Staphylococcus aureus LytM, a Zn2+-dependent glycyl-glycine endopeptidase of the M23 family, is one of the peptidoglycan hydrolases. It has a specificity towards staphylococcal peptidoglycan, making it an interesting target for antimicrobial studies. LytM hydrolyses the cell wall of S. aureus, a common pathogen with multi-resistant strains that are difficult to treat, such as the methicillin-resistant S. aureus, MRSA. Here we report the 1H, 15N and 13C chemical shift assignments of S. aureus LytM N-terminal domain and linker region, residues 26–184. These resonance assignments can provide the basis for further studies such as elucidation of structure and interactions.Antibiotic resistance is a growing problem and a global threat for modern healthcare. New approaches complementing the traditional antibiotic drugs are urgently needed to secure the ability to treat bacterial infections also in the future. Among the promising alternatives are bacteriolytic enzymes, such as the cell wall degrading peptidoglycan hydrolases.  The increasing prevalence of antimicrobial resistance in bacteria is among the most pressing threats to public health  is reported to have gained resistance against many of the first-line antibiotics, including the traditional β-lactam antibiotics . PGHs kill bacteria via degradation of peptidoglycan, an essential component of the bacterial cell wall  and N-terminal domain with the linker region (residues 26–184) were cloned in pET-15b vectors. To improve protein solubility the LytM constructs were tagged with GB1 in the N-terminus connected with linker region containing the TEV protease site. Proteins were expressed using Escherichia coli strain BL21(DE3). 50 ml precultures were grown in standard M9 supplemented with 100 µg/ml ampicillin at 30 °C with 230 rpm shaking for 20 h. Cultures were expanded to 1 l and the cells were grown at 37 °C with 250 rpm shaking in standard M9 minimal medium supplemented with 100 µg/ml ampicillin and 15NH4Cl (1 g/l) and 13 C-D-glucose (2 g/l) as the nitrogen and carbon sources for uniform 15N and 13C labelling, respectively, until the OD at 600 nm was 0.55–0.6. Cells were cooled down to 16 °C, and the protein expression was induced by adding 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 16 °C for 20 h, at 230 rpm.Two constructs of Cells were harvested by centrifugation, resuspended in 50 mM sodium phosphate pH 8, 300 mM NaCl buffer and lysed by sonication . Clarified lysates were purified with 1 ml His GraviTrap (Cytiva) column according to manufacturer’s instructions. GB1 was cleaved using TEV protease, overnight incubation in dialysis  at 4 °C. Cleaved proteins were purified in 20 mM sodium phosphate pH 6.5, 50 mM NaCl, 1 mM DTT buffer by size exclusion chromatography using ÄKTA pure chromatography system  with HiLoad Superdex S75 (16/60) column . Proteins were concentrated using Amicon Ultra-15 centrifugal filter units (Millipore).15N,13C labelled LytM(26–107) and LytM(26–184) protein fragments in a buffer composed of 20 mM sodium phosphate, 50 mM NaCl, 1 mM DTT, pH 6.5 in 95/5% H2O/ D2O. The concentrations of LytM(26–107) and LytM(26–184) used for the data collection were 1.0 mM and 0.2 mM, respectively. NMR spectra were acquired at 308 K using a Bruker Avance III HD 800 MHz NMR spectrometer equipped with a 1H, 13C, 15N TCI cryoprobe.The NMR data acquisition was done using uniformly 1H,15N HSQC . Backbone resonances were assigned using The Hα, Cα, N and C′ chemical shifts of the LytM linker region (residues 108–184) were assigned using the longer construct LytM(26–184) and by employing Hα-detected 4D HACANCOi and HACACON  and analysed using CcpNmr Analysis v.2.4.2 software  , on the other hand, are all found within 7.8–8.3 ppm in the 1HN dimension, and the signals overlap considerably. This discrepancy in the distribution of peaks between the two regions of the protein was also reflected in the extent of chemical shift assignment.The conclusion of intrinsic disorder is supported also by the experimental data. An overview of the 1H-15N pairs were assigned, omitting the N-terminal glycine not belonging to the native sequence. 98.8% of C′ and all Cα, Hα, Cβ and Hβ resonances were assigned. The aliphatic side chain assignment was 73.6% complete, and aromatic side chain assignment completeness was 93.2%. The low aliphatic side chain assignment percentage was largely due to the unassigned Asn δ2 (55.6% assigned) and Gln ε2 (12.6%) resonances. These resonances could not be reliably identified due to overlap of signals in the 1H,15N HSQC spectrum.The chemical shifts of the N-terminal domain (residues 26–107) were assigned more comprehensively. The backbone chemical shifts were assigned nearly completely, with an assignment percentage of 98.8%. Out of the non-proline residues, 97.5% of 1H,15N HSQC spectrum. The process is described in Fig. The backbone chemical shifts of the disordered linker region (residues 108–184) were assigned using additional 3D HA(CA)NCOi and HA(CA)CON, and 4D HACANCOi and HACACON experiments to overcome the issue of signal overlap and missing amides in the www.bmrb.wisc.edu) under accession number 51662.These data have been deposited in BioMagResBank (2) values derived from TALOS-N. These analyses outline a similar increase of order and decrease of flexibility in the mid-N-terminal domain.For further characterization, the chemical shifts were used to investigate the secondary structure content of LytM N-terminal domain and linker region. Two approaches, TALOS-N (Shen and Bax Comparison with the secondary structure elements of the crystal structure of full-length LytM (PDB ID 1QWY, Odintsov et al. Here we have presented the chemical shift assignment of LytM residues 26–184. A nearly complete assignment was obtained for N-terminal residues 26–107, which are likely to form a structured domain. Based on chemical shift dispersion and bioinformatics, residues downstream 107 are mainly disordered. Hα, HN, Cα, Cβ, C, and NH resonances were assigned for these residues. These assignments allow further studies such as structural characterization and investigation of interactions.


Cell migration and soft agar colony formation assays assessed cellular transformation potential. We found that all three A3 enzymes had similar γH2AX foci formation, despite different deamination activities in vitro. Notably, in nuclear lysates, the in vitro deaminase activity of A3A, A3B, and A3H did not require digestion of cellular RNA, in contrast to that of A3B and A3H in whole-cell lysates. Their similar activities in cells, nonetheless, resulted in distinct phenotypes where A3A decreased colony formation in soft agar, A3B decreased colony formation in soft agar after hydroxyurea treatment, and A3H Hap I promoted cell migration. Overall, we show that in vitro deamination data do not always reflect cell DNA damage, all three A3s induce DNA damage, and the impact of each is different.APOBEC3 (A3) enzymes deaminate cytosine to uracil in viral single-stranded DNA as a mutagenic barrier for some viruses. A3-induced deaminations can also occur in human genomes resulting in an endogenous source of somatic mutations in multiple cancers. However, the roles of each A3 are unclear since few studies have assessed these enzymes in parallel. Thus, we developed stable cell lines expressing A3A, A3B, or A3H Hap I using non-tumorigenic MCF10A and tumorigenic MCF7 breast epithelial cells to assess their mutagenic potential and cancer phenotypes in breast cells. The activity of these enzymes was characterized by γH2AX foci formation and  The human APOBEC3 (A3) enzymes belong to the APOBEC family and consist of seven members  that deaminate cytosine to form uracil on single-stranded (ss) DNA . Since uTo induce mutations in genomic DNA, it is important that the enzymes localize to the nucleus, but the enzymes must also be able to compete for ssDNA with Replication Protein A (RPA) during dynamic and transient processes such as replication, transcription, and DNA repair . Only A3To date, only A3A, A3B, and A3H Hap I have been implicated in processes related to somatic mutagenesis and cancer using retrospective data from The Cancer Genome Atlas (TCGA) or other methods where whole-genome sequencing and mRNA expression can be analyzed . SuppresThe formation of promutagenic uracils through the intrinsic deaminase activity of these enzymes occurs in a sequence-specific manner, and those involved in cancer preferentially deaminate the central cytosine in 5′TCW trinucleotide motifs (where W = A or T on ssDNA) to form uracil . Uracil-in vitro, suggesting that less steady-state A3A is needed in cells to induce somatic mutagenesis  but was later identified in other cancer types, including bladder, cervical, lung, and head-and-neck cancers, where it was evident that there was a positive correlation between A3B expression and the total APOBEC-mutation load . A3B is agenesis . This isagenesis . Anotheragenesis . In addiagenesis .in silico, but recently there has been direct evidence of A3A tumorigenic potential in vivo  lysates, where there may be less inhibitory RNA than in WC lysates. In addition, there have been no studies determining if A3-induced mutations cause any specific cancer cell phenotype. Here, we aimed to compare in parallel A3A, A3B, and A3H Hap I. We chose BC as a model because all three of the A3s have been implicated . Using tThe 293T, HCC1428, MCF10A, and MCF7 cell lines were obtained from ATCC and cultured as recommended by ATCC, excluding antibiotics. Lentiviruses were generated by transfection of a pLVX-3x Flag vector with psPAX2 and pMD2.G into 293T cells. The transduction of MCF10A and MCF7 with the resulting lentiviruses expressing Flag-tagged-A3A, -A3B, and -A3H Hap I was performed for 16 h in a medium containing 8 μg/mL polybrene. Transduced MCF10A and MCF7 cells were selected with 1 μg/mL and 2 μg/mL puromycin, respectively, for 7 days and maintained as polyclonal cell lines with 0.25 μg/mL puromycin. Experiments with dox-induced cells were controlled with either transduced cells that were not dox induced or dox-induced non-transduced parental cells.