TDP-43 proteinopathy in Alzheimer’s disease (AD) patients is recently emerging as a relevant pathomolecular event that may have been overlooked. Recent results in immortalized lymphocytes from AD patients have shown not only an increase of post-translational modifications in TDP-43, such as hyperphosphorylation and fragmentation, but also its prionic behaviour and cell-to-cell disease transmission. With the main goal to advance therapeutic interventions, we present in this work different kinase inhibitors with potential to restore this pathological mechanism.We have used immortalized lymphocytes from healthy controls and AD severe patients to evaluate the correction of TDP-43 pathology after the treatment with previously synthetized TTBK1 and CK1 inhibitors. Moreover we used the conditioned mediums of these cells to perform different disease propagation experiments.TDP-43 pathology observed in lymphoblasts from severe AD patients is reduced after the treatment with TTBK1 and CK1 inhibitors , Furthermore, the significant increase in TDP-43 phosphorylation, cytoplasmic accumulation and aberrant F-actin protrusions (TNT-like structures) observed in control cells growing in CM from AD lymphoblasts were abolished when the CM from AD lymphoblasts treated with previously reported TTBK1 and CK1 inhibitors were used. In addition, the cytosolic transport mediated by molecular motors of the receptor cells was altered with the induced TDP-43 pathology, but it was not produced with the abovementioned pretreated CMs.TTBK1 and CK1 inhibitors, specially VNG1.47 and IGS2.7 compounds, restore TDP-43 pathology and avoid cell-to-cell propagation in immortalized lymphocytes from AD patients, being excellent candidates for the future therapy of this prevalent and devastating disease. N-methyl-D-aspartate (memantine) (Alzheimer’s disease (AD) is the most common form of dementia accounting for more than 35 million people affected worldwide. It is considered a chronic illness and also a highly concerning world health problem. As the main risk factor is age and different studies predict an increase of the life expectancy of the population, an important escalation of the number of AD patients is expected in the following years . In thismantine) .In the last years, a new protein has attracted the focus in the field of neurodegenerative diseases: transactive response DNA-binding protein of 43 kDa (TDP-43). Hyperphosphorylated TDP-43 was identified as the main component of ubiquitinated protein aggregates found in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTLD) patients . Since tParticularly in AD, several studies have determined the impact of the deposition of TDP-43 in patients’ brain, stablishing that it aggravates the memory loss and the hippocampal atrophy. Furthermore, TDP-43 pathology has been found in half of the patients that were considered to havepure Alzheimer’s disease . MeanwhiTDP-43 is an important protein involved in the transcription, splicing and transport of numerous RNAs and it is found mainly in the nucleus where it exerts its main functions. However, at physiological conditions, it presents a localisation equilibrium with the cytoplasm, where it can be found in a 5–20% of the total amount of the protein . In pathin cellulo and in vivo models , cell division cycle kinase 7 (CDC7), glycogen synthase kinase 3 (GSK3) and tau tubulin kinase 1 (TTBK1) the better characterized . Brain po models .in vivo pharmacokinetic profiles with a 2 to 3:1 ratio regarding brain to plasma penetration and show motor neuron protection and decrease of TDP-43 phosphorylation in the spinal cord of TDP-43-transgenic mice after chronic treatment , penicillin/streptomycin , Fetal Bovine Serum (FBS) , polyvinylidene difluoride (PVDF) membranes for Western blots , Chemiluminescence (ECL) system , Pierce BCA Protein Assay kit , protease inhibitor complete mini mixture .50 values regarding kinase inhibition together with effective permeability values that predict their ability to cross the blood–brain barrier (BBB), are provided in Kinase inhibitors IGS2.7 and VNG1.47 were synthetized in our laboratory following previous described procedures . Their cAntibodies used in this study are listed in Blood samples from two healthy controls and two patients suffering from severe AD were obtained after written informed consent. Patients were diagnosed in the Hospital 12 de Octubre  as severe Alzheimer’s disease patients based on the criteria of the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRDA) . ClassifPeripheral blood mononuclear cells (PBMCs) were isolated on Lymphoprep™ density-gradient centrifugation following the manufacturer’s instructions . Lymphoblastoid cell lines (LCLs) were established by infecting peripheral blood lymphocytes with the Epstein Barr virus (EBV) as previously described .6 cells/mL) that contained 1% penicillin/streptomycin and 10% (v/v) fetal bovine serum (FBS). U2OS cells were obtained from the American Type Culture Collection and grown in DMEM media, supplemented with 10% (v/v) fetal bovine serum and 1% penicillin/streptomycin. All cell lines were grown at 37°C in a humidified 5% CO2 atmosphere.Lymphoblastoid cell lines were grown in suspension in T flasks in an upright position, in RPMI 1640 medium  or IGS2.7 (5 μM). Conditioned medium was collected after 72–96 h and added to healthy cells in a ratio 3:1 with fresh medium. After 72 h cells were harvested and processed for Western blotting or immunofluorescence analysis.2, 0.5% (vol/vol) sodium deoxycholate, 0.5% (vol/vol) NP-40 and 0.1% (vol/vol) SDS), containing 1 mM phenylmethylsulfonylfluride (PMSF), 1 mM sodium orthovanadate, 1 mM sodium pyrophosphate and protease inhibitor mixture). Pierce BCA Protein Assay kit was used to determine the protein content of the extracts.Cells were collected by centrifugation, washed with PBS, and then lysed in ice-cold RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 15 mM MgClEqual amounts of proteins (50 μg) were resolved by SDS–polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride (PVDF) membrane and blocked with 5% BSA in TTBS 1X. The antibodies used are listed in Immunofluorescence analysis was performed on cells grown on coverslips. For the attachment of lymphoblastoid cells, the coverslips were previously coated with a solution of 0.025% Gelatin  for 30 min at room temperature followed by a solution of 1 mg/mL poly-L-lysine  diluted 1:50 in Borax buffer  overnight at 37°C. Cells were then fixed for 25 min with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 10 min. Then samples were blocked with PBS-1% BSA for 60 min at 37°C.Cells were incubated with the primary and secondary antibodies described in N-termini with Cy5 to provide a fluorescent kinesin binding peptide. Their synthesis and labelling has been previously described  and VNG1.47 (10 μM). After the CM treatments, the probe Cy5-KBP was added to the cells at 2.5 μM for 15 min at 37°C. Peptides were washed using DMEM without phenol red and imaged using a confocal laser scanning microscope Leica TCS SP8 with a 63x oil immersion objective that included a humidified incubation chamber, a CO–1. Track mean displacement and track mean velocity were calculated and normalized to control cells in each experiment. Data were analyzed by one-way ANOVA using GraphPad Prism 8 software. For multiple comparisons, Bonferroni’s correction was applied.Single-particle trajectories were analyzed with the TrackMate plugin from the ImageJ software. Specifically, the LoG detector and the linear motion LAP tracker were used to detect and link particles with an estimated blob diameter of 1 μm and at a maximum distance of 2 μM. More filters were included to discard trajectories with less than 5 spots and with a maximum speed of 1 μm sp-value <0.05” was considered statistically significant.Statistical analyses were performed using Graph Pad Prism software version 6 . All the statistical data are presented as mean ± standard error of the mean (SEM). Statistical significance was estimated using two-tailed Student’s t-test for statistical comparisons between groups, or one-way ANOVA followed by the Bonferroni test for multiple comparators. A “VNG1.47 and IGS2.7 have shown to be effective in the experimental treatment of lymphoblasts from ALS patients which are characterized by presenting TDP-43 hyperphosphorylated cytosolic aggregates, the main pathological hallmark of ALS . We haveWe next investigated whether the reduction of this post-translational modification restores the nucleus-cytoplasmic functional balance of TDP-43 and reduces the aberrant F-actin cytoskeleton protrusions, reminiscent of tunnelling nanotubes or TNT-like structures that characterized these AD lymphoblasts . With thThe prion-like properties of TDP-43 have been described in several TDP-43-linked diseases. We have recently documented the prionic behaviour of this protein in AD lymphoblasts throughout conditioned medium (CM) experiments which contains a 25 kDa TDP-43’s prionic fragment . To furtFirst, we cultured AD lymphoblasts treated with and without the kinase inhibitors for 72 h. These pre-treated (ptCM) and non-treated (CM) mediums from AD lymphoblasts were collected and added to healthy control lymphoblasts for 72 h. Immunofluorescence staining with an anti-TDP-43, revealed the presence of TDP-43 pathological changes such as cytosolic localisation and generation of TDP-43 aggregates in healthy cells exposed to CM from AD lymphoblasts compared to control cells  . HoweverFinally, we analysed the phosphorylation status of TDP-43 in these CM studies. TDP-43 phosphorylation was shown to be higher in healthy cells exposed to CM from AD lymphoblast than in those exposed to CM from healthy cells in both, the full-length protein and in the 35 kDa fragment . HoweverIn previous studies, we have shown that human osteosarcoma U2OS cells treated with the conditioned medium from lymphoblasts from severe AD patients also showed increased phosphorylation and mislocalization of TDP-43 . BecauseU2OS cells were treated with conditioned medium from healthy lymphoblasts and lymphoblasts from two different severe AD patients for 72 h. A significant reduction on the average displacement of Cy5-KBP particles was found compared to U2OS treated with conditioned medium from healthy controls, showing a deficit in the intracellular transport in AD samples . FurtherIncreasing amount of evidence indicates that TDP-43 pathology may play an important role in AD. TDP-43 inclusions have been found in approximately 50% of necropsies, most often in severe cases of AD, suggesting that TDP-43 can influence AD pathology and neurodegeneration. The presence of TDP-43 pathology in AD brain has been associated with greater memory loss and brain atrophy.To date no effective treatment for AD exists, pre-clinical pharmacological approaches aimed at blocking disease progression had been focused at the clearance of extracellular deposits of β-amyloid, formation of intracellular neurofibrillary tangles, or preventing inflammation and oxidative stress among other processes . On the in vivo and in vitro  and the Spanish National Research Council Institutional Review Board (15 March 2007).ÁM-R, AM, and CG: conceptualization. LM-G, EC, and CT-B: methodology. LM-G, EC, CT-B, VP, and VN: investigation. ÁM-R and AM: resources. AM, LM-G, and VN: writing—original draft preparation. EC, CT-B, LM-G, CG, VP, ÁM-R, and AM: writing—review and editing. AM and VP: funding acquisition. All authors have read and agreed to the published version of the manuscript.


