organic layers and hydrogen‐bonded inorganic layers by the assembly methods of layered intercalation. Noticeably, the 2D intercalated materials (≈1.3 nm) exhibit the proton conductivity and electron conductivity, which are 2.02 × 10 The guest particles of inorganic ions and/or molecules insert into the interlamination of neutral host framework 2D MOFs, manufacturing the host–guest architecture of 2D metal–organic layers and hydrogen‐bonded inorganic layers. The intercalated layer provides the proton source and reduces the bandgap of hybrid architecture, which leads to the high‐efficiency mixed protonic–electronic transport. However, the materials of these traditional MPECs are limited by the extreme incompatibility of proton and electron conduction, the narrow range of working environmental conditions , and poor stability to the environment , which limit their practical application. Furthermore, these traditional proton–electron dual‐conductive materials are usually prepared by physical blending, which often results in an unclear structure and transmission path with a large degree of phase boundary, resulting in low transmission efficiency. Although some MPECs composites (such as graphene oxides and new fashioned perovskite) exhibit excellent properties, their accurately structural information is unclear, resulting in the uncertain structure–property relationship and unable to elucidate the mixed proton and electron conduction mechanism and interaction.Mixed protonic–electronic conductors (MPECs) possess a wide range of technical applications in devices that rely on mixed ion and electron transport, such as fuel cells, supercapacitors, sensors, flexible electrochromic devices, etc.1 Typi With the high surface area and porosity, abundant metal centers, regulable structure/composition, MOFs have been widely used in the investigation of gas adsorption and separation, catalysis, sensors, and medicine. Notably, a growing number of researches have focused on their role as proton or electron conduction materials, whereas these two developments are in parallel. According to the survey, there has been a few MOF‐based MPECs due to the design strategies and components. The conduction of MOFs originates from the conduction of carrying charges (protons and electrons) in the host frame and/or the conduction of protons and electrons realized by guest carriers  based on the host–guest interaction. However, The design of MOF‐based MPECs adopts a single conduction mode, that is, both ion and electron conduction originate from the same host frameworks or guest carrier, which not only leads to low transmission efficiency, but also involves components that interfere with each other. Therefore, the construction of individually functionalized host–guest architectures to integrate the mixed conductive active sites into a single structure, which is an efficient strategy for the preparation of MOF‐derived MPECs.Metal–organic frameworks (MOFs), also known as porous coordination polymers (PCPs), is a supramolecular compound constructed by coordination bonds between metal ions/clusters as nodes and organic linking units.8 Withπ–π stacking, multiple interactions between the wrinkled surface and/or other noncovalent effects are connected together. These metal–organic layers may be electrically neutral, positively, or negatively charged, and the interlayer space of which is a network of empty lattice sites, easy for filling by the guests, such as a combination of protons, solvent molecules and counter ions. Especially, the interlayer force of 2D MOFs is weak and the interlayer spacing possesses self‐coordination, which not only breaks through the pore nature of traditional 3D MOFs and restricts the species and components of the guests, but also exhibits high mass permeability coefficient. It indicates that 2D MOFs may be one of the best candidates for rational design and synthesis of high performance MPECs. Nevertheless, it is still a great challenge to entry guests into the interlamination of 2D MOFs in a controllable operation and determine their accurately structural information.Analogous to other 2D materials, the layers of 2D MOFs  is interconnected through hydrogen bonds, together.13 The2 Based on the interaction/reaction between the host and the guest of 2D materials, the predesigned guests stacking networks structure is inserted into the interlamination of parent layers to obtain desired properties and broaden its application range. The controlled preparation of the layer‐like stacking engineering is helpful for the construction of Van der Waals heterostructures (HSs) based on crystal engineering. In this work, a neutral 2D MOF with (–Co–O–)∞ chairs was constructed with Co2+ and succinic acid, which was exfoliated into ultrathin MOF nanosheets by immersed in a mixed solution of K2HPO4 and KH2PO4 . The inclusion of PBS solution particles  are inserted into the interlayer of the 2D MOFs to form hydrogen‐bonded inorganic layers, which not only provides proton source and conduction network, but also effectively regulates the energy states and band electron delocalization of the hybrid structure, realizing high‐efficiency mixed protonic–electronic electron conduction 6(H2O)3(C4H4O4)4]·7H2O (denoted as 2D‐Co), which crystallizes in the monoclinic space group P21/c and its independent unit contains seven metal centers Co(II), four fully deprotonated ligands C4H4O42−, six bridging OH− and three coordinated H2O molecules, and some disordered guests H2O molecules 14(OH)6(H2O)3] is connected into (–Co–O–)∞ chair by the bridged of Co7. Then, the cobalt oxide chain (–Co–O–)∞ is connected by C4H4O42− to form 2D layer with a thickness of 9.8 Å corresponding to the  crystal face or bc plane, and the monolayer {Co7(OH)6(H2O)3(C4H4O4)4}n is