α-tubulin, which was detected in parallel on the same blot. The antibodies purchased from Sigma-Aldrich were anti-α-Tubulin antibody, mouse monoclonal ; anti-α-Tubulin antibody, rabbit monoclonal ; ANTI-FLAG® antibody produced in rabbit ; monoclonal ANTI-FLAG® M2 antibody produced in mouse ; and anti-HA antibody produced in rabbit . The antibody purchased from Thermo Fisher Scientific was Histone H2B monoclonal antibody . The antibodies purchased from LI-COR Biosciences were IRDye 680RD donkey anti-rabbit IgG secondary antibody  and IRDye® 800CW donkey anti-mouse IgG secondary antibody .The MCF10A- and MCF7-derived stable cell lines were incubated for 72 h with doxycycline (dox) concentrations ranging from 0.05 to 2 μg/mL to induce the expression of A3-Flag enzymes. Cells were lysed with 2X Laemmli buffer, and WC and cytoplasmic (Cyt) and Nuc lysates obtained after fractionation were resolved by SDS-PAGE. Antibodies used are labeled on each immunoblot. Secondary detection was performed using LI-COR IRDye antibodies produced in donkey (IRDye 680-labeled anti-rabbit and IRDye 800-labeled anti-mouse). Quantification of band intensities was performed using Odyssey software with normalization of each experimental lane to its respective The RNA preparation, cDNA synthesis, and qPCR were carried out according to the work of ® M2 antibody produced in mouse .MCF10A- and MCF7-derived stable and transduced cell lines were grown on glass slides and treated for 24 or 72 h with dox for the expression of the A3-Flag enzymes and fixed with 100% cold methanol for 20 min. Where indicated, the cells were transfected with an expression vector encoding the bacteriophage PBS2 UGI protein 24 h before the dox induction . In addi6 cells in 150 µL HED buffer . Additionally, 0.1, 0.5, or 5 mM EDTA was added to inhibit nuclease activity. Samples were then centrifuged, and the supernatant was used in 20 µL deamination reactions with 1 µM (293T cells transfected with A3B or A3H Hap I expression plasmids), 0.1 µM (for MCF10A cell lines), or 0.05 µM (293T cells transfected with A3A expression plasmid) fluorescein-labeled 43 nt ssDNA substrate that was reported by Deamination assays were performed using a protocol adapted from the work of The 293T cells were or were not transfected in a T75 flask using GeneJuice (EMD Millipore) with 1 µg of pcDNA, pcDNA-A3A-3xHA, and pVIVO-A3H Hap I-HA for 40 h or 2 µg of pcDNA-A3B-3xHA for 48 h to obtain similar protein levels. For either non-transfected or transfected 293T cells, the RNase A  was added or not directly to the reaction mixture. MCF10A-derived stable cell lines were treated with dox for 72 h. Dox concentrations were used to achieve similar steady-state protein levels . Prior to performing the deaminase assay for the MCF10A stable cell lines, WC lysates were either pre-treated or not with RNase A (100 μg/mL) for 15 min at 37°C.For the Nuc lysates from the non-transfected 293T cells, the purified enzymes A3A, A3B, or A3H Hap VII were added to the reactions together with 40 U of murine RNase inhibitor . The activity of cell lysates was evaluated in reactions containing 85 nt linear or 21 nt hairpin substrates . For A3Ag for 10 min. The cells used were transduced MCF10A or MCF7 cells that were or were not treated with dox and 293T cells that were or were not transfected with A3-expression plasmids. These conditions to prepare the MCF10A and 293T cells are detailed in Cells were harvested and either lysed by sonication in HED buffer or with 2X Laemmli buffer to get the WC lysates for deamination or immunoblot, respectively, or were fractionated. For fractionation, cells were washed with 1X PBS and pelleted by centrifugation at 500 2, 340 mM sucrose, 0.5 mM EDTA, 1 mM DTT, and EDTA-free protease inhibitor (Roche)). After 10 min of incubation on ice, 12.5 µL of 10% NP-40 per 1 × 106 cells was added and followed by vortex mixing at high speed for 15 s. The samples were centrifuged at 3,300 x g for 10 min, and the supernatant was saved as Cyt lysate. The remaining pellet was washed once with Cyt extraction buffer and lysed with Nuc extraction buffer  for 30 min at 4°C. After centrifugation at 13,000 x g, the resulting supernatant was saved as Nuc lysate.The cell pellet was resuspended in Cyt extraction buffer  on anchorage-independent growth, MCF10A and MCF7  were either untreated or treated for 6 h with 2 mM or 12 mM HU, respectively, to stall replication . Followi5 cells) onto the upper chamber in media containing 0.5% serum. Media containing either 0.5% or 10% serum was loaded into the lower chambers. Migration during 48 h toward the lower chamber was determined by colorimetric measurement at 560 nm.The effect of A3 expression on the cell migration, in the presence or absence of HU, was evaluated using the QCM™ 24-well colorimetric cell migration assay . MCF10A- and MCF7-derived stable cell lines were untreated, treated with dox, or treated with dox + HU. The cells were treated with HU twice, on days 3 and 6, and at HU concentrations of 2 mM and 12 mM for MCF10A and MCF7 stable cell lines, respectively. After 7 days of dox treatment, cells were starved for 16 h in media containing 0.5% serum and then seeded  followed by the Holm–Sidak post hoc test was used for multiple groups. The results were considered statistically significant at a p-value of <0.05. Statistical analyses were performed using SigmaPlot 11.0 software.The results are presented as mean ± standard deviation (S.D.). A two-tailed α-tubulin in the same lane. The MCF10A- and MCF7- relative protein levels are comparable within each set but not to each other since they were detected on different immunoblots . Endogenous low-level A3B expression has previously been detected in these cell lines (x-axis), the level of A3A mRNA for both MCF10A and MCF7 cells was low, showing only 0.75- and 1.42-fold increases, respectively, although the steady-state protein expression was similar to A3B .Using the conditions that had equal A3-Flag expression levels, the mRNA expression levels were assessed by qRT-PCR to determine if there was any expression of endogenous A3 enzymes that could access the nucleus . For thell lines . After dr to A3B . In cont 60-fold . A3H HapF7 cells . Even thF7 cells   . HoweverWe also tested the tumorigenic MCF7 breast cancer cell lines and did not observe differences in the number of colonies between the uninduced and induced condition for parental cells or cells that expressed either A3A, A3B, or A3H Hap I . During We also investigated if the expression of A3A, A3B, and A3H Hap I, either in MCF10A or MCF7 cells, could influence cell migration in the absence or presence of HU. The cell lines were not or were dox induced for 7 days, and HU was added on day 3 and day 6, as indicated in Materials and Methods. Induction of A3s with or without HU treatment had no effect on the non-tumorigenic MCF10A cells . The expin vitro deamination assays using WC lysates do not directly correlate with accumulation of DNA damage in cells or the extent of inhibition by cellular RNA. Altogether, the data support a model in which A3A, A3B, and A3H Hap I all induce DNA damage in normal or tumorigenic breast epithelial cells, but the phenotypic effects of the induced damage are unique for each A3.The existing data implicating A3A, A3B, and A3H Hap I in cancer have led to an unclear understanding of the relative contributions of individual A3s to mutagenesis in BC . To overin vitro deamination in cell lysates is often used to gauge A3 activity in cells, we found inconsistencies with this method. Importantly, despite reports that A3A is a more potent inducer of DNA damage than A3B (in vitro deamination assay, we conducted deamination assays under various conditions. A3B and A3H Hap VII deaminated the ssDNA in the Nuc lysates as A3A does, either in the presence or absence of RNase A (While than A3B , our resthan A3B , 3. To i RNase A . This isin vitro as processive enzymes, but we found in this study that they were non-processive on ssDNA in Nuc lysates (in vitro when incubated for a sufficient time (A3B and A3H Hap VII have been previously characterized  lysates  (Feng et lysates . When ha lysates . In addi lysates . Studies lysates , and som lysates . Howeverent time . Since kent time . While oent time . If procin vitro, all three enzymes had equal ability to induce γH2AX foci in cells (Despite A3A being more active in Nuc lysates in cells , 3. Overin cells  and morein cells . This main cells . In contin cells . AlthougIn this study, we looked for phenotypic markers of A3 expression. We observed that A3A expression could delay the anchorage-independent growth of cells in soft agar . A3A wasThere have been few studies that have examined A3H Hap I and its effect on cancer. The A3H mutation signature is found in lung adenocarcinomas . A genetin vitro activities. These data demonstrate the importance of using accurate in vitro systems to determine cytidine deaminase activity and necessitate a reassessment of the contributions of A3A, A3B, and A3H Hap I to somatic mutagenesis during tumorigenesis. These data also provide more detailed deamination activity analysis for A3A, A3B, and A3H Hap I in the context of nuclear DNA that can be used in the design of base editing technologies.In summary, we demonstrated that all three A3s are able to induce cellular phenotypes as a result of their induced DNA damage. Importantly, we showed that cytidine deamination activity for all A3s was not inhibited by RNA in Nuc lysates, and deamination-induced DNA damage was similar in cells, even with differing


Glycine max L.) is an important crop with high agronomic value. The improvement of agronomic traits through gene editing techniques has broad application prospects in soybean. The polyethylene glycol (PEG)-mediated cell transfection has been successfully used to deliver the CRISPR/Cas9-based ribonucleoprotein (RNP) into soybean protoplasts. However, several downstream analyses or further cell regeneration protocols might be hampered by PEG contamination within the samples. Here in this study, we attempted to transfect CRISPR/Cas9 RNPs into trifoliate leaf-derived soybean protoplasts using Neon electroporation to overcome the need for PEG transfection for the first time. We investigated different electroporation parameters including pulsing voltage (V), strength and duration of pulses regarding protoplast morphology, viability, and delivery of CRISPR/Cas9. Electroporation at various pulsing voltages with 3 pulses and 10 ms per pulse was found optimal for protoplast electro-transfection. Following electro-transfection at various pulsing voltages , intact protoplasts were observed at all treatments. However, the relative frequency of cell viability and initial cell divisions decreased with increasing voltages. Confocal laser scanning microscopy (CLSM) confirmed that the green fluorescent protein (GFP)-tagged Cas9 was successfully internalized into the protoplasts. Targeted deep sequencing results revealed that on-target insertion/deletion (InDel) frequencies were increased with increasing voltages in protoplasts electro-transfected with CRISPR/Cas9 RNPs targeting constitutive pathogen response 5 (CPR5). InDel patterns ranged from +1 bp to −6 bp at three different target sites in CPR5 locus with frequencies ranging from 3.8% to 8.1% following electro-transfection at 1,300 V and 2.1% to 3.8% for 700 V and 1,000 V, respectively. Taken together, our results demonstrate that the CRISPR/Cas9 RNP system can be delivered into soybean protoplasts by the Neon electroporation system for efficient and effective gene editing. The electro-transfection system developed in this study would also further facilitate and serve as an alternative delivery method for DNA-free genome editing of soybean and other related species for genetic screens and potential trait improvement.The soybean ( The CRISPR/Cas9 system has become a versatile technology in plant breeding and functional genomics due to its design simplicity and high efficiency in genome editing . Genome Agrobacterium-mediated  that is associated with soybean trichome growth, we validated the mutations at three different sgRNA targeted sites and determined the mutagenesis efficiency of CRISPR/Cas9 in soybean protoplasts by targeted deep sequencing.In the present study, we report the establishment of a CRISPR/Cas9 RNP delivery system that facilitates efficient RNA-based genome editing in soybean protoplasts using electro−transfection. Using the