Streptococcal species.Rapid diagnostic testing (RDT) allows antimicrobial stewardship programs (ASP) to intervene early in the setting of sepsis and septic shock leading to lower rates of death. Combining RDT with ASP results in faster antibiotic de-escalation compared to RDT alone; however, the utility of this approach for Streptococcal bacteremia is unproven. The objective of this study was to evaluate the impact of RDT with real-time ASP intervention on blood cultures growing Streptococcus spp. bacteremia over two 6-month periods. During the pre-RDT period, patients were prospectively followed by ASP who provided recommendations based on gram-stain and organism identification results. During the post-RDT period, blood cultures were tested by GenMark ePlex BCID-GP that identifies S. agalactiae, S. anginosis group, S. pneumoniae, S. pyogenes and Streptococcus spp. within 90 minutes which were reported directly to ASP for recommendations on antibiotic discontinuation, de-escalation, and additional diagnostic testing.Retrospective, observational study comparing patients with Table 1). The median ages were 58 and 56, 62% and 68% were men, and a total of 12% and 16% were immunocompromised. The mean qSOFA scores were 1.9 and 2. The most common Streptococcus spp. was S. mitis (figure 1). Prior to RDT, the median time to antibiotic de-escalation was 88 hours. Following implementation of RDT, median time to de-escalation was 42 hours (P=0.002). De-escalation to targeted therapy improved from 50% to 86% in post-RDT (P< 0.01). Transition to oral antibiotics increased from 14% to 30% (P=0.08). There were no differences in hospital re-admission or acute kidney injury. A trend toward decreased 30-day mortality was identified in the post-RDT period (P=0.06).94 patients were included in the analysis, 50 pre- and 44 post-RDT. Across cohorts, patient demographics, clinical characteristics, and severity of illness were comparable (Streptococcal bacteremia. We noted significant reductions in broad-spectrum antibiotic use and a trend towards lower mortality. Larger studies are needed to demonstrate the full potential of this approach.Our study highlights the impact of RDT with ASP intervention on clinical outcomes for patients with Ryan K. Shields, PharmD, MS, Allergan: Advisor/Consultant|Cidara: Advisor/Consultant|Entasis: Advisor/Consultant|GSK: Advisor/Consultant|Melinta: Advisor/Consultant|Melinta: Grant/Research Support|Menarini: Advisor/Consultant|Merck: Advisor/Consultant|Merck: Grant/Research Support|Pfizer: Advisor/Consultant|Roche: Grant/Research Support|Shionogi: Advisor/Consultant|Shionogi: Grant/Research Support|Utility: Advisor/Consultant|Venatorx: Advisor/Consultant|Venatorx: Grant/Research Support


Graphene is a two-dimensional (2D) material with a single atomic crystal structure of carbon that has the potential to create next-generation devices for photonic, optoelectronic, thermoelectric, sensing, wearable electronics, etc., owing to its excellent electron mobility, large surface-to-volume ratio, adjustable optics, and high mechanical strength. In contrast, owing to their light-induced conformations, fast response, photochemical stability, and surface-relief structures, azobenzene (AZO) polymers have been used as temperature sensors and photo-switchable molecules and are recognized as excellent candidates for a new generation of light-controllable molecular electronics. They can withstand trans-cis isomerization by conducting light irradiation or heating but have poor photon lifetime and energy density and are prone to agglomeration even at mild doping levels, reducing their optical sensitivity. Graphene derivatives, including graphene oxide (GO) and reduced graphene oxide (RGO), are an excellent platform that, combined with AZO-based polymers, could generate a new type of hybrid structure with interesting properties of ordered molecules. AZO derivatives may modify the energy density, optical responsiveness, and photon storage capacity, potentially preventing aggregation and strengthening the AZO complexes. They are potential candidates for sensors, photocatalysts, photodetectors, photocurrent switching, and other optical applications. This review aimed to provide an overview of the recent progress in graphene-related 2D materials (Gr2MS) and AZO polymer AZO-GO/RGO hybrid structures and their synthesis and applications. The review concludes with remarks based on the findings of this study. Graphenarticles . It has perature ,11,12. GThe GO and RGO are used as supporting platforms for biological binding processes due to their ease of functionalization, non-covalent binding, and variable hydrophobicity ,14,15. B1, sp2, and sp3 [2 carbons  with relatively large surface areas exhibits novel properties and is used in different industrial sectors [Among different materials, carbon-based materials play an important role in civilization . Carbon, and sp3 . The dif and sp3 . Carbon  and sp3 . Carbon  sectors ,45,46,47 sectors ,50,51,52 sectors . Recent  sectors  on the f sectors ,56. Grap sectors . CNTs we sectors . In 1991 sectors  was the  sectors . CNTs, wOrganic dyes are one of the biggest families in the synthetic organic domain. For example, in the textile domain, the most common dyes can be classified in two ways: (i) based on their application characteristics or (ii) based on their main chemical structure ,61,62. TThe azobenzene family is the photochromic molecule capable of changing shape and polarity upon light irradiation. Azobenzene is a molecule able to change conformation from trans to cis upon light  and reveThis section discusses existing graphene-based materials that can serve as the basis for functionalization and GO syntheses available in the literature. Various functionalization methods for graphene and the azobenzenic functionalization of GO are also discussed.−1 (relativistic speed) [Graphene oxide, which has better oxidative characteristics than graphene, or nanoporous graphene (NPG) could be employed instead of graphene alone ,67. Becac speed) . Graphenc speed) . Graphenc speed) . Becausec speed) . Graphenc speed) . A schemc speed) .The functionalization of graphene and graphene derivatives has increased the number of potential applications of graphene-based materials. Covalent functionalization, non-covalent functionalization, substitutional doping of graphene, and hybridization with nanoparticles, nanowires, and other materials have been classified as functionalization modes based on the methods and materials used . These v4 and HNO3 to oxidize graphite. Hummer’s approach combines graphite, potassium permanganate, and H2SO4 in a solution. The conjugation of stacked graphene sheets is broken down into nanoscale sp2 graphitic domains surrounded by extremely disordered oxidized domains (sp3 C/C) and carbon vacancy defects when graphite is oxidized [There are two approaches:top–down and bottom–up, to synthesize GO. In thetop–down approach, physical methods such as mechanical/ball milling, sputtering, laser ablation, etc., are used. In chemical methods, there are two approaches such as chemical and green routes. It is necessary to define graphene composites before proceeding with the analysis. Graphene composites are any graphene-based materials that have been changed . The GO is the most well-known graphene composite material, produced via the chemical exfoliation of graphite. Graphene has been severely oxidized and contains a range of oxygen functions. Many theories ,78 have oxidized . The pheoxidized .4 with fuming HNO3) [2SO4, HNO3, and KClO4 [2SO4 and KMnO4. There are two ways to prepare RGO: chemical reduction and annealing at a high temperature. The most effective thermal annealing method [2 hybridized carbon atoms. Graphene can easily be converted to GO and RGO. G is hydrophobic, whereas GO is hydrophilic. As a result, GO is easily dispersible in water. GO contains aliphatic (sp3) and aromatic (sp2) domains, increasing surface interactions. A range of approaches for surface modification of graphene has been developed in response to the increased interest in employing graphene as a reinforcing filler in polymer matrices to construct multifunctional materials. The filler-matrix bonding interaction considerably impacts the final composite properties. Most dispersion processes result in non-covalent composites in which the polymer matrix and filler connect via relatively weak dispersion forces. To enhance stronger interfacial interaction, researchers are increasingly focusing on creating covalent connections between graphene-based filler and the supporting polymer. Graphene becomes best functionalized via sp2 stacking complexation or by inserting particular moieties on edge or basal planes (2-conjugated carbons [Hummers and Offeman  developeng HNO3) . The fornd KClO4 . Later, nd KClO4  developeg method  is thermg method ,90. Howel planes  due to t carbons .AZO chromophore has received extensive research as a molecular solar thermal storage material because of its ability to absorb light in the ultraviolet-visible range and release heat based on reversible isomerization . AZO preElectrostatic interactions between GOscis, a negatively charged material (carboxylate groups) on basal planes and edges , and theLi et al.  describeMost AZO-functionalized GO is mainly described for energy storage; very few investigations used the capability of AZO-GO for pollutant sensing and removal. The pesticide residues and metabolites in food, water, and soil are major issues. Fenitrothion (FT)  phosphorothioate) and modifying graphite pencil electrodes with AZO-GO displays excellent performance for the detection of these pollutants . The lowThe removal of bacteria from water is also of great interest, as the WHO declared in 2017 that ESKAPEE bacteria pose a serious public health problem . In thisA variety of photo energy conversion or storage devices may be made using photo-induced variations in microstructures, electrical characteristics, steric effects, and optical response of AZO moieties. AZO moieties functionalized with GO/RGO may reflect, prolong, and boost the optically controlled conductance, absorption, catalytic behaviour, and electrostatic response of the composite’s constituents. AZO-GO composites have many advantages: high quantum yields, charge transfer in nanoseconds at the interface, energy storage in chemical bonds, electrochemical catalytic activity, the regulated electrostatic environment surrounding carbon-conjugated structures, and ultrafast isomerization in a few picoseconds (10–12 s) . Table 1Photoswitches are molecules that alter structurally when exposed to light irradiation. Depending on the light irradiated wavelength, AZO can stay in two different isomeric forms, such as trans and cis. Its typical isomerization characteristics, including a moderate change in dipole moment, a low photobleaching rate, and a substantial conformational change, make it an excellent choice for the coupling counterpart in light-driven molecular switches. For instance, Kizhisseri et al.  found thH) and cis-to-trans thermal barrier (aE). RGO-cis-ortho-AZO remains thermally stabilized for a prolonged half-life (5400 h) through intramolecular hydrogen bonds, substantially longer than RGO-para-AZO (116 h). The RGO-para-AZO has a thermal storage capacity of 269.8 kJ kg−1 with a single intermolecular hydrogen bond, while RGO-ortho-AZO has a thermal storage capacity of 149.6 kJ kg−1 (44.55% less) with several inter- and intra-molecular hydrogen-bonds of AZO. The AZO-GO hybrid has the potential to use as energy storage material with the good thermal stability of cis-hybrid and substantial energy density of around 240 Wh kg−1 because of the lightweight structure and significant grafting density of AZO moieties, as stated by Pang et al. [−1 and 2230 Wkg−1, respectively. Optimizing molecular interactions with high-density, adjustable RGO-bis-AZO storage allows for the creation of solar-heat conversion materials. Luo et al. [−1 due to steric hindrance, intermolecular H-bonds, extended storage lifespan (52 days), and an outstanding cycling performance for 50 visible light-irradiated cycles at 520 nm, making substituted AZO-based graphene a stable, high-energy, and recyclable molecular solar thermal storage material. One of the key difficulties is developing multifunctional materials to harness solar energy as a sustainable resource. Solar thermal fuels have the potential to store solar energy in chemical bonds or structures and then release that energy as heat. AZO derivatives have a high potential for solar energy storage owing to their reversible isomerization, high light absorption, and thermal reversion regulated by steric structure and functional group. Feng et al.  developeg et al. . They deg et al. . Due to o et al.  reported2 g−1), AZO-GO materials have the potential to be used in electrochemical capacitors [Due to their inherent conductivity and extraordinarily high specific area  with AZO molecules can meet ever-growing and -challenging multifunctional system demands. Oxygen-containing groups on GO/RGO enable covalent and non-covalent composite synthesis, producing well-defined and customizable physical and chemical characteristics. These characteristics include increased quantum effects, effective charge transfer at the interface, electrostatic changes surrounding π-conjugated structures, optically controlled conductance, heat storage in chemical bonds, and steric conformation. These materials can be used in various devices such as photoswitches, photodetectors, phototherapy, molecular junctions, sensors, flexible photonics and electronics, solar thermal storage, smart devices, and biological-based recognition. In addition, these solar-powered materials might lead to eco-friendly, innovative, and sustainable goods. However, there are still some challenges, such as ensuring effective linkages between GO/RGO and AZO moieties, a comprehensive understanding of complex interactions between GO/RGO and AZO, low quantum and thermal storage yields of photoswitching isomers, modulating photochromic and electronic properties to enhance light sensitivity and shorten response time need to address for applying such multifunctional responsive materials with great success. A detailed analysis of the working process of GD-AZO composite materials, focusing on the molecular interaction between these structures, is also required to increase our understanding for future advancement.