stacked along the direction of d001 to form 2D‐Co with the interlamellar spacing of 7.5 Å 4]·7H2O19  can be also exfoliated into small nanosheets (denoted as 2D‐Co‐NS), which maintains the structure of precursor of 2D‐Co  image showed that the lamella of 2D‐Co‐NS has large lateral dimension up to the micrometer size and the thickness is ≈1.0 nm  for 12 h without ultrasound, which can transform to nanosheets similar to the morphology of 2D‐Co‐NS, named as 2D‐Co‐NS‐PBS  and 1HNMR, respectively  is larger than that of 2D‐Co‐NS (≈7.5 Å), and both of them are along the d001 of the referential 2D‐Co crystal  or the thickness of single layer of 2D‐Co. Combining with infrared spectra , extended X‐ray absorption fine structure (EXAFS), and the corresponding R‐space analysis. As shown in Figure2D‐Co‐NS‐PBS exhibited characteristic edge absorptions at 7709.5 and 7724.8 eV, corresponding to the Co pre‐edge  and Co1s–Co3d features, which is almost exactly coincident with the exfoliating precursor 2D‐Co‐NS (7709.8 and 7724.6 eV). In addition, the valance state of Co are in the oxidation state of +2 before and after intercalation, which is consistent with the XPS result. The Fourier‐transformed (FT) EXAFS indicates that the coordination structure of Co(II) do not change 6(H2O)3(C4H4O4)4}n of 2D‐Co‐NS‐PBS does not change at all, the introduced ions K+, H2PO4−, and HPO42− are intercalated with the metal–organic layers.The refined structure of 2D‐Co‐NS‐PBS  with cell parameter list as Table 2D‐Co‐NS‐PBS implies the crystal symmetry of the intercalated layer  is matched with that of {Co7(OH)6(H2O)3(C4H4O4)4}n, the metal–organic layers and intercalation can form a double‐layer heterojunction as the construct unit layer of 2D structure with the thickness and interlayer distance of 12.8 and 9.8 Å, respectively . PXRD showed that the mixture contained 2D‐Co‐Bulk diffraction peaks and inorganic salts diffraction peaks , (321), and (312) crystal planes of KH2PO4, respectively) , which increases as the temperature increases, obtaining the optimal conductivity of 2.02 × 10−5 S cm−1 at 100 °C and 99% RH. The activation energies (Ea) of the materials derived from Arrhenius plots is found to be 0.04 eV for 2D‐Co‐NS‐PBS . Based on the proton conduction mechanism of 2D‐Co‐NS‐PBS, the protons originate from PBS‐NS and migrate directionally along the 2D hydrogen bond networks constructed by metal–organic layer and inorganic layer, which verifies the rationality of 2D heterojunction structure.In order to simulate practical application, we prepared the ion intercalation MOFs of coin cell as the ion separator device Figure4a, and l Figure . The proS Figure , which l In Figure 2D‐Co‐NS and 2D‐Co‐NS‐PBS increases with the rising of temperature in the range of 25–100 °C, corresponding to the electron conductivity increased from 2.09 × 10−10 to 2.12 × 10−8 S cm−1 and 2.03 × 10−5 to 3.84 × 10−4 S cm−1, respectively. The Arrhenius plots of 2D‐Co‐NS and 2D‐Co‐NS‐PBS belongs to linear relationships and the Ea is 285 eV and 156 eV 6(H2O)3(C4H4O4)4}n effectively tuned the structure's electron conductivity, increasing the conductivity by 4–5 orders of magnitude. The MOFs with inorganic ions intercalation exhibits excellent proton–electron dual conduction, which is comparable to most of the other material that has been reported (see in the Table Eg) of 2D‐Co‐NS (2.16 eV) and 2D‐Co‐NS‐PBS (1.80 eV) can be derived from their UV–vis–NIR spectra  after twice electrochemical testing; corresponding to the electron conductivity decreases from 2.03 × 10−5–3.84×10−4 to 7.12 × 10−6–4.85 × 10−5 S cm−1. Due to the disorder of layer‐layer stacking leads to the hydrogen bond chain be destroyed and the proton conductivity decreases, and the non‐crystallizing also leads to the decrease of its electron conductivity. Meanwhile, the XPS profile and EPR analysis reveal that the valence states and spin states of metal center in 2D‐Co‐NS‐PBS does not change before and after the electrochemical test , which is consistent with the optical band gap obtained by electronic spectroscopy. Furthermore, the density of states (DOS) indicates that the valence bond (VB) is mainly composed of Co and O centered orbitals  decreases from 5.14 to 4.78 eV (Figure PBS‐NS intercalation, which is consistent with the Ea obtained from the variable‐temperature electronic conductivity measurement. It indicates that intercalation effectively regulates the surface electronic structure of the metal–organic layer and indirectly regulates the intrinsic electron conduction ability of the structure.The mechanism of electron band conduction in the metal‐organic layer before and after PBS intercalation was further investigated by density functional calculation. It reveals the intrinsic electronic band structure of BS Figure5, the 3In summary, we developed a new strategy that the guest particles of inorganic ions and/or molecules insert into the interlamination of neutral host framework 2D MOFs, manufacturing the host–guest architecture of 2D metal–organic layers and hydrogen‐bonded inorganic layers. In this architecture, the hydrogen‐bonded inorganic layers offers the proton source and proton transfer path, while the 2D metal–organic layers provides the electron conduction path to ensure the proton and electron conductivity of the 2D hybrid networks, respectively. Moreover, the electronic structure of host frameworks of MOFs can be accurately regulated by the intercalation, to increase the electronic conductivity of intrinsic MOFs. This work expands the category of MPECs materials, as well as the fundamental understanding of structure–property relationship involved mixed conduction has further investigated.The authors declare no conflict of interest.Supporting InformationClick here for additional data file.