Neon electroporation system , we investigated the important electrical factors including voltage strength and pulse duration on protoplasts, and we analyzed protoplasts of post-electroporation. With the established electrical parameters, we successfully demonstrated the delivery of exogenous green fluorescent protein (GFP)-Cas9 into the nucleus of soybean protoplasts using electro-transfection. By targeting the G. max) seeds cv. OAC Bayfield was planted and grown in soil pots under a photoperiod of 8-h light and 16-h dark at 25°C in a growth chamber (Enviro Plant®) for 3 weeks. The newly expanded trifoliate leaves from 14–16-day- old soybean seedlings were used for protoplast isolation. The extraction of protoplasts was carried out according to our previous study  against CPR5 coding region in our previous study , MMG solution , and HEPES electroporation buffer (10 mM of Hepes (pH 7.2), 0.2 M of mannitol, 5 mm of CaCl2, and 150 mM of NaCl). Next, 20 µl of RNP complex was added to 80 µl of protoplast resuspension to bring a final volume of 100 µl. Then, the electroporation was carried out using the Neon™ Transfection System  following the manufacturer’s instructions in 100-µl volumes. Protoplasts were electroporated with various voltages , pulse (1 and 3), and width  to optimize and obtain efficient transfection conditions as described in the Results section. With the optimized electro-transfection conditions, 20 µl of RNP complexes (1:3 molar ratio) consisted of 10 µg of ready-to-use GFP-tagged Cas9 from Sigma-Aldrich (CAS9GFPPRO), and 30 µg of sgRNA was electrophoretically introduced into 80 µl of resuspended protoplasts. Control and electroporated cells were immediately transferred in a 12-well poly-d-lysine-coated cell plate with 0.5 ml of precooled KP8 liquid medium . The viability of control and electro-transfected cells was assessed by 0.5% fluorescein diacetate (FDA) staining, observed under reflected light with Axio Vert.A1 (FL-LED Stand) inverted light microscope, and captured via Axiocam 202 mono . The fluorescence images of GFP-tagged Cas9 were acquired under bright field and electronically switchable illumination and detection module (ESID) using confocal laser scanning microscopy (CLSM)  using a diode laser (488-nm line) with a 40× objective lens. The viability and transfection efficiency were calculated as the number of fluorescent protoplasts divided by the total number of protoplasts in one representative microscope field .GmCPR5 loci  . For each sgRNA , the target-specific nested PCR primers were designed in PR5 loci . CRISPR/alyzer/) . Briefly6 cells per gram fresh weight. Isolated protoplasts were 10 μm to 50 μm in size, and most were spherical in shape  for 500 V, 700 V, 1,000 V, and 1,300 V. As shown in To establish an electroporation-mediated soybean protoplast transformation using the Neon system, the electro-transfection method was optimized based on cabbage and wheat protocols where transfection efficiency reaches 3.4%  and 2.2% , respectively . FollowiThe calculated relative frequency of cell viability was 86.5% ± 2.4% under 0 V compared to the non-treatment control of 100%. Under various voltage treatments, the viability of cells was decreased with the increase in pulsing voltage. The observed relative frequency of viable rates for treatments under 500 V, 700 V, 1,000 V, and 1,300 V were 76.7% ± 2.8%, 66% ± 1.4%, 59.1% ± 1.2%, and 57.6% ± 1.1%, respectively Figure 2To further optimize and validate the delivery of Cas9 protein, a fixed amount of 10 µg of GFP-conjugated Cas9 was electro-transfected into protoplasts with the abovementioned pulsing voltages. After 24 h of electroporation, the internalization of GFP-tagged Cas9 inside the protoplasts was confirmed under the CLSM microscope Figure 3CPR5 gene, respectively  under the accession number PRJNA983990. Targeted deep sequencing results confirmed that the InDels present at all three target sites including T1, T3, and T5 under different pulsing voltages, similar to that in the T7E1 cleavage assay. As shown in CPR5 target sequences using the Cas-Analyzer tool. The target sites of T1, T3, and T5 have InDel frequencies that ranged from a minimum of 2.1% to a maximum of 8.1% with an average frequency of 4.21% ± 0.73% in the GmCPR5 locus. It was found that the estimated InDel frequencies were increased with the increase in pulsing voltage. For the T1 target site, InDel frequency following electro-transfection at 700 V, 1,000 V, and 1,300 V were 2.1%, 3.7%, and 8.1%, respectively. Similarly, in T3 and T5, the frequency was increased from 2.1% and 3.8%  to 3.8% and 5.7% at 1,300 V, respectively  Bioproject  and viability of protoplasts (70% ± 2.1%) in this study. While ensuring high transfection efficiency in protoplast cells, and at the same time retaining viability and ability to differentiate (cell division), we have tested the protoplasts of both unifoliate and trifoliate to the different pulsing voltages of 500 V to 1,000 V in a pilot study. It was found that trifoliate cells endure and survive under electrophoretic conditions compared to the unifoliate cells, as they were greatly damaged after electroporation, even at 500 V. In addition, the isolated trifoliate cells had exhibited first protoplast divisions at 5 days after culture initiation as noted for the unifoliate cells in our previous study  transfection was increased with increasing pulse voltage from 700 V or 1,000 V to 1,300 V. In this study, the calculated InDel frequencies of T1 (700 V) and T5  were slightly higher than the observed InDel frequencies of 1.2% (750 V) and 3.4%  for PDS1 gene in cabbage protoplasts following electro-transfection  can be found below: NCBI Bioproject accession PRJNA983990.SS: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. SA-T: Conceptualization, Formal Analysis, Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing.


Notably, most cancer cells have been found to have active hTERT mRNA transcription, resulting in continuous cell division, which is crucial for malignant transformation. Therefore, discovering mechanisms underlying the regulation of hTERT transcription is an attractive target for cancer-specific treatments.Telomerase is a ribonucleoprotein ribonucleic enzyme that elongates telomere repeat sequences at the ends of chromosomes and contributes to cellular immortalization. The catalytic component of telomerase, human telomerase reverse transcriptase  vector (3p21.3-HAC) with only the 3p21.3-p22.2 region and performed functional analysis using the 3p21.3-HAC. HSC3 microcell hybrid clones with an introduced 3p21.3-HAC exhibited significant suppression of hTERT transcription, similar to the microcell hybrid clones with an intact chromosome 3. In contrast, HSC3 clones with truncated chromosome 3 with deletion of the 3p21.3 region (3delp21.3) showed no effect on hTERT expression levels. These results provide direct evidence that hTERT suppressor gene(s) were retained in the 3p21.3 region, suggesting that the presence of regulatory factors that control telomerase enzyme activity may be involved in the development of OSCC.Loss of heterozygosity (LOH) of chromosome 3p21.3 has been frequently observed in human oral squamous cell carcinoma (OSCC). Moreover, we previously reported that HSC3 OSCC microcell hybrid clones with an introduced human chromosome 3 (HSC3#3) showed inhibition of The online version contains supplementary material available at 10.1007/s10577-023-09726-8. Indeed, ectopic hTERT expression in telomerase-negative normal cells can extend the lifespan and establish immortalized cell lines via telomere elongation   , exogenous mini-chromosomes, are artificially created by chromosome engineering  for gene therapy targeting Duchenne muscular dystrophy (DMD)  were retained in the 3p21.3 region and could be involved in developing OSCC. Our results can provide important information regarding the functional significance of the LOH at the 3p21.3 region in OSSC. They may lead to identifying novel hTERT suppression genes encoded by the 3p21.3 locus.This study investigated whether telomerase regulatory factors such as 2. CHO K1 cells containing truncated chromosome 3 were cultured in Ham’s F-12 nutrient mixture (Invitrogen) supplemented with 10% FBS and 800 μg/ml G418 at 37 °C in a humidified incubator with 5% CO2. CHO (hprt − / −) cells containing HAC were cultured in a Ham’s F-12 nutrient mixture (Invitrogen) supplemented with 10% FBS and 8 μg/ml blasticidin S hydrochloride  at 37 °C in a humidified incubator with 5% CO2, as previously described. A9 and HSC3 cells containing a truncated chromosome 3 were cultured in Dulbecco’s Modified Eagle Medium  supplemented with 10% FBS and G418  at 37 °C in a humidified incubator with 5% CO2. A9 and HSC3 cells were cultured containing a HAC vector in DMEM supplemented with 10% FBS and blasticidin S hydrochloride  at 37 °C in a humidified incubator with 5% CO2. All cell lines were confirmed to be mycoplasma-free using a MycoAlert Mycoplasma Detection Kit  and were not passaged more than 20 times from the validated stocks.Chicken DT40 cells were obtained from Takeda . A9, HSC3, and Hprt-deficient CHO (hprt − / −) cells were obtained from the Japanese Collection of Research Bioresources Cell Bank . CHO K1 cells were obtained from ATCC . Chicken DT40 cells containing truncated human chromosome 3 were cultured in RPMI medium 1640  supplemented with 10% fetal bovine serum , 1% chicken serum, 50 µM 2-mercaptoethanol, and 1.5 mg/mL G418  at 40 °C in a humidified incubator with 5% COThe 3p21.3-loxP targeting vector was constructed as an X3.2 vector, which contains additional restriction enzyme sites  of the AgeI site in the X3.1 vector . The DNA oligomer was inserted into the BbsI site of pX330-U6-Chimeric_BB-CBh-hSpCas9, which was a gift from Feng Zhang (Massachusetts Institute of Technology) .sgRNA target1 (D3S1568) targeting 5′-GATTGAGCGGCCCTCACTTCAGG-3′ was constructed with 5′-caccGATTGAGCGGCCCTCACTTC-3′ and 5′-aaacGAAGTGAGGGCCGCTCAATC-3′ primers. sgRNA Target 2, targeting 5′-GCATCGTACGCGTACGTGTTTGG-3′ was constructed with 5′-caccGCATCGTACGCGTACGTGTT-3′ and 5′-aaacAACACGTACGCGTACGATGC-3′.The CRISPR/Cas9 vector construction was performed as previously described  containing the truncated chromosome 3 were transfected with the loxP vector and Cas9 vectors by lipofection with a 0.2 µg loxP plasmid, and each of 0.8 µg Cas9 vector with 7.5 µl Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. After 24 h in a basic growth medium, the cells were cultured in a medium containing G418. Fourteen days later, drug-resistant colonies were selected and expanded for further analysis.The CHO hybrids (4 × 105 cells) containing HAC were transfected with the Cre expression vector pBS185 CMV-Cre by lipofection with 2 µg loxP plasmid with 7.5 µl Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. After 24 h in a basic growth medium, the cells were cultured in a HAT (Sigma) medium. Fourteen days later, drug-resistant colonies were selected and expanded for further analysis.CHO hybrids  Marker sequences are described in Supplementary Table hTERT was analyzed using the following primers: forward, 5′-GCCTTCAAGAGCCACGTC, reverse: 5′-CCACGAACTGTCGCATGT. cDNA was amplified using an Applied Biosystems StepOne thermal cycler system and a SYBR Green PCR kit . The