Staphylococcus aureus is one of the most common organisms isolated from respiratory secretions in lung transplant donors and recipients perioperatively. Within the first 90 days after lung transplantation, methicillin-susceptible Staphylococcus aureus (MSSA) infections have been associated with increased mortality and acute and chronic rejection. However, it is unclear whether respiratory cultures positive for MSSA at the time of transplantation can lead to clinically significant infection. The aim of this study was to assess the microbiological and clinical outcomes for lung transplant recipients (LTRs) with positive perioperative donor or/and recipient respiratory cultures for MSSA. A retrospective study was conducted evaluating MSSA-positive respiratory cultures at the time of lung transplantation from donors and/or recipients from January 1, 2008, to December 30, 2019. Patients who did not have a bronchoalveolar lavage at 2 weeks after the lung transplant or died within 2 weeks of lung transplant were excluded. The main outcome was MSSA eradication at 2-week bronchoscopy. Recipients were evaluated for MSSA infections at the 12-week period after the transplant. Of the 1,678 individuals who underwent lung transplantation, 218 LTRs had S. aureus isolated in perioperative donor or recipient respiratory cultures, and 29 were subsequently excluded. Of the remaining 189 LTRs, MSSA eradication at the 2-week bronchoscopy was achieved in 186 (98.4%) recipients. During the 12-week follow-up, 15 (7.9%) recipients were diagnosed with MSSA pneumonia; concurrent MSSA bacteremia was noted in one recipient. No anastomotic infection, empyema, or lung abscess related to MSSA was diagnosed during the follow-up period.In LTRs, the rate of MSSA eradication at 2-week post-transplant recipients is high, and it is associated with a low rate of infectious complication within the first 12 weeks after transplant. Most of the recipients received a combination therapy with at least one agent active against MSSA. More studies to evaluate the optimal antimicrobial stewardship policies regarding the regimen and duration of antibiotic therapy for these patients are needed. Impaired graft mucociliary function, weak cough reflex, airway ischemia, perioperative invasive procedures, and the level of immunosuppression put LTRs at increased risk of infection, particularly pneumonia. During the early post-transplant period, pneumonia develops mainly due to nosocomial acquisition, recipient colonization, or donor-derived infection6. One of the most common organisms that has been implicated in positive respiratory cultures at the time of transplant is Staphylococcus aureus. Methicillin-susceptible Staphylococcus aureus (MSSA) contributes up to 25% of positive respiratory cultures in LTRs and 48% in donors8.Infection in lung transplant recipients (LTRs) is a leading cause of morbidity and mortalityS. aureus (MRSA) colonization at the time of lung transplant has been found to be a risk factor for progression to infection in early post-lung-transplant period, but there is no established correlation between MSSA isolated in peri-transplant respiratory culture and progression to infection. At our center, piperacillin-tazobactam is used empirically for prophylaxis after lung transplantation for 72 hours until intraoperative culture results are available. The practice of most transplant physicians is to switch antibiotics to cefazolin or cloxacillin if MSSA is isolated in respiratory cultures and treat for at least 14 days with intravenous antibiotic therapy. In the case of multiple organisms being recovered, or the recipient is known to be colonized with Pseudomonas, cefazolin or cloxacillin is merely added to piperacillin-tazobactam. This practice is mainly derived from studies of patients with MSSA bacteremia, where using an anti-staphyloccocal penicillin or cefazolin is associated with significantly lower 30-day mortality compared to piperacillin-tazobactam.Methicillin-resistant The aim of the study was to assess the rate of eradication of MSSA isolated from recipient or donor respiratory cultures at the time of lung transplantation and to assess the development of MSSA-related infection and complications during the first 12 weeks after the transplant.We conducted a retrospective observational cohort study at the Toronto General Hospital, a 456-bed hospital in Toronto, Ontario, Canada. LTRs with MSSA cultured from bronchoalveolar lavage (BAL) from recipients, donors, or both at the time of lung transplantation were recruited from January 1, 2008, to December 30, 2019. During the study period, the routine practice at our center consisted of performing surveillance bronchoscopy for all patients at 2 weeks, 6 weeks, and 12 weeks. All transplanted patients during the study time frame were eligible and were included if they met all the following criteria: (1) aged 18 years or older, (2) had follow-up surveillance bronchoscopy at 2 weeks, (3) survived for 2 weeks after lung transplant, and (4) if MSSA was isolated in the recipient, donor, or both. As mentioned, we categorized the source of the positive MSSA BAL culture into three categories: recipient, donor, and both donor and recipient. Patients were excluded if MRSA was cultured at the time of lung transplantation, in either the donor or recipient. The results of BAL cultures at 2 weeks, 6 weeks, and 12 weeks were assessed for MSSA eradication, persistence, recurrence, or reinfection, as defined in the following sections. Antibiotics used as a prophylaxis or treatment were assessed from the transplantation date through discharge date or 2 weeks after transplantation, whichever was longer.The primary endpoint was MSSA eradication from BAL culture at the 2-week bronchoscopy. The secondary endpoints included (1) MSSA eradication at 6 weeks and 12 weeks; (2) clinical outcome at the 2-week follow-up as assessed by clinical improvement, stability, or deterioration; and (3) clinical criteria of MSSA infection at follow-up that occurred by 12 weeks after lung transplantation (see Definitions)MSSA eradication was defined as a negative BAL culture for MSSA in the follow-up bronchoscopy at 2 weeks. MSSA persistence was defined as a continuously positive BAL culture since the time of transplantation. MSSA recurrence was defined as a positive BAL culture after eradication for MSSA with the same antibiotic susceptibility testing (assumed to be the same strain) compared with the initial MSSA strain isolated at the time of transplantation. Reinfection was defined as a positive BAL culture for MSSA after eradication with a different antibiotic susceptibility (assumed to be a different strain) in comparison to the MSSA strain isolated at the time of transplantation..The clinical assessment at 2 weeks was determined by improvement defined by a decrease in oxygen requirement, no ongoing signs of sepsis, and no new infiltration in the Chest X-ray (CXR); stability was defined by stable oxygen supplement since starting the targeted therapy, no ongoing signs of sepsis, and no new infiltration in the CXR; and deterioration by development of respiratory failure, fever or sepsis, or new infiltration in the CXR. MSSA infection within the first 12 weeks after lung transplant was defined according to the International Society for Heart and Lung Transplantation consensus criteriaData were abstracted from the database of the Toronto Lung Transplant Program of Toronto General Hospital for all patients’ cultures obtained at the time of transplantation and at 2 weeks, 6 weeks, and 12 weeks after transplantation. Paper and electronic patient charts were also examined for clinical data, laboratory data, vital signs, and diagnostic imaging. Collected data included patient demographics, underlying lung disease for lung transplant, comorbidities, antibiotic treatment, and clinical outcome.t test for continuous variables and chi-square or Fisher’s exact tests for categorical variables. All analyses were performed using GraphPad Prism version 9.4.1 Characteristics were compared using Student’s The study protocol was approved by the Research Ethics Board of the University Health Network, Toronto, Ontario, Canada . Patient consent was waived for this retrospective cohort study.S. aureus from BAL fluid at the time of transplantation. Twenty-nine recipients were excluded due to death within 14 days of transplant (5), lack of follow-up bronchoscopy at 2 weeks (5), or isolation of MRSA (7 in the recipient and 5 in the donor)  had positive respiratory cultures for e donor) . The basAll LTRs in our study had received empirical prophylactic antibiotics after transplantation. Piperacillin-tazobactam was the most frequently administered empirical therapy (70.3%), followed by meropenem (18.5%), and then ceftazidime (8.5%). One hundred and eighteen (62.4%) recipients received at least one dose of vancomycin empirically, in addition to a beta-lactam antibiotic, prior to the culture results being finalized.With regard to targeted antibiotic therapy for MSSA, 98 (51.9%) received a combination of antibiotics, whereas 91 (48.1%) received monotherapy. The mean duration of antibiotic therapy for MSSA was 12.5 days. The empirical and targeted antibiotic regimens for the 189 recipients are shown in The microbiological outcomes of the 189 patients are shown in P. aeruginosa pneumonia, acute rejection (6 out of 27), or myocardial infarction (3 out of 27); while four LTRs were treated for both rejection and pneumonia. Eventually two of the patients who developed myocardial infarction died.At the 2-week assessment, 150 (79.4%) recipients exhibited clinical improvement, while 29 (15.3%) recipients deteriorated, and 9 (4.8%) recipients were clinically stable. Of the 29 LTRs who deteriorated, MSSA pneumonia was confirmed in two patients. One received 14 days of vancomycin, and the second patient was on cefazolin since transplantation, which was switched to cloxacillin after a result of 2-week BAL due to progression of pneumonia. Both patients achieved full recovery. The causes of deterioration in the other 27 LTRs were attributed to non-MSSA pneumonia (14 out of 27), of which 6 were due to During the following 10 weeks of the observation period, an additional six recipients had MSSA-confirmed infection. Five of them had MSSA pneumonia, while one patient had concomitant MSSA pneumonia and bacteremia. The latter patient received 4 weeks of cefazolin for persistent MSSA bacteremia, but infective endocarditis was ruled out by transesophageal echocardiogram. Eventually, he achieved eradication of the blood stream infection and was discharged home. The other five patients received 2 weeks of different antimicrobial agents including cefazolin, piperacillin-tazobactam, and amoxicillin-clavulanic acid, and they all achieved full recovery. None of the recipients developed MSSA-related anastomotic infection, empyema, lung abscess, or infective endocarditis during the follow-up time. The details of the patient characteristics, treatment, and clinical outcomes in the recipients who developed MSSA infection are shown in We conducted a retrospective evaluation of the rate of microbiological eradication of MSSA isolated in BAL fluid at the time of transplant with follow-up to 12 weeks after lung transplant. We also looked at the development of MSSA infection and clinical outcomes in these recipients up to 12 weeks after transplant.This study demonstrated a high rate of MSSA eradication at the 2-week post-transplant bronchoscopy in the LTRs irrespective of it being of donor or/and recipient origin. To the best of our knowledge, our study is the first of its kind evaluating the microbiological outcomes associated with MSSA isolated in the respiratory cultures of LTRs perioperatively. Interestingly, more recipients had positive respiratory cultures for MSSA at 6-week (5 of 173) and 12-week (7 of 174) bronchoscopy than at the 2-week bronchoscopy assessment (3 of 189). This could be due to the fact that most patients had been treated with antibiotics within the 2-week time frame prior to the 2-week bronchoscopy, which may have contributed to the negative respiratory cultures. At the 2-week and 6-week assessment bronchoscopies, LTRs with positive respiratory cultures were more likely to have a similar strain of MSSA than positive cultures at 12 weeks, when acquisition of new MSSA strains contributed to a 57% positivity rate of the MSSA in respiratory cultures (4 of 7). However, the overall rate of eradication remained high up to the 12-week evaluation (96.1%).S. aureus is the second most common organism isolated in lung transplants perioperatively, with 74% of the isolates being MSSA. However, whether LTRs with positive MSSA cultures at the time of transplant will subsequently progress to clinically significant infection is unclear. We demonstrated a low prevalence of MSSA-related infection in those patients. Over the 12-week study period, only 8 (4.2%) recipients were diagnosed with MSSA pneumonia, and concurrent MSSA bacteremia was diagnosed in only one of these patients (0.5%). Shields et al. reported that 18% of LTRs developed S. aureus infection in the first 90 days after transplant, and 62% of them were due to MSSA. Although they found that the presence of MRSA in recipient sterility cultures and nasal swabs at the time of transplant was indeed a risk factor for early S. aureus infection, no specific risk factors were identified for MSSA infection. Our low rate of infectious complications due to MSSA compared with the previous cohort may reflect the results of aggressive antibiotic management approaches taken in our LTRs from the time of transplantation. We observed that more than half of LTRs were treated with a dual antibiotic therapy. This is likely owing to the presence of polymicrobial respiratory cultures with concomitant Pseudomonas and other gram-negative organisms . It should be noted that most of the recipients who received a dual antibiotic therapy had received cloxacillin or cefazolin in combination with an anti-pseudomonal antibiotic . This practice is derived from studies on MSSA bacteremia, which indicate that treating MSSA bacteremia with piperacillin-tazobactam, vancomycin, or ceftriaxone alone was associated with a poor outcome14. However, there is limited evidence of the impact of using beta-lactam, other than anti-staphylococcal beta-lactam, in clinical outcomes of patients with MSSA pneumonia. A meta-analysis by Whiddon et al. found that using ceftriaxone to treat MSSA pneumonia is associated with higher clinical failure than ceftaroline or ceftobiprole, which are highly active against S. aureus. In LTRs, such data are lacking. However, in contrary, recent data showed that antibiotic appropriateness at the time of lung transplantation has no impact on 30-day mortality, hospital length of stay, or intensive care unit length of stay.Previous studies have demonstrated that . On the other hand, the duration of antimicrobials for the treatment of gram-positive infection in this population has been associated with developing drug-resistant organisms. Presently, there are no studies that delineate the optimal perioperative antibiotic regimen or the duration of therapy for patients with MSSA isolated in respiratory secretions at the time of lung transplantation. More studies are needed to identify whether treatment of polymicrobial flora isolated from the BAL fluid cultures that may include MSSA at the time of transplantation requires prolonged broad-spectrum coverage, or whether a shorter duration would be adequate to diminish the threat of the emergence of resistance in keeping with the principles of antimicrobial stewardship.In our study, the mean duration of therapy was 12.5 days, which is considered a longer duration of antibiotic therapy. Previously, using a long-course perioperative antibiotic regimen had no mortality benefit in LTRs with positive cultures compared to a shorter courseOur study, however, has several limitations. The retrospective cohort design excluding patients who missed follow-up or died prior to the 2-week bronchoscopy hindered the assessment of the primary outcome and may have limited enrollment. Although none of the excluded recipients had MSSA infection as the cause of death, we still cannot comment on the short-term mortality related to MSSA due to the limited number of patients. In addition, we excluded those individuals who had positive respiratory cultures for MRSA (isolated from the donor or recipient) as well as concomitant MSSA and MRSA due to concerns about biasing the primary endpoint results for MSSA as MRSA may be harder to eradicate. Moreover, as with all retrospective studies, missing data at times posed a problem. This prevented us from comparing antibiotic regimens between those included and those excluded. Third, due to the low number of patients who received piperacillin-tazobactam or meropenem monotherapy, we could not evaluate whether the use of a broad-spectrum antibiotic alone versus an anti-staphylococcal beta-lactam may have exerted an impact on clinical or microbiological outcomes or on mortality associated with MSSA infection. Consistent with this, the number of recipients who developed clinical MSSA infection was low precluding any analysis of risk-identifying factors for MSSA infection.In conclusion, MSSA is a commonly isolated organism in the respiratory secretion cultures of donors and recipients at the time of lung transplantation. The rate of eradication by 2 weeks was high and was associated with a low rate of infectious complication within the first 12 weeks after transplant. Most of the recipients received a combination therapy with at least one agent active against MSSA. However, it was not clear whether this is an appropriate practice or not. More studies to evaluate the optimal regimen and its duration are needed to enhance antimicrobial stewardship in LTRs.


N-acetylglucosamine kinase (NAGK) has been identified as an anchor protein that facilitates neurodevelopment with its non-canonical structural role. Similarly, small nuclear ribonucleoprotein polypeptide N (SNRPN) regulates neurodevelopment and cognitive ability. In our previous study, we revealed the interaction between NAGK and SNRPN in the neuron. However, the precise role in neurodevelopment is elusive. In this study, we investigate the role of NAGK and SNRPN in the axodendritic development of neurons. NAGK and SNRPN interaction is significantly increased in neurons at the crucial stages of neurodevelopment. Furthermore, overexpression of the NAGK and SNRPN proteins increases axodendritic branching and neuronal complexity, whereas the knockdown inhibits neurodevelopment. We also observe the interaction of NAGK and SNRPN with the dynein light-chain roadblock type 1 (DYNLRB1) protein variably during neurodevelopment, revealing the microtubule-associated delivery of the complex. Interestingly, NAGK and SNRPN proteins rescued impaired axodendritic development in an SNRPN depletion model of Prader–Willi syndrome (PWS) patient-derived induced pluripotent stem cell neurons. Taken together, these findings are crucial in developing therapeutic approaches for neurodegenerative diseases. SNRPN) gene located on the 15q11-q13 region of the human chromosome, encodes for the SmN protein and is also associated with Prader–Willi syndrome/Angelman syndrome (PWS/AS) [Neurodevelopment is a complex process that involves the interaction of various genes and proteins. N-acetylglucosamine kinase (NAGK) is an enzyme that phosphorylates N-acetylglucosamine (GlcNAc) to produce GlcNAc-6-phosphate (GlcNAc-6-P), which is further utilized to synthesize oligosaccharide chains, glycolipids ,2, and g(PWS/AS) . In our (PWS/AS) . SNRPN, (PWS/AS) ,9,10. NA(PWS/AS) . In thisReports also suggested the crucial role of SNRPN in the regulation of neuronal-specific alternative splicing events . Since nNAGK also interacts with the dynein light-chain roadblock type 1 (DYNLRB1) protein in developing neurons . HoweverSNURF-SNRPN, MKRN3, MAGEL2, and NECDIN [PWS is a genetic disorder characterized by intellectual disability, hyperphagia, hypotonia, and other developmental abnormalities . The patd NECDIN . The 15qd NECDIN ,28. The d NECDIN . The used NECDIN ,30. The Our findings indicate that the dynein-mediated microtubule transport of the NAGK–SNRPN complex is involved in the axodendritic branching of the neuron.p < 0.001) (p < 0.001) from DIV 1–5 in neuronal processes (p < 0.001) and reduced significantly at DIV 21 (p < 0.001)  with antibodies specific to the NAGK and SNRPN proteins to observe NAGK and SNRPN interaction in primary hippocampal culture categorized as early (DIV 1/3/5) and late stages (DIV 12/16/21) . The neu< 0.001) A,B, and rocesses A,B. Simirocesses C. The NA< 0.001) C,D. Howe< 0.001) C,D.p < 0.001) (p < 0.05) (p < 0.001) at DIV 12–16/P12–16 compared to the DIV 21/P 21 lysates  assay from the protein lysates collected during the different developmental stages of the neuron, i.e., the early  and late  phases. The protein lysate was collected from the primary hippocampal culture at DIV 1/3/5/12/16/21 as the mouse pup’s brains from P1/3/5/12/16/21. NAGK and SNRPN expression at different stages of neurodevelopment was confirmed via the WB analysis . Express< 0.001)  and mous < 0.05) . In the  lysates .Although NAGK and SNRPN interaction was verified via yeast-two hybrid assay previously , in vitrp < 0.001) the neurite mean area calculated via the NeurphologyJ plugins in Image J software Version 1.43 (p < 0.001) through the knockdown of the NAGK and SNRPN proteins as compared to the control plasmid (p < 0.001) in the cell body (DAPI) and the process (GFP) of the neurons with NAGK, SNRPN, and NAGK + SNRPN plasmid overexpression in the neuron (p < 0.001) of the mean NAGK–SNRPN PLA dots was observed with NAGK, SNRPN, and NAGK + SNRPN protein knockdown in the cell body and processes of DIV 4 neurons  the frequency of axodendritic branching (p < 0.001) axodendritic branching compared to the control plasmid (p < 0.001) in the cell body and processes of neurons with NAGK, SNRPN, and NAGK + SNRPN overexpression (p < 0.001) in the mean number of NAGK–SNRPN PLA dots was observed with NAGK, SNRPN, and NAGK + SNRPN knockdown in the cell body and processes of DIV 14 neurons  at the early stages of development (p < 0.001) at DIV 3 (p < 0.001) and reduced significantly at DIV 21 (p < 0.001)  C,D.p < 0.001) during the early stages of development, as shown in p < 0.001) at DIV 3, as shown in p < 0.001), but reduced significantly at DIV 12 and 21 (p < 0.001), as shown in Similarly, we observed the interaction between SNRPN and DYNLRB1 proteins in primary hippocampal cultures at different developmental stages . NeuronaTo confirm the direct interaction between NAGK and SNRPN proteins with DYNLRB1, we conducted an in vitro binding assay using the His pull-down assay, as shown in To describe NAGK–SNRPN complex variability in the cell body and process of the neuron, we observed the microtubule transport machinery of NAGK and SNRPN in a melanoma cell line model. We performed ICC to observe the colocalization between NAGK, SNRPN, and DYNLRB1 on microtubules using SK-MEL-31 cells. The NAGK proteins were colocalized on the microtubule , which hWe further confirmed the dynein-mediated transport of the NAGK–SNRPN complex via two techniques in the SK-MEL-31 cells: (a) blocking dynein movement using the dynein inhibitor (ciliobrevin D) and (b) disrupting the microtubule using nocodazole. The microtubule transport mechanism of NAGK and SNRPN was observed using the dynein inhibitor  and microtubule-disrupting compound nocodazole (10 µM for 1 h) in SK-MEL-31 cells . The conWe attempted to demonstrate melanosome colocalization with the NAGK and SNRPN proteins since we used the melanosome trafficking pathway as a model to study NAGK, SNRPN, and DYNLRB1 interaction. We analyzed the colocalization of the NAGK and SNRPN