mRNA levels were normalized against GAPDH mRNA .RNA isolation and reverse transcriptase (RT)-PCR were performed as described previously  and truncated chromosomes that have lost this region and performed functional analysis using those chromosomes. The overall strategy for this approach is shown in Fig. To investigate the gene(s) involved in regulating the expression of the HAC can clone megabase-sized genomic regions using the Cre/loxP-mediated gene cloning system  gene for a positive selection resistance gene  clones (clone 3 and 4) showed the expected homologous recombination bands (product size of 473 bp and 1958 bp) by detecting specific primers for the 5′ junction sequence (primer pair#1) , and blasticidin S (To clone the 3p21.3-p22.2 locus in 3delp22 onto a HAC (HAC2)   in HSC cells. To further investigate whether suppression of hTERT transcription affects the proliferative rate, we performed a comparative analysis of the proliferation rate of the parental cells, 3p21.3-HAC clones, 3delp21.3 clones, and intact3 clones. The results showed that the 3p21.3-HAC clones showed a significantly decreased growth rate compared with the parental cells and 3delp21.3 clones. In addition, compared with intact3 and 3p21.3-HAC, there was a tendency for an even lower proliferative rate in intact3; however, the difference was insignificant. These results suggest that the suppression of hTERT expression by 3p21.3-HAC negatively regulates cell proliferation.To investigate hTERT suppressor gene encodes human chromosome 3 in RCC and OSCC cell lines  is a tumor suppressor gene in the 3p21.3 region. Overexpression of RASSF1 induces cell cycle arrest in lung carcinoma cell line H1299. RASSF1A inhibits cell cycle progression by preventing the accumulation of Cyclin D1  is a tumor suppressor gene in the 3p21.3 region. Ectopic expression of ZMYND10 in breast cancer cells induces apoptosis and inhibits cell growth, migration, and invasion in vitro and in vivo. ZMYND10 suppresses NEDD9, a known oncogene related to metastasis and mRNA translation, by upregulating miR-145-5p  are long (> 200 bp) non-coding RNAs that do not overlap regions of protein-coding and are categorized as long non-coding RNAs (lncRNAs) (Ulitsky and Bartel https://genome.ucsc.edu/). This indicates that lncRNAs, including lincRNAs, may be involved in regulating hTERT.Non-coding RNAs may potentially affect the transcriptional regulation of hTERT protein levels but not hTERT mRNA in human cervical cancer and thyroid carcinoma cell lines  are non-coding RNAs. The overexpression of miR-138, coded on the 3p21.3 region, was reported to downregulate CYP3A) is the most abundant P450 isozyme in the human liver and small intestine, metabolizing approximately 50% of commercially available medical drugs. Using the HAC vector system, we cloned approximately 700 kb of the human CYP3A gene cluster  containing its regulatory region into the HAC vector and generated mice transferred with the CYP3A-HAC vector. To generate fully CYP3A humanized mice (CYP3A-HAC/Cyp3a-KO), we also generated mice in which the endogenous mouse Cyp3a gene cluster was disrupted (Cyp3a-KO mice) and crossed them with CYP3A-HAC mice. In these mice, tissue-specific expression of CYP3A4 was observed in the liver and small intestine, similar to that in humans. In addition, CYP3A4, specifically expressed in the adult stage, and CYP3A7, specifically expressed in the fetal stage, were expressed in the adult and fetal stages, respectively  contribute to gene regulation by restricting chromatin interactions between target genes and regulatory sequences such as enhancers. TADs are the basic units of 3D nuclear organizations  genes at the sequence level, suggesting that the transcriptional regulator CTCF binding to the insulator of the ZMYND10 gene results in the formation of a barrier within the separate epigenetic domains of the RASSF1A and ZMYND10 genes juxtaposed in the 3p21.3 gene cluster region Supplementary file2 (XLSX 13 KB)Below is the link to the electronic supplementary material.


Mycobacterium tuberculosis (Mtb) F-ATP synthase generates most of the biological energy currency ATP. Previously, we identified the mycobacterium-specific loop of the F-ATP synthase subunit γ as a new anti-tuberculosis target and discovered the novel diaminopyrimidine GaMF1, whose potency was improved by structure-activity relationship studies leading to the analog GaMF1.39. Here, we report that GaMF1.39 depletes cellular ATP formation by targeting the mycobacterial F-ATP synthase without affecting proton coupling or oxygen consumption. The antimycobacterial compound is bactericidal and potent against Mtb in macrophages without inducing phenotypic changes in biofilm formation, planktonic bacteria, or being toxic to zebrafish larvae. Combining GaMF1.39 with the NADH dehydrogenase inhibitor clofazimine, the cyt-3bcc:aa inhibitor Telacebec, or the F-ATP synthase inhibitor TBAJ-876 showed enhanced whole ATP synthesis inhibition and anti-tuberculosis activity. These results suggest that GaMF1.39 may add value to a compound combination targeting oxidative phosphorylation for tuberculosis treatment.The 1FO-ATP synthase elements opened the door for a renaissance of structure-based target identification and inhibitor design. In this study, we present the GaMF1.39 antimycobacterial compound, targeting the rotary subunit γ of the biological engine. The compound is bactericidal, inhibits infection ex vivo, and displays enhanced anti-tuberculosis activity in combination with ETC inhibitors, which promises new strategies to shorten tuberculosis chemotherapy.New drugs are needed to combat multidrug-resistant tuberculosis. The electron transport chain (ETC) maintains the electrochemical potential across the cytoplasmic membrane and allows the production of ATP, the energy currency of any living cell. The mycobacterial engine F-ATP synthase catalyzes the formation of ATP and has come into focus as an attractive and rich drug target. Recent deep insights into these mycobacterial F World Health Organization’s 2021 Global TB report (Mycobacterium tuberculosis (Mtb), which causes TB, are spreading worldwide, causing major global health issues. Drug tolerance of Mtb is proposed to be due to the ability of the pathogen to enter a metabolically quiescent state, in which it is phenotypically tolerant to drug challenge with conventional chemotherapeutics (1FO ATP synthase and bringing oxidative phosphorylation (OXPHOS) and the electron transport chain (ETC) complexes (Tuberculosis (TB) is one of the major bacterial-caused infectious diseases and resulted in 1.5 million deaths in 2020, which, for the first time in over a decade, has increased according to the omplexes  into theomplexes –10. Hoomplexes , along womplexes . The antomplexes , 13–161FO ATP synthase, which is a latent ATPase and essential for growth (3:β3:γ:ε:9b-δ:b′:a:c (+-translocating FO domain (subunits 9a:c) uses the proton motive force (PMF), generated by the ETC complexes NADH dehydrogenase (in the case of NDH-1) and cytochrome 3bcc:aa (cyt-3bcc:aa), to drive rotation of the central stalk subunits γ-ε. The latter causes sequential binding, entrapment, and phosphorylation of ADP to ATP within the nucleotide-binding and catalytic α3:β3-headpiece. The peripheral stalk subunits b-δ:b′ smooth the transmission of power between the rotary c-ring and the α3:β3:γ:ε domain ..Mycobact3bcc:aa inhibitor Telacebec (Q203) alone and in combination with the cyt-bd inhibitor ND-011992, as well as TBAJ-876, respectively.Here, we present a detailed antimycobacterial activity study of the bactericidal GaMF1.39, evidence for its specific targeting of the mycobacterial F-ATP synthase, and anti-TB potency in macrophages, without altering biofilm formation or being toxic to zebrafish larvae. GaMF1.39 potentiates the anti-TB activity of the NADH dehydrogenase (NDH) inhibitor clofazimine (CFZ), the cyt-50) of 6.8 µM and MIC90 of 12.2 µM  and Mtb (3.0 µM)  of M. bovis BCG and M. smegmatis was inhibited at an IC50 of 51.6 ± 1.35 and 90 ± 1.1 nM  (IC50 = 0.14 nM and MIC50 = 1 nM)   −1 −1) was calculated (Since GaMF1.39 lacked the ability to induce uncoupled proton pumping  and doesotein)−1 . When th in IMVs , a 51% ilculated , and incMtb γ-loop ], a molecular docking approach was performed , maintained the hydrogen bonding interactions with R71 main chain amide atoms  and γD174 residues (3.8 Å) (2) atom mediated the hydrogen-bonded interaction with γ-loop residue Q171 . The benzimidazole ring was positioned to maintain van der Vaal’s contacts (cyan dotted lines) with H65 , E169 (3.18 Å), and R172 side chain amide atoms (3.4 Å). Finally, the 4-chlorophenyl group (ring A) was also in close anchorage  to residue Q46 of the c subunit (light brown).Recent NMR titration experiments and docking studies of the parent molecule GaMF1 demonstrated its binding to the b γ-loop . To obtaerformed . Overall (3.8 Å) , likewisb′ , where the inhibitor is interacting mainly with the γ-loop residues and its surrounding subunit γ residues. The data underline that GaMF1.39 binds specifically to the mycobacterial γ-loop.During 360° rotation, the γ-loop of the rotary γ subunit reaches one conformation, where it comes near the peripheral stalk subunit b′ . To testa, b, b′, δ, and ε, the recombinant M. smegmatis F1-αβγH65W (MsF1-αβγH65W) mutant was generated  of 0.7 ± 0.09 µM. Considering GaMF1.39’s 4-chlorophenyl group proximity to the c subunit residue Q46, which would be missing in our MsF1-αβγH65W:GaMF1.39 bound complex, the determined DK is in a similar range as the IC50, determined for the M. smegmatis IMVs (90 ± 1.1 nM)  for binding affinity measurements. The γH65 residue lies in close proximity (4.6 Å) to the ligand binding site. Homology models by mutation of γH65 to tryptophan did not show any steric clashes with its neighboring residues or alter the secondary structure. Furthermore, this variation should not alter ligand binding as the closest carbon atoms of the indole ring of tryptophan residue are spaced at 2.33–2.35 Å from the chloro-benzene ring of GaMF1.39 . To avoienerated . The fluGaMF1.39 . As a coM. bovis BCG was tested at eightfold its MIC50, which is shown clearly by broth experiments and on 7H10 agar plates . At that concentration, GaMF1.39  when compared with the initial inoculum (day 0). The infected macrophages did not show any visible optical alteration of the membrane, cell form, or size as tested microscopically. This is in line with experiments performed on zebrafish embryos, where no signs of toxicity-induced killing were recorded in the presence of 3, 9, or 30 µM (10× MIC50 against Mtb) of GaMF1.39 , and under growth conditions, simulated by the addition of fresh growth medium  while being slightly less lipophilic (clogP = 6.51) than BDQ (clogP = 7.25).The killing efficiency of GaMF1.39 against GaMF1.39  is more GaMF1.39 . To explGaMF1.39 . Similarh medium . These rMtb, the ETC has been demonstrated to be a druggable complex with modulators such as (i) CFZ, proposed to compete with the mycobacterial specific electron acceptor menaquinone for its reduction by the NDH-2 complex , achieving a minimum fractional inhibitory concentration (FIC) index of 0.75 in M. bovis BCG (Table S1). In comparison, the combination of the bc1 inhibitor Q203 and GaMF1.39 showed a significant growth reduction . In addition, Q203 lowered the MIC50 of GaMF1.39 from 6.8 to 0.23 µM (Table S1). Since GaMF1.39 showed no cross resistance to BDQ (see above), we tested whether GaMF1.39 would antagonize or potentiate the activity of the clinical development candidate and BDQ analog TBAJ-876, which is less lipophilic (clogP = 5.80), has higher clearance, and displays lower cardiotoxic potential than BDQ  thiostatic , or  of the GaMF1.39 + Q203 than of the GaMF1.39 + CFZ combination, we tested whether the increase in growth reduction of the GaMF1.39 + Q203 regimen  would al2 from central carbon metabolism, thereby facilitating redox balance and generating a PMF essential for maintaining transmembrane electrochemical gradients to regulate PMF-driven pumps and to drive ATP synthesis via OXPHOS  strains were used in this study. The cultures were maintained in complete Middlebrook 7H9 medium  supplemented with 0.5% (vol/vol) glycerol , 0.05% Tween-80 (Sigma-Aldrich), and 10% Middlebrook albumin-dextrose-catalase (ADC) (Sigma-Aldrich). Mtb H37Rv (ATCC #27294) cultures were maintained in Middlebrook 7H9 broth medium supplemented with 0.2% glycerol, 0.05% Tween 80, and 10% ADC at 37°C with 5% CO2. Bacterial colonies were grown on Middlebrook 7H10 agar (Sigma-Aldrich) supplemented with 0.5% (vol/vol) glycerol, 0.05% Tween-80, and 10% Middlebrook oleic acid-albumin-dextrose-catalase (OADC) (Sigma-Aldrich). BDQ and Q203 were purchased from MedChem Express  and freshly dissolved in 100% dimethyl sulfoxide . Compound TBAJ-876 was synthesized by Bioduro LLC  as described previously . Test compounds were dispensed into each flask and incubated at 37°C with shaking at 110 rpm for 5–6 days. Approximately 10 µL of culture was taken out of each flask, followed by a serial dilution with PBS. Twenty-five microliters of cultures of respective dilutions was plated on each quadrant of 7H10 agar plates. M. smegmatis mc .matis mc  155 and 2). Confluent THP-1 cells were then treated with 200 nM phorbol myristate acetate (Sigma-Aldrich) and distributed at a density of 3 × 106 cells per well in 24-well plates. After 24 h of differentiation, the cell monolayers were infected with Mtb at a multiplicity of infection of 10 for 60 min. Pre-warmed complete RPMI medium, with or without the test drugs, was added. BDQ at 2 µM was used as a positive control, whereas the DMSO-treated group served as a drug-free control. Mycobacterial viability was determined after 5 days of infection by CFU determination on agar plates.THP-1 cells  were grown to reach confluence in Roswell Park Memorial Institute (RPMI) media (Sigma-Aldrich) under standard cell culture conditions  is calculated using the following equation: CLint (µL/min/mg protein) = ln(2) × 1,000 / t1 / 2 / protein conc.The microsomal stability assay was outsourced and performed by Bioduro-Sundia Ltd., USA, based on the following protocol described previously , 37. WorM. smegmatis and M. bovis BCG, cells were grown overnight at 37°C in 7H9 medium supplemented with 10% ADC, 0.5% glycerol, and 0.05% Tween-80 until they reached an optical density (OD) of 0.6–0.7 at 600 nm. The culture was expanded in 200 mL of supplemented 7H9 medium and grown in 1 L shake flasks (180 rpm) until it reached an OD of 0.6–0.7. This culture was used to inoculate a 500-mL culture that was then grown overnight in 2 L shake flasks (180 rpm) until it reached an OD of 0.6–0.7. Approximately 5 g (wet weight) of WT M. smegmatis, M. smegmatis I66M c-subunit mutant strain, and M. bovis (BCG) was resuspended in 20 mL membrane preparation buffer (50 mm (3-(N-morpholino)propanesulfonic acid) (MOPS), 2 mm MgCl2, pH 7.5) containing EDTA-free protease inhibitor cocktail  and 1.2 mg·mL−1 lysozyme. The suspension was stirred at room temperature for 45 min and additionally supplemented with 300 µL of 1 M MgCl2 and 50 µL DNase I, and stirring was continued for another 15 min at room temperature. All subsequent steps were performed on ice. Cells were lysed by three passages through an ice-cooled microfluidizer  at 18,000 psi. The suspension containing lysed cells was centrifuged at 4,200 × g at 4°C for 20 min. The supernatant containing the membrane fraction was further subjected to ultracentrifugation at 45,000 × g at 4°C for 1 h. The supernatant was discarded, and the precipitated membrane fraction was resuspended in membrane preparation buffer containing 15% glycerol, separated into aliquots, snap-frozen, and stored at −80°C. The concentrations of the proteins in the vesicles were determined by the bicinchoinic acid assay . IMVs were stored at −80°C.In order to purify the IMVs of M. smegmatis IMVs was measured on the basis of a decrease in 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence using a Cary Eclipse fluorescence spectrophotometer  as described previously (−1) were pre-incubated at 37°C in 10 mm HEPES/KOH (pH 7.5), 100 mm KCl, and 5 mm MgCl2 containing 2 µM ACMA, and a baseline was obtained by monitoring for 5 min. The reaction was started by adding 2 mM ATP or 2 mM NADH. After 8 min, any proton gradient was collapsed by the addition of 2 µM of the uncoupler SF6847 . The excitation and emission wavelengths were 410 and 480 nm, respectively.ATP-driven proton translocation into eviously . IMVs  containing 10 µM ADP, 250 µM Pi, and 1 mM NADH. The concentration of Pi was adjusted by adding 100 mM KH2PO4 to the assay buffer. ATP synthesis was started by adding IMVs containing M. smegmatis WT, M. bovis BCG, and the I66M c-subunit mutant to a final concentration of 5 µg·mL−1. The reaction mixture was incubated at room temperature for 30 min before adding 50 µL of CellTiter-Glo reagent, followed by incubation for another 10 min in the dark at room temperature. The luminescence produced, which correlates with the amount of ATP synthesized, was measured by an Infinite 200 Pro plate reader (Tecan), using the following parameters: luminescence; integration time, 500 ms; and attenuation, none.ATP synthesis was measured in flat-bottomed white 96-well microtiter plates (Corning). The reaction mix (50 µL) comprised assay buffer , and the liposomes were resuspended in ATP synthesis buffer . ATP synthesis was measured at 37°C by a continuous luciferase assay, monitoring the emitted light in a luminometer . The ATP synthesis measurement was carried out on white flat-bottomed 96-well microtiter plates. A total of 375 µL of the proteoliposomes, containing the reconstituted MsF-ATP synthase and 20 µL ATP Bioluminescence Assay Kit CLS II , were mixed in the individual wells for 3 min at 37°C, and a baseline was recorded for 3 min at 37°C. After pre-incubation, ATP synthesis was started by the addition of 2 µM valinomycin (Sigma-Aldrich) to induce a ΔΨ and 5 mM ADP . For inhibitor studies with GaMF1.39 (90- and 180 nM) or BDQ (4 nM), proteoliposomes containing the reconstituted F-ATP synthase were additionally pre-incubated for 10 min at 4°C with the respective inhibitor before the ATP synthesis measurements were carried out.Recombinant w et al. . The purw et al. . ProteolMsF1-αβγH65W mutant was generated via site-directed mutagenesis using the following two primers: 5′-CTG GAC TGG CCG CTG CTC GTG GAG-3′ and 5′-CAG CGG CCA GTC CAG CGC ACT GGC-3′, and pYUB1049-MsF1-αβγ as template , and DNA sequencing  was performed to confirm the integrity of the plasmid. Plasmids carrying the gene of interest were then transformed into M. smegmatis mc24517 electrocompetent cells for large-scale protein production and purification  was titrated in 50 mM Tris-HCl, pH 7.5, and 150 mM NaCl with increasing concentrations of GaMF1.39, while quenching of tryptophan fluorescence was monitored at 320 nm following excitation at 295 nm. Dissociation constant (DK) and maximum fluorescence (ΔFmax) values were determined following the fitting of the data to an equation describing binding to a single affinity site (Steady-state fluorescence measurements were performed on a Cary Varian Eclipse fluorescence spectrophotometer (Leine) using a 10-mm path length quartz cuvette. Both excitation and emission slit widths were set to 5 nm. The binding affinity of GaMF1.39 to ity site .Mtb subunit γ was generated to obtain the missing coordinates for missing γ-loop residues as described in Hotra et al.    with a γLigand preparation: The 3D coordinates of GaMF1.39 were prepared using ligand preparation using the OPLS4 force field. All possible conformation states at pH 7.0 were generated using the Epik Classic tool during ligand preparation, and energy was minimized using the Macromodel tool from the Schrödinger suite of programs .Docking studies: The residues within 5 Å vicinity of the γ166–179 loop were used to define the site point to dock the ligands with default parameters, and the inbuilt ChemPLP and gold scoring functions in the gold program  were usen = 120) were used. The embryos were immersed in 5 mL of E3 medium containing GaMF1.39 in a 6-well plate for 5 days. DMSO was used as vehicle control. The embryos were observed under a microscope. Abnormal phenotypes and survival rates in each treatment group were observed for 5 consecutive days. The experiment was repeated twice, with three replicates each.Zebrafish care and ethics statement: All zebrafish experiments were approved by the NTU Institutional Animal Care and Use Committee under reference no. A20038. Experiments were done using wild-type AB zebrafish. The ages of the embryos are shown in hours post fertilization (hpf). Experiment procedure: De-chorionated zebrafish wild-type embryos at 48 hpf  to a final OD600 of 0.05. Then, 1 mL of the diluted culture was then added to each well of the 24-well plate and incubated at 37°C with 100 rpm shaking. Following 3 h of incubation, each well was washed once, and the culture media was replaced with 1 mL of 1× PBS (pH 7.4) or fresh M9 glucose medium containing 1–3 µM of BDQ or 3–9 µM of GaMF1.39. The treated samples were further incubated under the same conditions for 3 h. At t = 6 h, the samples were collected. The 1× PBS (pH 7.4) buffer containing suspended bacteria cells with treatment was collected into 1.5 mL Eppendorf tubes and considered to be “planktonic samples.” Subsequently, each well was washed once with 1 mL of 1× PBS (pH 7.4) before resuspending biofilm cells in the same volume of 1× PBS (pH 7.4). Biofilm cells were dislodged into the buffer by means of a cell scraper, and 1 mL of the sample was collected into 1.5 mL Eppendorf tubes labeled “biofilm samples.” The samples contained in Eppendorf tubes were sonicated in a water bath using the following settings: 5 min degas mode, 37 Hz, 100%, followed by 5 min pulse mode, 37 Hz, 100%. Subsequently, the samples are serially diluted and used for CFU counts. The experiment was repeated independently at least two times, with two technical replicates per independent experiment. CFU counts were analyzed using GraphPad Prism V9.3.0  was done to analyze the results. The FICI is calculated as  + . This calculation was done only for wells that showed 50% inhibition of bacterial culture growth compared to drug-free bacterial culture wells. A FICI of ≤0.5 indicates synergy, a FICI of >0.5 to 4 indicates additivity (no interaction), and a FICI of >4 indicates antagonism (A checkerboard titration assay was carried out as described previously , 47. Britagonism .