proteins via ICC in the melanosome trafficking process with the melanosome-specific primary antibody (PMEL). Melanosomes were treated with vehicle (DMSO), ciliobrevin D (80 µM for 6 h), and nocodazole (10 µM for 1 h). ICC was performed with NAGK (red) and PMEL (green) primary antibodies and colocalization (overlay) was observed on the SK-MEL-31 cells . SimilarAfter observing the colocalization of NAGK, SNRPN, and DYNLRB1 proteins, we attempted to describe the role of NAGK and SNRPN in dynein-mediated cargo transport in SK-MEL-31 cells via observing the distribution pattern of melanosomes in the cell. We performed the NAGK, SNRPN, and DYNLRB1 protein overexpression and knockdown experiment on SK-MEL-31 cells to observe the distribution of melanosomes . OverexpFurthermore, knockdown of the NAGK, SNRPN, and DYNLRB1 plasmids along with the control was performed in SK-MEL-31 cells B. In theWe were curious to observe the effect of SNRPN depletion on neurodevelopment. To observe the effect of SNRPN depletion on neurodevelopment, we obtained patient- derived iPSC (PWS-2-9) with SNRPN deletion and normal human iPSC (UCH-HF-YK-27). We differentiated the human fibroblast-derived iPSC into neurons until day 30 in vitro. The morphology of the neurons was observed after fixing the neurons at day 30 of neuronal differentiation . The numAfter observing the SNRPN depletion effect in human neurons, we decided to observe the effect of exogenous NAGK and SNRPN on the human-iPSC-derived neuron. The PWS-2-9-cell-derived iPSC neurons were overexpressed with the NAGK and SNRPN transgenes. ICC was performed with MAP2  and α-tubulin (red) staining, and DAPI (blue) was used to stain the cell body of the neuron . The oveThe non-canonical role of NAGK in axodendritic development was reported previously ,7,11. AlDefects in the SNRPN locus are pointed to as the crucial factor in the PWS phenotype ,36,37,38The interaction of NAGK with dynein motor protein DYNLRB1 was verified through different molecular biological analyses including PLA, yeast two-hybrid assay, and immunoprecipitation assays . StudiesIn this study, we introduce the SNRPN protein as a new interacting partner with NAGK and DYNLRB1 proteins and regulate the retrograde cargo transport in the neuron. SNRPN’s gene locus is located within the paternally imprinted region of the genome that is critical in Prader–Willi syndrome (PWS). However, little is known about its behavior, except for its incorporation into snRNPs . The SNRStudying neurological diseases is challenging due to limited access to brain material. However, generating induced pluripotent stem cells (iPSCs) from PWS patients can help overcome this obstacle ,49,50,51Dynein-mediated transport of NAGK and SNRPN may play a critical role in neuronal development, and the disruption of this transport can have deleterious effects on the developing brain. Moreover, it is unclear whether the loss of function of these genes affects other aspects of neuronal development and function beyond neurite outgrowth. Therefore, further research is needed to elucidate the role of SNRPN and NAGK in PWS and their potential as therapeutic targets for the disorder. Also, the precise mechanisms through which SNRPN and NAGK contribute to the neurological symptoms of PWS remain unclear. In vivo studies of the NAGK and SNRPN knock-in and knock-out mice model can help to strengthen the current findings, which could provide a fascinating future perspective.For this study, the following antibodies were used in the experiments at the specified dilutions: mouse monoclonal NAGK ; chicken polyclonal NAGK ; rabbit polyclonal NAGK ; rabbit polyclonal DYNLRB1 ; rabbit polyclonal SNRPN ; mouse polyclonal SNRPN ; mouse monoclonal His ; mouse monoclonal alpha-tubulin ; mouse monoclonal GFP ; chicken polyclonal MAP2 ; mouse monoclonal GST ; rabbit monoclonal glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ; rabbit polyclonal Actin . To detect the primary antibodies, Alexa Fluor 488 and 568  secondary antibodies were employed for fluorescent labeling.HEK293T cells were transfected with His-tagged pENTER-CMV-NAGK CH817585) and pENTER-CMV-DYNLRB1 (CH803465) plasmids from Vigene Biosciences, Hollister, CA, USA, as well as the Myc-DDK-SNRPN (RR201341) and Myc-DDK-DYNLRB1 plasmids RR210555) from Origene Technologies, Rockville, MD, USA. The NAGK gene was cloned on the pGSTag plasmid  from Addgene, Watertown, MA, USA, and pET-32a+ (69015-3) from EMD Biosciences, San Diego, CA, USA. The SNRPN gene from Myc-DDK-SNRPN plasmid was cloned using restriction digestion (BamHI-NotI) on the pET-32a+ vector for bacterial expression and pull-down assay. The NAGK and SNRPN overexpression and knockdown genes were cloned on the Adeno-associated virus (AAV) vector pAAV.CMV.PI.EGFP.WPRE.bGH 105,530). Cloned NAGK and SNRPN transgenes were packaged using the pAAV2/5  and pAdDeltaF6  plasmids [0. Cloned5 from Or5 and pEN2 in a serum-free neurobasal media supplemented with B27, glutamate, and β-mercaptoethanol preincubated wells at 37 °C under 5% CO2 and 95% air. All experiments were conducted in accordance with the guidelines and approval from the Animal Care and Use Committee of Dongguk University, College of Medicine.Pregnant Sprague Dawley rats were purchased from KOATECH, Pyeongtaek, Republic of Korea, and raised in a light/dark cycle of 12/12 h with access to food and water ad libitum. On the 19th day of pregnancy, the pregnant rat was euthanized with isofluorane and the hippocampus of the fetus’s brain was collected for primary neuronal culture as previously described ,60. The E. coli competent cells were used for plasmid production. Bacterial pure protein was expressed in BL-21 competent cells obtained from New England Biolabs, Rowley, MA, USA.HEK293T and SK-MEL-31 cells were acquired from the Korean Cell Line Bank located in Seoul, Republic of Korea. The culture media was DMEM and MEM  with 10% fetal bovine serum and 1% penicillin-streptomycin (P-S). HEK293T cells were transfected with the desired plasmid for NAGK, SNRPN, and DYNLRB1 overexpression used in the in vitro binding assays. DH5α and NEB Induced pluripotent stem cells (iPSCs) of a PWS patient (PWS 2-9) and control UCH-HF-YK-27 cell lines derived from fibroblast were purchased from PWS iPSC Biobank University of Connecticut (UConn Stem Cell Core), Farmington, CT, USA. The fibroblast cells were induced into the stem cell using the lentivirus reprogramming method . The PWSFixation of primary hippocampal cultures was performed using a previously described sequential paraformaldehyde/methanol fixation protocol . ImmunosThe PLA was conducted utilizing a Duolink kit  with slight modifications to the manufacturer’s instructions. Fixed cells were exposed to primary antibodies in a pre-blocking buffer  overnight at 4 °C. After washing the cells three times with pre-blocking buffer for 10 min each at room temperature (RT), secondary antibodies conjugated with oligonucleotides (PLA probe anti-mouse MINUS and PLA probe anti-rabbit PLUS) were diluted in pre-blocking buffer, added to the cells, and incubated for 2 h at 37 °C in a humidity chamber. The remaining steps of the assay were carried out according to the manufacturer’s instructions. Briefly, cells were washed with the wash buffer provided in the Duolink kit, and then exposed to the ligation mixture and ligase for 30 min at 37 °C to allow for hybridization and ligation of the DNA oligonucleotides. Subsequently, an amplification solution containing polymerase was added, resulting in a rolling circle amplification reaction. The amplified product was detected using complementary fluorescently labeled oligonucleotides. In the case of combining PLAs with ICC, the PLA reactions were performed first, followed by overnight incubation of primary antibodies with the cells at 4 °C, and then incubation with fluorophore-conjugated secondary antibodies according to the ICC procedure.For the pull-down assay, His-tagged NAGK, Myc-DDK-SNRPN, His-DYNLRB1, and Myc-DDK-DYNLRB1-tagged plasmids were transiently transfected into HEK293T cells using calcium-phosphate-mediated transfection. After 48 h of incubation, the cells were lysed and collected using Pierce™ IP Lysis Buffer   containing protease inhibitor cocktail , followed by washing with cold PBS. The supernatant was then allowed to bind to MagListo™ Ni-NTA magnetic silica resin  as per the manufacturer’s instructions for the pull-down assay.w/v) glycerol, 0.01% bromophenol blue) and the elution was collected for further analysis.The co-immunoprecipitation assay was performed with a His-tagged antibody using puredown Protein G-Agarose beads . The beads (40 µL) were incubated with the individual and co-transfected protein lysate (500 µg) of His-NAGK and Myc-DDK-SNRPN plasmids. The beads were incubated overnight with His-antibody and control IgG at 4 °C with rotation. After washing the beads four times in 100 mM phosphate-buffered saline (PBS), the beads were boiled in 50 µL of 2 × lamelli buffer  method. The protein sample of each group was separated using SDS-PAGE, transferred to 0.2 μm polyvinylidene difluoride (PVDF) membranes, and blocked in 5% skimmed milk for 1.5 h. Incubation of the membranes was performed with the desired primary antibodies overnight at 4 °C, followed by incubation with goat anti-mouse/rabbit/chicken HRP secondary antibodies for 1 h at room temperature. The PVDF membranes were soaked in ECL luminous solution and detected using the Tanon 5200 Luminescence imaging system. Images were analyzed using Image J software version 1.43. Each band was compared with the GAPDH band (loading control) in the study.® polarizing light microscope equipped with an Olympus DP72® camera, controlled by cellSens™ software   .The phase contrast microscopy images were acquired using a Leica DM IL LED microscope from Gyeongbuk Technopark Advanced Medical Convergence Textile Center, Daegu, South Korea, which was equipped with a Leica DFC3000 G digital monochrome camera (1296 × 966 pixels) featuring EX view HAD technology and controlled by Leica LAS X software  . Immunocytochemistry images were captured with an Olympus BX53http://biology.ucsd.edu/labs/ghosh/software (accessed on 12 March 2023)), as previously described. The neurite area for the treated and control neurons was evaluated using the Neurphology J plug-in with Image J software (version 1.43).Image analysis for morphometry and quantitative analysis was performed using Image J software (version 1.49) with the simple neurite tracer plug-in , concentric circle plug-in, and Sholl plug-in  from the three independent experiments unless otherwise specified. Statistical analysis was conducted using GraphPad Prism version 8.0.0 for Windows . The statistical significance was assessed using different tests, including Student’s t-test and one-way and two-way ANOVA, followed by multiple comparison tests such as Dunnett’s and Tukey, and statistical significance was defined as In conclusion, our study demonstrated a significant upregulation of NAGK and SNRPN interaction in the cell body and processes of neurons during the neurite outgrowth and axodendritic development stages of neurodevelopment. Additionally, we successfully confirmed the microtubule-mediated transport of NAGK–SNRPN through their interaction with DYNLRB1 in both neurons and the SK-MEL-31 cell line. Remarkably, we found that NAGK–SNRPN interaction rescued the compromised axodendritic outgrowth observed in the SNRPN deletion model of PWS-2-9-patient-derived iPSC neurons. These findings have important implications for developing therapeutic approaches for neurodegenerative diseases.


Since OpenAI released ChatGPT, with its strong capability in handling natural tasks and its user-friendly interface, it has garnered significant attention.A prospective analysis is required to evaluate the accuracy and appropriateness of medication consultation responses generated by ChatGPT.A prospective cross-sectional study was conducted by the pharmacy department of a medical center in Taiwan. The test data set comprised retrospective medication consultation questions collected from February 1, 2023, to February 28, 2023, along with common questions about drug-herb interactions. Two distinct sets of questions were tested: real-world medication consultation questions and common questions about interactions between traditional Chinese and Western medicines. We used the conventional double-review mechanism. The appropriateness of each response from ChatGPT was assessed by 2 experienced pharmacists. In the event of a discrepancy between the assessments, a third pharmacist stepped in to make the final decision.P=.01).Of 293 real-world medication consultation questions, a random selection of 80 was used to evaluate ChatGPT’s performance. ChatGPT exhibited a higher appropriateness rate in responding to public medication consultation questions compared to those asked by health care providers in a hospital setting (31/51, 61% vs 20/51, 39%; The findings from this study suggest that ChatGPT could potentially be used for answering basic medication consultation questions. Our analysis of the erroneous information allowed us to identify potential medical risks associated with certain questions; this problem deserves our close attention. With its impressive ability to perform natural language tasks and its user-friendly interface, ChatGPT has garnered significant attention since its release by OpenAI. ChatGPT is an extension of a generative pretrained transformer (GPT) natural language processing (NLP) model called GPT-3 developed by OpenAI; it represents an advanced iteration known as GPT-3.5. In addition to achieving human-level performance in entertainment-oriented conversations and writing tasks, ChatGPT can also provide satisfactory answers to questions involving many different professional knowledge domains. The field of NLP is experiencing rapid progress, largely due to extensive data from the Internet and computational power advancements in accordance with Moore’s law, and many language models with an even larger size than GPT-3 have been trained, released, and made publicly available . HoweverMany studies have designed specialized testing procedures to evaluate ChatGPT’s abilities and limitations. In medicine, it can achieve a nearly passing score of 60% accuracy on the US Medical Licensing Exam (USMLE) ,3. In prThis exploratory study aimed to understand better the suitability of ChatGPT for answering real-world medication consultation questions in pharmaceutical services. Additionally, we conducted an in-depth analysis of the accuracy of responses to drug-herb interaction questions to assess the potential of ChatGPT in medication education and consultation.We followed the STROBE  reporting guidelines for observational studies . Figure Our test questions were divided into 2 groups. The first included 293 open-ended queries; we excluded 103 questions related to chronic prescriptions and medication reservations, then randomly selected 80 of the remaining questions. The second group of questions comprised 8 queries concerning drug interactions between traditional Chinese and Western medicines. All of the questions we evaluated and the responses generated by ChatGPT were in Chinese.ChatGPT is a large language model that can interact with users in a conversational way. One of its most remarkable features is its capacity to perform well on many natural language tasks in few-shot and zero-shot settings, which is unprecedented in the field of NLP ,4. As stOur test questions comprised 2 groups. The first group was designed to assess the model’s ability to provide useful and accurate drug information. We selected 293 open-ended questions from medication consultations recorded in February 2023 by the Central Medical Center in Taiwan. A total of 103 questions related to chronic prescriptions and medication reservations were excluded. From the remaining 190 questions, 40 questions were randomly selected from both the general public and health care professionals  on traditional Chinese medicine . The test questions primarily covered 4 categories: medication information, dosage adjustment, drug-drug interactions, and intravenous injection compatibility (An assistant submitted both sets of questions (open-ended medication consultation questions and drug-herb interaction questions) to the ChatGPT interface, recording the initial response generated for each query.In the first part of the study, an example of a question (translated from Chinese) is “What is the recommended meropenem dosage for patients with renal dysfunction?” An example of a ChatGPT response  is “Meropenem dosing may require adjustment in patients with renal dysfunction, as the drug is primarily eliminated through the kidneys. Generally, for adult patients with renal impairment (creatinine clearance <50 mL/min), the recommended dose ranges between 500 mg and 1000 mg every 12 hours, depending on the severity of renal dysfunction” . The level of statistical significance for all tests was set at a 2-sided This survey study was deemed exempt from review by the Ethics Review Board of China Medical University because no personal identities were used.P=.01; From the 190 real-world medication questions, 80 open-ended questions were randomly selected and evaluated by 2 pharmacists. The responses generated by ChatGPT were initially reviewed for appropriateness by the 2 pharmacists, with a discordance rate of 12.5% (10/80). The questions with inconsistent annotations P=.01; . Upon fuThe final section of the test data set consisted of 8 questions selected by a pharmacist. Using aspirin as an example, we inquired about its potential interactions with ginseng, safflower, licorice, astragalus, coptis, hawthorn, angelica, and ginkgo. The results indicated that the rate of inconsistencies in the pharmacist’s annotations was 37.5%, (3/8) whereas ChatGPT displayed an inappropriateness rate of 50% (4/8) in its responses. Further analysis of ChatGPT’s answers revealed that the responses to the questions about whether aspirin could be used in combination with safflower, astragalus, coptis, and hawthorn lacked sufficient detail .Previous research primarily used multiple-choice data sets to test language models, with a smaller number using real-world consultation questions ,12. DespOur findings suggest that ChatGPT tends to produce analogous answers to similar queries. In our assessment of ChatGPT’s appropriate responses, most of them involved traditional Chinese medicinal materials that the general public is more familiar with, such as ginseng, licorice, angelica, and ginkgo. However, responses that were evaluated as inappropriate, such as those related to safflower, astragalus, coptis, and hawthorn, were deemed insufficient due to a lack of information, making it difficult to provide patients with clear recommendations. Therefore, we speculate that the machine learning model’s database may still lack sufficient information on traditional Chinese medicine.Regarding the ChatGPT response to the safety and efficacy of using saffron with aspirin, there may not be enough research evidence. However, clinical evidence shows that the components of saffron can affect platelet function and inhibit blood clotting. Therefore, patients who are scheduled for surgery in the near future should avoid using saffron. Furthermore, combination prescriptions with saffron are very rare in clinical practice, and in some individuals who are sensitive to it, bleeding may occur. Additionally, saffron is expensive. The interaction rate of traditional Chinese medicine may indeed be affected by factors such as the patient’s health condition, age, and other medications they are taking, which could impact its practicality in clinical practice. These are factors that ChatGPT cannot discern.We deem ChatGPT’s response on the interaction between aspirin and astragalus to be overly neutral. In traditional Chinese medicine theory, astragalus is characterized as a qi-tonifying herb with warming properties, believed to enhance qi, raise yang, nourish defensive qi, and consolidate the exterior. A study from China’s Shanxi Hospital suggests that concurrent use of astragalus injection and aspirin could potentially augment blood flow and elevate bleeding risk.huanglian) is classified as a bitter and cold herb with primary functions to clear heat, dry dampness, purge fire, and detoxify. Mechanistically, it does not interfere with anticoagulants. Huanglian contains berberine, known for its effect on relaxing vascular smooth muscle, but it does not directly influence the anticoagulant mechanism of aspirin. Hawthorn belongs to the category of resolving food stagnation, promoting digestion, regulating qi, and dispersing blood stasis. Hawthorn contains various organic acids, which can help to contract the uterus, strengthen the heart, counteract arrhythmia, increase coronary blood flow, dilate blood vessels, lower blood pressure, and reduce blood lipids. Hawthorn has the function of promoting blood circulation, removing blood stasis, and relieving pain. It is used to treat postpartum abdominal pain and lochia retention caused by blood stasis or dysmenorrhea due to blood stasis. Therefore, it is not recommended to use it together with aspirin.We consider the interaction responses between aspirin and coptis or hawthorn as inaccurate, attributable to the insufficiency of detail in ChatGPT’s responses. In traditional Chinese medicine theory, coptis . In Taiwan, a higher proportion of pharmacists practice in the Western medicine domain compared to the field of traditional Chinese medicine. We reasonably infer that pharmacists may be more susceptible to the influence of information generated by ChatGPT in this subspecialty. We should be aware of the potential risks and harms that may arise from relying too heavily on ChatGPT for medical information and providing inaccurate information to health care providers [In the 2 test sets, we found that the rate of inconsistency among pharmacists’ evaluations appeared to be higher in questions related to interactions between traditional Chinese and Western medicines (10/80, 12.5% vs 3/8, 37.5%; roviders .As ChatGPT acquires its medical knowledge from online resources, we may expect substantial improvements in AI model performance with the development of technology and growing availability of open-access academic research. However, medical errors are not tolerated . With thThis study has several limitations. First, although ChatGPT is multilingual, we speculate that its responses in English may be more accurate due to a larger data pool. Second, limited by our research period, we used GPT-3.5 as the test model. The technical report released by OpenAI for GPT-4 highlights the substantial research efforts aimed at reducing hallucinations. It shows that GPT-4 produces fewer instances of hallucinatory output compared to earlier models. However, OpenAI acknowledges that the issue of hallucinations remains a current limitation of GPT-4 . TherefoTo our knowledge, studies discussing and analyzing the reasons for errors in ChatGPT’s responses are relatively scarce. We found that ChatGPT provided largely appropriate responses to simple medication questions as evaluated by pharmacists. However, for more complex questions related to individual patient scenarios, the answers may be inaccurate or vague, thereby making it challenging for the person asking the question to obtain the necessary information. As pharmacists, we recognize that many patients and health care professionals continue to depend on us for medication information and education. While we are optimistic about ChatGPT’s potential in assisting pharmacists in providing medication consultations to the public and aiding pharmacy educators in identifying gaps in student knowledge, our study suggests that we must remain cognizant of the risks associated with the provision of incorrect information.