They are proved to be the mixture of normal CA2 pyramidal neurons, CA3-like neurons in CA2-CA3 mixed border, some CA2 interneurons and rarely few CA1-like neurons, which are probably the ones projecting to the revealed CA2 downstream targets, VMH, STHY and PMV in WT mice injecting this AAV/M1-Cre virus but not in Amigo2-Cre mice. Though it is still challenging to get a pure CA2 tracking and manipulation system, this tool provides a new, more flexible and extended strategy for in-depth CA2 functional study in the future.Mice hippocampus contains three prominent subregions, CA1, CA3 and DG and is well regarded as an essential multiple task processor for learning, memory and cognition based on tremendous studies on these three subregions. The narrow region sandwiched between CA1 and CA3 called CA2 has been neglected for a long time. But it raises great attentions recently since this region manifests the indispensable role in social memory. Its unique physical position connecting CA1 and CA3 suggests the potential novel functions besides social memory regulation. But the CA2 is too small to be accurately targeted. A flexible AAV tool capable of accurately and efficiently targeting this region is highly demanded. To fill this gap, we generate an AAV expressing Cre driven by the mini Map3k15 promoter, AAV/M1-Cre, which can be easily utilized to help tracing and manipulating CA2 pyramidal neurons. However, M1-Cre labeled a small percentage of M1The online version contains supplementary material available at 10.1186/s13041-023-01038-6. As one of the most studied brain regions, hippocampus is well known for its crucial roles in learning, memory and recognition due to the decades of unstopped study enthusiasm in this field. The best-known function of hippocampus is to act as the key regulator of episodic memory and spatial navigation , 2. ThesHowever, hippocampus contains another region, a narrow region sandwiched between CA1 and CA3, called CA2. CA2 has several distinguished features comparing to CA1 and CA3. The pyramidal neurons in CA2 have simple spines as CA1 pyramidal neurons but they have larger soma body size than CA1. While the pyramidal neurons in CA3 have thorny excrescences (complex spines) on their dendrites and similar soma body size as CA2 pyramidal neurons –10. CA2 The function of CA2 keeps mysterious for a long time. Until recently a bunch of studies indicate that this region actually plays unique roles in social memory formation –15. And To realize accurate targeting to CA2 regions, Hitti and Kohara et al. constructed two knock-in mouse lines respectively, Amigo2-Cre and Map3k15-Cre, specifically labeling CA2 pyramidal cells , 18. But6–8 week-old male C57BL/6 mice were kept group-housed in 4 per cage. Inbred mouse strain Tg (Amigo2-cre) 1Sieg (Amigo2-Cre) was acquired from the Jackson Laboratory and inbred in the Animal Core Facility of Nanjing Medical University  for experiments. The mice were housed under standard laboratory conditions with access to food and water ad libitum, stable temperature (22 ± 1 °C), and 12-h light–dark cycle (lights on at 07:00). All animal care and experimental procedures were followed by the Animal Experimental Ethical Guide of Southeast University and Animal Core Facility of Nanjing Medical University.The promoter of specific CA2 expression gene was identified based on Allen Brain Atlas and Mouse Genome Informatics. The sequences of mitogen-activated protein kinase kinase kinase 15  and regulator of G protein signaling 14  were downloaded from the NCBI. Map3k15 and RGS14 promoter were amplified using mouse DNA as a template. Activity of promoters was detected by OIBO company .And the following primers are used for the amplifications: 5′-gctggaactagggaaggatttc-3′ (Map3k15-2.3 kb promoter-F) & 5’-atcgtagaaggcatcgagcac-3’ (Map3k15-2.3kb promoter-R);5′-ggaggaagtcagggagggaggaaa-3′ (Map3k15-0.5 k promoter-F3) & 5’-gcggggctggcggcttcgaa-3’ (Map3k15-0.5k promoter-R3);5′-tgctccctatctctctgctttctg-3′ (RGS14-1.4 k promoter-F) & 5’-tctgccacaggaagtgtctcta-3’ (RGS14-1.4k promoter-R).The specific CA2 promoter construction plasmids  were packaged into Adeno-associated viruses (AAV)-M1-CRE (Map3k15-0.5 kb), AAV-M2-CRE (Map3k15-2.3 kb), and AAV-RGS14-CRE (RGS14-1.4 kb) by OBIO . AAV-CMV-EGFP (AOV022-1 AAV2/9), AAV-hSyn-DIO-mcherry (H4828 AAV2/9 CK1214 AAV2/9) were purchased from OBIO . AAV-Ef1a-DIO-EGFP , AAV-hSyn-CRE , AAV-Ef1a-DIO-hM4Di-mcherry (PT-0043 AAV2/9) were purchased from VTA . Clozapine-N-oxide  was purchased from APEXbio .12 vg/ml) and AAV-hSyn-CRE (1.09 × 1012 vg/ml) were injected with equal amounts of viral particles by controlling the injection volume, and the volume ratio of AAV-CRE and AAV-DIO was ensured to be 2:3. A syringe pump  was used to inject 100 nl of AAV into the dorsal CA2 region bilaterally at 10 nl/min. The needle was gradually withdrawn 20 min after injection. To prevent hypothermia after anesthesia, the mice were positioned on a heating pad through the procedure until fully awake. The injected mice were monitored daily for recovery status, body weight and infection.P30 mice were anesthetized with isoflurane (2–5%)  and had their scalp immobilized in a stereotaxic apparatus (RWD). After a precise craniotomy, the AAV was loaded in a glass pipette pulled by a heater, which was slowly targeted for injection into the dorsal hippocampus CA2 . AAV-M1-CRE , which consists of two parts, the center (29.7 cm diameter in the center of the box) and the periphery. Mice were allowed to move freely for 10 min in the box. The movement of mice was recorded and analyzed with a webcam and Ethovision XT software .stranger1-Timeempty (stage 1), and social difference score = Timestranger2-Timestranger1 (stage 2) were used to define the social novelty and social memory of the subject mice.The three-chamber behavior apparatus (60 cm long × 40 cm wide × 22 cm high) containing three compartments with equal size, which were separated by two walls with small doors (5 cm wide × 8 cm high). 20 min after intraperitoneal injection of CNO or vehicles, mice were placed in the device and allowed to explore freely for 10 min. The left and right chambers were placed with empty inverted uncapped cages before the experiment. Stranger mice  were placed in the inverted cages in the chamber where the subject mice were not habitually explored. During the social novelty stage, the time that the test mice spent sniffing the novel mice and the empty cage in the other chamber was compared. After 10 min, the test mice were isolated in the middle chamber for 10 min, and at the same time a second novel stranger mouse  was carefully put into the inverted cage that was originally empty in the previous stage. The subject mice were released from the middle chamber to explore the two compartments, and social preference was determined by comparing the time the mice took to sniff S2 and S1 in the last 10 min. Before introducing new subjects into the apparatus, the chambers and inverted cages were thoroughly cleaned with 25% ethanol. Behavior was monitored and scored for social novelty and memory which were represented by the anogenital and nose-to-nose sniffing time of the subject mice. Sniffing time was calculated by a trained observer, who was unaware of genotype and treatment of the mouse. The formulas, social difference score = Time2 bullets, animals were perfused intracardially with ice-cold PBS followed by 4% ice-cold PFA/PBS. Mouse Brains were fixed in 4% PFA overnight and then transferred into 30% sucrose for dehydration. After being embedded in Tissue-Tek® O.C.T., the brains were snap-frozen in liquid nitrogen and stored at − 80 °C. The brains were sectioned into the slices with the thickness of 30 µm using a Leica cryostat .After appropriate anesthesia with the COBrain serial sections were attached to glass slides coated with poly-L-lysine , and then washed for 15 min with PBS. Heated-antigen retrieval was performed for staining. In general, water-bathed sections were incubated at 96 °C for 5 min in 50 mM sodium citrate pH 8.5 . After being washed from the incubator for 5 min, the sections were permeabilized in 0.3% Triton X-100 in PBS (PBT) for 30 min. Subsequently, the sections were blocked at room temperature for 2 h with 10% Fetal Bovine serum (FBS) in 0.3% PBT (10% FBST) before overnight incubation with primary antibodies at 4 °C diluted with 10% FBST (1:500). The next day, the sections were washed 7 times for 35 min in 0.3% PBT and stained with secondary antibody (1:500) in 10% FBST for 1 h, followed by 5 times washing in 0.3% PBT for another 25 min. After the sections were stained with DAPI , the slices were mounted. Images were captured under a Zeiss confocal microscope LSM 700 or LSM 900 . The whole coronal brain image was taken by Palm Microbeam (Zeiss).+ cell number in RGS14 stained region was defined as total CA2 cells (Cell sum) and is normalized as 100%. The formulas for Fig. For RGS14, Calretintin, Calbindin, GAD67, Parvalbumin, NeuN, mcherry, CaMKII-α, PCP4 and GFP staining, the brain sections were incubated with the primary antibodies  and secondary antibodies . For CA2 spine imaging, RGS14 and CaMKII-α were costained on 1.5%PFA fixed brain slices with the thickness of 150 μm prepared by a vibratome. Sections were incubated with 5% FBS in 0.1% PBT for 1 h, then incubated with the primary antibodies against RGS14 (1:500) and CaMKII-α (1:500) in 3% FBS (0.05% PBT) overnight, and visualized using secondary antibody Alexa 555 (1:200) and Alexa 647 (1:200). The cells were counted on the complete coronal hippocampus images which were acquired through stitching the 10 × confocal microscopic diagrams together. RGS14ImageJ software  and ZEN  were used to count cells and measure soma size.Mice were perfused with 1.5% PFA, and the brains were postfixed by the same solution overnight. Then, 150 µm-thick sections were prepared by a vibratome and labeled by Dil-labeled Au particles using Gene gun. After 24 h at room temperature proceed with incubation with antibody to RGS14 (1:500) in 10% FBS for 16 h, and visualized using secondary antibody to mouse Alexa 647.Ordinary one-way ANOVA, post-hoc test Bonferroni’s test, and two-tailed unpaired Student’s test was used to assess statistical significance as described in the figure legends. GraphPad Prism 7  was used for data analysis. Photoshop CS6  was used to merge confocal images to fully present the hippocampus. All the statistical data in the graphs are presented as the mean ± SEM. p < 0.05 was considered significant. All p values were shown in the figures and n represented the number of animals.https://portal.brain-map.org), we decided to choose comparatively more restricted CA2 markers, RGS14 and Map3k15, for further promoter activity and specificity test. We wonder which gene and which promoter region are the best choice for CA2 highly preferential viral plasmid construction. Therefore, several vectors containing different promoter fragments from rgs14 and map3k15 with various lengths were constructed and tested. Using the luciferase assays for the promoter activity tests, we screened out a fragment from map3k15 with the length of around 500 bp, which displays the highest luciferase activity  in CA2  or hSyn driven EGFP expressed cells   mossy fibers from dorsal dentate gyrus  for M1-Cre treatment   mixture performed as before in dorsal CA2  layer also show the larger cell body size than CA1 pyramidal neurons in Py layers as previously reported [+RGS14− neurons from stratum oriens (Or) layer are also quantified and their body size is close to the body size of PV and CB interneurons but not CR interneurons in Py layer  layer of CA2 . But some of them are M1+RGS14− neurons. They can be detected in different area of CA2. Half of them are normal CA2 pyramidal neurons. But half of them are CA3-like neurons (grey) within CA2–CA3 mixed border (Z4 area) or CA2b region tightly close to the border (Z4–Z3 border). The case to detect CA1-like M1+RGS14− neurons in CA1–CA2 border is very rare comparing to the possibility to detect CA3-like M1+RGS14− neurons in CA2–CA3 border.We summarize what we found about CA2 M1The mixed usage of two viruses usually has a lower site accuracy than the single viral usage. It is consistent with our results in Fig. To test if M1 driven system can be utilized for anterograde neural tracing, AAV9/M1-Cre & AAV9/EF1α-DIO-mcherry or AAV9/M1-Cre & AAV9/EF1α-DIO-EGFP were injected and fluorescent signals were traced Fig. A. We sucLast we wonder if AAV9/M1-Cre virus suitable to be applied into neural activity regulation through chemogenetic manipulation in CA2 region. To test chemogenetic efficiency, AAV9/M1-Cre & AAV9/EF1α-DIO-hM4Di-mcherry were co-injected into CA2 Fig. A and sucOverall, we successfully developed a CA2 highly preferential Cre viral system to label and manipulate neural activity in CA2 region. It can efficiently drive CA2 pyramidal neurons as what Amigo2-Cre transgenic mice does. Meanwhile, it even displayed an extraordinary CA2 targeting accuracy comparing to most of current available targeting systems for CA2 and it is much more flexible to be utilized into different mice to realize the CA2 specific manipulation and gene deletion.+RGS14+ and M1+RGS14− pyramidal neurons in Py layers of CA2. M1+RGS14+ neurons occupy around 80% of total M1+ cells and they are all typical CA2 pyramidal neurons. For those M1+RGS14− neurons in Py layers of CA2, their cell body size and CaMKII presence still announce their typical CA2 pyramidal neuron identity. Further studies on spine morphology indicate that the M1+RGS14− CA2 like pyramidal neurons in CA2-CA3 border actually are the mixtures of normal CA2 neurons (with simple spines) and CA3-like neurons (with complex spines). Half of M1+RGS14− neurons are the normal CA2 pyramidal neurons, which occupy around 10% of total M1+ neurons in CA2. Some of M1+RGS14− cells  does localize in Or or SLu layer of CA2. They are probably GAD67+ interneurons with other novel markers need to be further explored. Meanwhile CA2 neurons displayed diverse morphology of pyramidal neuron cell bodies not only in M1+RGS14− neurons in CA2 Py layers, but also in M1+RGS14+ or regular CA2 neurons in Py layer sometimes. This finding transform our former impression on pyramidal neurons (more homogenic in morphology and function comparing to the interneurons) in hippocampus. But it is consistent with the previous reports that CA2 does have multiple pyramidal neuron types [Moreover, our mini-Map3k15 driving Cre system enriched pyramidal neuron types able to be manipulated in CA2. Using this system, we highly preferentially labeled CA2 neuronal cells containing both M1on types , 23.+ neurons in CA2 Py layers cannot colocalize with Amigo2. That is, this portion of M1+RGS14− pyramidal neurons could not be labeled by Amigo2-Cre mice. And it won’t be surprised if this group of pyramidal neurons have different axonal projections with M1+RGS14+ or Amigo2+RGS14+ pyramidal neurons in CA2. And this also could explain why we could trace CA2 projecting axons to VMH, STHY and PMV using our M1-Cre system whereas this result could not be recapitulated in Amigo2-Cre mice. Similar phenomena were observed in other Cell-type specific Cre models [One more thing needs to be noticed. Given the 97% overlapping between RGS14 with Amigo2 as previously reported, ~ 10% of total M1e models , 25. Thee models . But in e models , 27.To further increase the accuracy and usage of the CA2 specific viruses, the following strategies should be considered in the future. First, a more restricted AAV serotypes such as AAV2 or AAV5 is worth testing. Second, the viral tools for different purposes in CA2 region should be developed. An AAV/M1-Cre-EGFP (mcherry) for neural labeling/tracing and an AAV/M1-Cre-hM4Di (or hM3Dq) for neural activity regulation should be generated separately so that one viral usage can be applied instead of using mixed viruses. Third, it is uncertain if AAV/M1-Cre or Amigo2-Cre can accurately label ventral CA2. But the in-situ hybridization (ISH) data for Amigo2, RGS14 and Map3k15 in Allen Brain Atlas display the expression discrepancy of these genes in anterior and posterior part of the hippocampus which means they might not be able to correctly label the posterior CA2 as well as the anterior CA2. Otherwise, CA2 interneurons definitely need to be labeled and manipulated separately to study their own functions in the future. It is necessary to be mentioned that M1-Cre virus is obviously hard to label most of interneurons. GAD67/Map3k15 colocalization events observed in Fig. Additional file 1: Figure S1. for Figure 3 Quantitative analysis for soma size in different types of hippocampus neurons, and M1/PCP4 and M1/RGS14 co-staining comparisons. A Quantitative column chart displaying the soma size of cells in hippocampus. For measurements, images were examined using image J by delineating the soma fluorescence edge. The surface areas of CA1 pyramidal neurons, CA3 PN and CA2-EGFP+ were quantified by measuring AAV9/M1-EGFP-expressing neurons in IHC images. The surfaced areas of PV, CB and CR were quantified by measuring antibodies stained in IHC images. All means were calculated from no less than 5 brain slices of each biological sample. Data are presented as mean ± SEM. B Scatter chart displaying the proportions of M1+PCP4- and M1+RGS14- neurons in the total Map3k15-EGFP neurons. The proportions of PCP4-failed expressed and RGS14-failed expressed neurons in the EGFP were similar. C Pie charts displaying the proportions of M1+PCP4- and M1+PCP4+ in Map3k15-EGFP, and the proportions of M1+RGS14- and M1+RGS14+ in M1-EGFP. D Pie charts displaying the proportions of M1+PCP4- and M1+RGS14- neurons located in CA2 Or and Py. The proportions of M1+PCP4- in Py and Or are the number of PCP4-in Py or Or divided by total PCP4-failedexpressed neurons number. Figure S2. for Figure 5 Description of M1-EGFP+ neurons outside CA2 region, and expression pattern of the AAV9/hSyn-DIO-mcherry and M1-CRE co-injection system. A1-A3 Images of M1-EGFP co-stained with RGS14. Box in A1 displays RGS14-EGFP+ neurons considered as the neuron outside RGS14 stained region. Dash-line boxes in A2 and A3 display virus leakage in the case of small probability. D1-D4, E1-E4 Coronal serial sections of dCA2 from two different mice infected with AAV9/hSyn-DIO-mcherry and M1-CREfrom head to tail. Scale bars: 200 μm, 500 μm.