Enterobacterales are increasing in the US. Recommended treatment (tx) options include ceftazidime-avibactam (CZA) plus ATM or FDC. We assessed the in vitro activity of tx options in the setting of a multi-species NDM outbreak.New Delhi metallo-β-lactamase (NDM)-producing Patient isolates were identified through active carbapenemase testing and characterized by whole-genome sequencing. Minimum inhibitory concentrations (MICs) for ATM, ATM-AVI, ceftazidime, CZA, FDC, FDC-AVI, FEP and FEP-TAN were determined by broth microdilution. AVI and TAN were tested at 4 mg/L. Index isolates were defined as the first NDM-producing isolate per species per patient.Table 1) comprising 10 species, the most common species were E. cloacae complex (15), E. coli (3), and K. aerogenes (3). Isolates harbored NDM-5 (72%) or NDM-1 (28%). Other β-lactamases included ACT (15), SHV (7), TEM (7), and CTX-M (6) variants. 93%, 93%, and 69% of index isolates were susceptible to ATM-AVI, FEP-TAN, and FDC, respectively; corresponding MIC50 values were 0.06, 0.5, and 2 mg/L, respectively (Table 2). FDC susceptibility improved to 86% with addition of AVI.48 isolates from 24 patients were included from 2018-2023; 73% of isolates were collected in the last year. 17% of patients were infected with >1 NDM-producing species. Among index isolates . Two patients were treated with CZA-ATM. In 1 case ATM-AVI MICs increased against E. coli from 16 to >128 mg/L post-CZA-AVI tx, attributed to an insertion (YRIN) and substitution (A417V) in ftsI (PBP3). Overall, 4 isolates  from 2 patients were non-susceptible to ATM-AVI, FEP-TAN, and FDC. All were related by WGS and harbored CMY-145, NDM-5, and a YRIN duplication in PBP3.Among serial isolates (n=19), rates of susceptibility were 89%, 84%, and 16% for ATM-AVI, FEP-TAN, and FDC, respectively (in vitro activity against clinical isolates. Susceptibility rates for both β-lactam/β-lactamase inhibitors was higher than FDC, suggesting they may be preferred for tx in the absence of timely susceptibility testing. The activity of each agent is compromised by NDM-producing strains that harbor PBP3 mutations.During a multi-species NDM outbreak we found ATM-AVI and FEP-TAN demonstrated potent Ghady Haidar, MD, Allovir: Grant/Research Support|AstraZeneca: Advisor/Consultant|AstraZeneca: Grant/Research Support|Karius: Advisor/Consultant|Karius: Grant/Research Support|NIH: Grant/Research Support Ryan K. Shields, PharmD, MS, Allergan: Advisor/Consultant|Cidara: Advisor/Consultant|Entasis: Advisor/Consultant|GSK: Advisor/Consultant|Melinta: Advisor/Consultant|Melinta: Grant/Research Support|Menarini: Advisor/Consultant|Merck: Advisor/Consultant|Merck: Grant/Research Support|Pfizer: Advisor/Consultant|Roche: Grant/Research Support|Shionogi: Advisor/Consultant|Shionogi: Grant/Research Support|Utility: Advisor/Consultant|Venatorx: Advisor/Consultant|Venatorx: Grant/Research Support


The suggested method includes the graphene synthesis via the template method, followed by the deposition of an additional graphene layer doped with boron in an autoclave at 650 °C, using a mixture of phenylboronic acid, acetone, and ethanol. After this carbonization procedure, the mass of the graphene sample increased by 70%. The properties of B-carbon nanomaterial were studied using X-ray photoelectron spectroscopy (XPS), high-resolution transmission electron microscopy (HRTEM), Raman spectroscopy, and adsorption-desorption techniques. The deposition of an additional graphene layer doped with boron led to an increase of the graphene layer thickness from 2–4 to 3–8 monolayers, and a decrease of the specific surface area from 1300 to 800 m2/g. The boron concentration in B-carbon nanomaterial determined by different physical methods was about 4 wt.%.A new method for the synthesis of boron-doped carbon nanomaterial  has been developed. First, graphene was synthesized using the template method. Magnesium oxide was used as the template that was dissolved with hydrochloric acid after the graphene deposition on its surface. The specific surface area of the synthesized graphene was equal to 1300 m Graphene attracted the attention of researchers due to its exceptional structural, mechanical, and electronic properties ,2,3,4. TThe properties of graphene-based materials can be improved by doping with other “I have checked”.elements . Boron, The doping of carbon nanomaterials with boron atoms can alter their electronic properties. Recently, it was found that boron-doped carbon nanomaterials have remarkable properties in electrocatalytic oxygen reduction reactions (ORRs) ,18,19,20In this article , orderedThere are several reports in the literature on the synthesis of boron-doped graphene. In these studies, the conventional CVD method was used for growing graphene on different polycrystalline foils. It was reported that boron-doped graphene could be synthesized using phenylboronic acid as a source of carbon and boron . The effIn the article , graphen2 using NaBH4 as a reducing agent, followed by thermal treatment. It was demonstrated that the presence of O–B–C fragments increased the activity of porous carbon in ORRs, and led to high stability in cycles. The effect of oxygen and boron co-introduction in porous carbon on the activity of ORRs was studied in . The synIn this study, graphene obtained via the template synthesis ,26,27 waMagnesium oxide  was subjected to carbonization with 1,3-bitadiene at 600 °C in a quartz flow reactor. After this reaction the MgO particles were covered with a thin carbon film. Then, the carbon film was cleaned from the MgO template by treatment in a hydrochloric acid solution .The synthesized graphene was used for doping with boron. 6H5-B-(OH)2)  was used as a boron-containing precursor. Its structure containing a phenyl group favored the introduction of boron into the synthesized graphene framework. Phenylboronic acid  using a ThemisZ  electron microscope with an accelerating voltage of 200 kV and maximum lattice resolution of 0.07 nm was used for the investigation of the structure and microstructure of the catalysts. The TEM images were recorded with a Ceta 16  CCD matrix. Elemental analysis was performed with Super-X EDS detector . High-angle annular dark-field images (HAADF STEM image) were recorded using a standard ThemisZ detector. The samples for the HRTEM study were deposited on a holey carbon film mounted on a copper grid by the ultrasonic dispersal of the B-graphene suspension in ethanol.α radiation, hν = 1253.6 eV, 150 W). The binding energy (BE) scale was pre-calibrated using the positions of the peaks of Au4f7/2 (BE = 84.0 eV) and Cu2p3/2 (BE = 932.67 eV) core levels. The samples in the form of powder were loaded onto a conducting double-sided copper scotch. The survey and the narrow spectra were registered at the analyzer pass energy 20 eV. Atomic ratios of the elements were calculated from the integral photoelectron peak intensities, which were corrected by corresponding sensitivity factors based on Scofield photoionization cross-sections [X-ray photoelectron spectra were recorded on a SPECS (Germany) photoelectron spectrometer using a hemispherical PHOIBOS-150-MCD-9 analyzer  with 2 cmThe surface areas and porosity of the synthesized graphene and B-carbon nanomaterial were determined using an ASAP-2400  instrument .The boron concentration in B-carbon nanomaterial was determined by the atomic emission spectroscopy. Graphene synthesized by the template method was studied by electron microscopy. A HRTEM image of graphene is shown in 2/g. The graphene aggregates have dimensions of about 1 μm a. The siGraphene was doped with boron atoms by heating in an autoclave in the presence of phenylboronic acid, acetone, and ethanol at 650 °C. After doping with boron, the mass of the graphene sample increased by 70%. HRTEM images of B-carbon nanomaterial are shown in The boron concentration in the B-carbon nanomaterial sample determined by EDX was equal to 4 wt.%. This boron concentration is in agreement with the results of the elemental analysis by atomic emission spectroscopy. According to the latter, the content of atomic boron in B-carbon nanomaterial was 4.3 wt.%.The nitrogen adsorption-desorption isotherms measured on pristine graphene and B- carbon nanomaterial are presented in 2/g. The deposition of the additional layer of graphene doped with boron led to the surface area decreasing from 1300 to 800 m2/g. Specific surface area of the synthesized B-carbon nanomaterial was equal to 800 mThe pore size distributions of pristine graphene and of the B-carbon nanomaterial sample are presented in The data presented in B-carbon nanomaterial takes the shape of hollow spheres. According to the pore size distribution , its maiB-carbon nanomaterial samples were studied by Raman spectroscopy to determine specific features of their structure. The first-order Raman spectra of graphene and B-carbon nanomaterial are shown in −1), related to vibrations of carbon atoms in the graphene layer, and D mode (1355 cm−1), related to the presence of carbon atoms in sp3 hybridization [−1), D + D′ (≈2950 cm−1). The Raman spectrum confirmed that the synthesized carbon material had a graphene-like local structure. Therefore, according to the Raman data, B-carbon nanomaterial is a defective graphene-like carbon nanomaterial.Two typical modes were observed in the Raman spectra of the synthesized carbon nanomaterial samples: G mode (1597 cmdization . The appThe data presented in Based on the results of B-carbon nanomaterial characterization by physical methods, the following general scheme of its synthesis can be suggested .2/g. The suggested method includes the graphene synthesis using the template method, followed by deposition of an additional graphene layer doped with boron in an autoclave at 650 °C using a mixture of phenylboronic acid, acetone, and ethanol. After this carbonization procedure, the mass of the graphene sample increased by 70%. The boron concentration in B-carbon nanomaterial determined by different physical methods was about 4 wt.%. The deposition of an additional graphene layer doped with boron led to an increase of the graphene layer thickness from 2–4 to 3–8 monolayers, and a decrease of the specific surface area from 1300 to 800 mThe state of carbon and boron in B-carbon nanomaterial samples was studied by X-ray photoelectron spectroscopy. Survey scans of pristine and B-carbon nanomaterial samples are presented in The C1s spectrum of the B-carbon nanomaterial sample is presented in The concentrations of different elements in B-carbon nanomaterial were determined by XPS. The results are presented in 2 bond [A more accurate conclusion on the state of boron can be made from the binding energy for the B1s peak of the B-carbon nanomaterial. The B1s peak of B-graphene sample is shown in 2 bond ,36,37. 