N-acetylneuraminic acid, its γ-lactone derivative, and N-acetylneuraminic acid that overcomes the limitations of the previous analytical procedure for their identification.A subclass of the sialic acid family consists of intramolecular lactones that may function as key indicators of physiological and pathological states. However, the existence of these compounds in free form is highly improbable, since they are unlikely to exist in an aqueous solution due to their lability. Current analytical method used to detect them in biological fluids has not recognized their reactivity in solution and is prone to misidentification. However, recent advances in synthetic methods for 1,7-lactones have allowed the preparation of these sialic acid derivatives as authentic reference standards. We report here the development of a new HPLC–MS method for the simultaneous detection of the 1,7-lactone of The online version contains supplementary material available at 10.1007/s10719-023-10114-x. N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), and 2-keto-3-deoxy-D-glycero-D-galactonononic acid  are a family of naturally occurring α-keto acids with nine carbon atoms derived from three members, Due to the importance and diversity of Sias, efforts have been made to develop appropriate analytical methods for their identification in biological samples of different origins. The approach that has contributed most to the mapping of these sugars to date is based on the derivatization of their hydroxyl groups as heptafluorobutyrates (HFB) prior to GC–MS analysis . About f8 reversed-phase column and [13C3]Neu5Ac as an internal standard. A stability study was also performed on the different solvents and conditions used for the analysis and purification steps from the biological matrix (plasma). Finally, we have performed experiments in human plasma that completely rule out the possibility that the free 1,7-lactone of Neu5Ac can survive in free form in this biological fluid, even as a more stable γ-lactone.Therefore, in this work we have developed a new analytical HPLC–MS approach that overcomes the flaws of the GC–MS method and allows the simultaneous detection of underivatized Neu5Ac 1,7-lactone, its γ-lactone, and Neu5Ac using a CN-acetylneuraminic acid (Neu5Ac) was purchased from Carbosynth . 1,2,3-[13C3]-N-acetylneuraminic acid  was purchased from Isotec (Sigma Aldrich). N-acetylneuraminic acid 1,7-lactone  and N-acetylneuraminic acid γ-lactone (Neu5Ac-γ-lactone) were prepared in our laboratory following the procedures previously published – 290.0881, found 290.0882. Other physico-chemical properties were identical to those previously reported – 290.0881, found 290.0882. Other physico-chemical properties were identical to those previously reported N-acetylneuraminic acid (311.11605 m/z), N-acetylneuraminic acid 1,7-lactone and N-acetylneuraminic acid γ-lactone (290.08814 m/z). Calibration was performed before the analysis using the Pierce™ Negative Ion Calibration Solutions .Mass spectrometry analysis was performed using a Q-Exactive Plus Orbitrap mass spectrometer equipped with an Ion Max API source fitted with an electrospray probe (HESI-II) . The mass spectrometer was set with ESI spray voltage of 2.50 kV, capillary temperature of 320 °C, sheath and auxiliary gas flow of 35 and 10 units, respectively and S-lens RF value of 50%. All spectra were acquired in negative ion mode with a full-scan MS spectra visualizing, from 70 to 400 mData acquisition was carried out by the Excalibur software  and raw files elaboration was accomplished using Freestyle 1.8 SP2 software (Thermo Fisher Scientific).13C3]Neu5Ac, Neu5Ac-1,7-lactone and Neu5Ac-γ-lactone were prepared accurately dissolving 1 mg of the standard compound in 1 ml of deionized ultrapure water (LC–MS grade) for Neu5Ac and [13C3]Neu5Ac, and acetonitrile (LC–MS grade) for the two lactones, obtaining solutions with a concentration of 1000 mg/L. The stock solutions were aliquoted and stored at -20 °C. Neu5Ac and [13C3]Neu5Ac were found unchanged after up to 1 month, unlike the 1,7-lactone stock solution that showed, after 2 weeks, a 35% of degradation. 10 mg/L working solution of Neu5Ac and [13C3]Neu5Ac were prepared making a 1:100 dilution of the stock solution in ultrapure water; 50 mg/L Neu5Ac-1,7-lactone and Neu5Ac-γ-lactone solutions were prepared making 1:20 dilution of their stock solutions in acetonitrile (LC–MS grade).Standard stock solutions of Neu5Ac, Neu5Ac 10 mg/L at 0 °C and vortexed for 30 s. Successively, a series of injections for HPLC–MS analyses were performed at different times . Notably, the stability has been evaluated both at RT and 4 °C. In addition, to test the stability to the drying process, a replicate of the sample at t = 0 has been incubated in the SpeedVac at 40 °C for 40 min, then resuspended in CH3CN/water  mixture and injected for the analysis.90 µL of a selected solvent: CH3CN starting from the 50 mg/L working solution and stored at -20 °C until use. Successively, 5 µL of the analyte and 5 µL of 10 mg/L [13C3]Neu5Ac standard have been added to 90 µL of a CH3CN/water, 1:1 v/v mixture (to ensure the complete solubilization of both the molecules) to obtain the final 1,7-lactone concentrations of 5, 1, 0.5, 0.25, 0.1 mg/L. Acquisitions using full scan and PRM modes have been collected. All the steps have been performed at 4 °C in accordance with the results reported and discussed in the “Some sequential dilutions of Neu5Ac 1,7-lactone have been prepared in CH13C3]Neu5Ac 10 mg/L and 5uL of 1,7-lactone 50 mg/L (or 5uL of water for the controls) were added to 100uL of the related matrix and the final solution was vortexed for 1 min. Then, 100 μL of deionized ultrapure water was added, followed by a 30 s vortex (this step was exclusively applied to BSA and plasma matrices). Successively, after the addition of 300 μL of acetonitrile (to precipitate proteins), all samples were vortexed for another 1 min and, finally, centrifugated at 15,000 g × 5 min. The water sample was directly injected for HPLC–MS analysis  before analysis  before analysis  or plasma. In particular, for the last two, was used a 5% BSA solution prepared in ultrapure water and a pooled blank human plasma stored at -80 °C and melted to room temperature. All the preparation steps were executed operating rigorously in ice. Regardless of the matrix, 5 µL of [t-test has been performed. Values of p < 0.05 were considered statistically significant.GraphPad Prism 8  was used for graphical display and statistical analysis of the obtained data. The results were presented as the mean of two or more independent experiments carried out in duplicate. An unpaired 1H NMR tabulation in the “13C3]Neu5Ac) used as an internal standard. The Thermo Fisher C8 aQ column proved to be the best choice, allowing the separation of the three analytes with high resolution and very short elution time (see Table 3CN (or other solvents) used to resuspend the sample affects the retention time of the analytes, as observed for the two lactones  at 4.09 min corresponded to the intact 1,7-lactone, while the minor peak (2) at 4.27 min corresponded to the γ-lactone, which conceivably formed from the 1,7-lactone due to the presence of water. This hypothesis was confirmed after injection of pure standard γ-lactone (50 mg/L) solubilized in CH3CN, yielding a single peak that eluted at 4.27  obtained by a 1:20 dilution of the 1 mg/ml stock solution in pure CH3CN. In this case, two additional peaks (3 and 4) were observed, distinct from those of 1,7-lactone (1) and γ-lactone (2)  Fig. b. Peak 35Ac Fig. c, wheream/z (Neu5Ac), 311.11605 m/z ([13C3]Neu5Ac), and 290.08814 m/z . This type of acquisition is based on the isolation of the different precursor ions and their successive fragmentation with a specific collision energy (NCE = 42.5), resulting in a specific fingerprint  was found.The method also provided parallel reaction monitoring (PRM) analysis in negative ion mode with an inclusion list consisting of 308.09870 nt Figs. . Under tterature . In mostm/z) showed a highly overlapping fragmentation pattern  was occasionally detected.Moreover, the two peaks corresponding to the molecular weight of two lactones  and analyzed using the method settings reported previously. The resulting curve , calculated by linear regression using the peak areas in full-scan mode and weighted by a factor of 1/x2, gave an R2 value of 0.990 . Briefly, lower amounts of 1,7-lactone  were injected in the presence of 10 mg/L Neu5Ac as an internal standard. Specifically, 5 µL of 50 mg/L pure (> 95%) 1,7-lactone was dissolved in ultrapure water, and the purification protocol  before analysis. As shown in Fig. First, the purification protocol was simulated in pure water to investigate the feasibility of the different steps in a simplified aqueous environment, using Neu5Ac 1,7-lactone and [13C3]Neu5Ac as an internal standard). This experiment was key to understanding the possible effects of the protein environment on the stability of our analyte. A 5% BSA solution (pH 7.2/7.3) was chosen as the plasma surrogate matrix, as described in the literature [13C3]-Neu5Ac ratio of 0.30 in full scan analysis and 0.36 in PRM. This evidence definitively confirmed the instability of 1,7-lactone, at least in its free form, and suggested that 1,7-lactone-derived γ-lactone should be sought as a potentially more suitable biomarker because it is more stable in the surrogate matrix, especially in a protein-rich environment with a pH equivalent to that of plasma.The purification method was then applied to a surrogate matrix spiked with Neu5Ac 1,7-lactone (and [terature . Specifi13C3]Neu5Ac as a standard), and the purification protocol was applied. Surprisingly, no traces of 1,7- or γ-lactone were found in all experiments performed under either condition (40 °C SpeedVac and direct injection after precipitation)  Figs. c and S3cn) Figs. c, confirIn light of these data, it seems that it is very difficult to detect this metabolite in this biological fluid, not even as an intermediate γ-lactone; it is likely that it converts rapidly to Neu5Ac through a variety of unstable intermediates. All assumptions about 1,7-lactone's existence and its behavior in the biological matrix are purely speculative. Therefore, it is likely that the complex protein and enzyme environment in plasma greatly affect the stability of both lactones, which means 1,7-lactone, if it exists, can only be detected if glycoconjugates are present as its more stable form.N-acetylneuraminic acid, its γ-lactone derivative, and N-acetylneuraminic acid without derivatization. The method overcomes the limitations of existing analytical procedures that lead to the misidentification of these compounds. In addition, the best conditions to keep the 1,7-lactone stable in solution were investigated. Nevertheless, only Neu5Ac could be detected in plasma, suggesting that the free 1,7-lactone cannot be identified in this biological fluid, even in its more stable γ-lactone intermediate.In this work, we have developed an HPLC–MS method that allows the simultaneous separation of the 1,7-lactone of Supplementary file1 (DOCX 754 KB)Below is the link to the electronic supplementary material.