2 fragment in B-carbon nanomaterial (Note that the binding energy of the B1s peak for the C–B bond is in the range of 188–189 eV. The shift of the B1s peak to higher binding energies indicates that the boron atom is bonded with an oxygen atom. So, the following model structure can be suggested for the C–BOmaterial .We believe that the incorporation of boron atoms into a formed carbon nanomaterial is difficult because boron atoms are larger that carbon atoms. It was shown  that theThe proposed structure of a fragment of the boron-doped carbon layer deposited on the surface of the initial graphene is shown in 2/g. The Raman spectrum confirmed that the synthesized carbon material had a graphene-like local structure. According to the XPS data, boron in B-carbon nanomaterials is present in the form of C–BO2 fragments. A new method for the synthesis of B-carbon nanomaterials has been developed. The method includes graphene synthesis using the template method, followed by the deposition of an additional layer of boron-doped graphene in an autoclave at 650 °C using a mixture of phenylboronic acid, acetone, and ethanol. Following the graphene carbonization in the autoclave, its weight increased by 70%. The presence of boron atoms in the graphene framework was confirmed by atomic emission spectroscopy, XPS, and EDX. The boron concentration in B- carbon nanomaterials was found to be about 4 wt.%. The deposition of an additional graphene layer doped with boron led to an increase of the graphene layer thickness from 2–4 to 3–8 monolayers, and a decrease of the surface area from 1300 to 800 m


Graphene, with its excellent properties and unique structure, has been extensively studied in the context of modifiable cement-based materials. However, a systematic summary of the status of numerous experimental results and applications is lacking. Therefore, this paper reviews the graphene materials that improve the properties of cement-based materials, including workability, mechanical properties, and durability. The influence of graphene material properties, mass ratio, and curing time on the mechanical properties and durability of concrete is discussed. Furthermore, graphene’s applications in improving interfacial adhesion, enhancing electrical and thermal conductivity of concrete, absorbing heavy metal ions, and collecting building energy are introduced. Finally, the existing issues in current study are analyzed, and the future development trends are foreseen. Cement-based materials are widely used in infrastructure construction due to their high production efficiency, abundant raw material sources and good workability. As a result, they are the most commonly used construction materials in the world . HoweverWith the rapid development of nanomaterial science, researchers have used nanomaterials combined with industrial solid waste materials  in When fly ash concrete is mixed with GO of 0.25% mass and 24.3% oxygen, its chloride ion penetration resistance is the strongest , and wheGenerally, the factors affecting concrete carbonization can be divided into three aspects: material factors, environmental factors , and construction factors. Regarding material factors, graphene materials inhibit the carbonation rate of concrete by enhancing its compactness (microscopic pore structure) and alkalinity. Devi  exposed In terms of bending fatigue, Cho  proposedResearchers have also explored the application of graphene materials in concrete abrasion resistance, antibacterial properties, and crack reinforcement. Liu  found thResearchers have used protective coatings or carbon fiber-reinforced composites (CFRP) to enhance the durability and strength of concrete. The introduction of graphene can enhance the bonding between concrete and coating materials, steel bars, and CFRP, thus enhancing the protection and strengthening effect. The following section reviews the application of graphene materials in improving the interfacial bonding between concrete and coating materials, steel reinforcement, and CFRP.To improve the bonding effect between concrete and CFRP, Al-Saadi  used oxiAs shown in In order to improve the adhesion between the concrete and the protective coating, the scholars found that the use of GO to enhance the adhesion is a reliable new approach. Bahraq  believedGraphene can enhance the electrical and thermal conductivity of cement-based materials, and the modified materials play an essential role in areas such as road snow and ice melting, structural health monitoring, and building energy harvesting, bringing benefits such as reducing energy consumption, improving structural safety, and protecting the environment.In response to road snow and ice melting, researchers have designed the optimal mixing quantities of graphene materials considering concrete’s mechanical strength and resistivity. Wang et al. ,96 belieCombined with the improved electrical conductivity of the concrete, the researchers also studied the health monitoring of concrete structures. As shown in In recent years, the problems of cities’ ecological environment have become worse. With the continuous implementation of the goal of “double carbon” and the continuous promotion of “sponge city” construction, recycled concrete and thermoelectric composite concrete have become a global research hotspot. It is found that graphene-permeable concrete can meet the requirements of light-load road pavement in sponge cities and has good adsorption function for common heavy metal ions (such as lead ions) in water. Zhang Yubin et al.  preparedIn addition, graphene cement-based composite materials can convert the abundant solar energy in concrete structures into electrical energy for energy harvesting in urban buildings. Its principle is shown in Although graphene and its derivatives have broad research prospects in improving the properties of cement-based materials, some issues and challenges still need to be overcome before they can be generalized to practical engineering applications.(1) The production cost of graphene materials is high, and it is still challenging to accurately control the size, thickness, number of layers, and oxygen content during the preparation process.(2) The dispersion of graphene materials is terrible, and the chemical reactivity between cement-based materials and graphene is limited, resulting in a small amount of graphene and its derivatives (the amount of addition in the research paper is no more than 0.3%), which makes it difficult to give full play to its strengthening effect. In addition, the high cost and low efficiency of the commonly used ultrasonic dispersion methods limit their wide application in practical engineering.(3) The strengthening mechanism of graphene’s different sizes, thicknesses, layers, and oxygen content on cement-based composites is not precise. Researchers have different opinions on the optimal dosage of graphene materials, and no consensus has been reached.(4) Under the influence of temperature, humidity, alkaline environment, stress, and other factors, the long-term performance stability of graphene and its derivatives in cement has not been fully verified.In this paper, we have summarized and systematically analyzed the research status of graphene and its derivatives in improving the cement base’s operational performance, mechanical properties, durability, and other applications of cement substrates. The conclusions are as follows:(1) The graphene material adsorbs the free water in the mix, reducing the cement-based material’s fluidity and slowing the concrete’s shrinkage and creep. At the same time, the good thermal conductivity of the graphene materials also promotes hygroscopic rate and heat diffusion in cement-based materials.(2) There are three main reasons for using graphene to improve the overall mechanical properties of cementitious materials: the structural characteristics of graphene materials give high strength, the ability to promote the hydration reaction of cement, and the ability to fill the cavity gaps. In addition, the oxygen content, diameter, thickness, specific surface area of graphene materials, and the curing time of concrete will indirectly affect the intrinsic mechanical properties and reasonable material dispersion is a prerequisite for improving the overall mechanical properties.(3) Introducing graphene improves concrete’s impermeability, chloride ion penetration resistance, freeze–thaw resistance, and inhibits fatigue cracks’ expansion. In addition, the antimicrobial, hydrophobic, electrically and thermally conductive properties of graphene materials provide new ideas for the durable protection of marine building structures.(4) Graphene materials have great potential in improving the bond between cement-based materials, melting snow on a concrete surface, monitoring concrete stress state, absorbing heavy metal ions, and collecting building energy, etc. Continued exploration in these areas will promote the development of concrete structures in the direction of high durability, functionality, intelligence, environmental protection, and energy conservation.To further advance the engineering application of graphene materials for improving concrete properties, this paper provides the following outlook on the development direction of this field.(1) Future research should further explore the interaction mechanism between graphene and cement-based materials and develop more diverse graphene derivative materials. Meanwhile, it is necessary to find low-cost and efficient graphene dispersion methods to realize the engineering applications of graphene materials.(2) To determine the optimal mix range of graphene materials under multiple factors, it is also necessary to conduct systematic tests and simulations to explore the strengthening mechanism of graphene size, thickness, layer number, and oxygen content on cement-based composites.(3) Further research on the toxicity, environmental impact, and long-term stability of graphene concrete is a vital prerequisite for promoting the widespread use of graphene materials.(4) Relevant research institutions or departments can also standardize the preparation technology of graphene materials and formulate classification standards for the properties of different graphene materials, which can improve its application effect in cement-based materials and help promote the comparative analysis of different subsequent research results.