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.


Despite the presence of significant cortical pTDP-43 inclusions of heterogeneous morphologies in patients diagnosed with amyotrophic lateral sclerosis (ALS), pathological subclassification is routinely performed in the minority of patients with concomitant frontotemporal dementia (FTD).In order to improve current understanding of the presence and relevance of pathological pTDP-43 subtypes in ALS, the present study examined the pattern of cortical pTDP-43 aggregates in 61 ALS cases without FTD.Based on the presence, morphology and composition of pTDP-43 pathology, three distinct ALS-TDP subtypes were delineated: (1) A predominant pattern of pTDP-43 granulofilamentous neuronal inclusions (GFNIs) and grains that were immuno-negative for p62 was identified in 18% of cases designated ALS-TDP type E; (2) neuronal cytoplasmic inclusions (NCIs) that were immuno-positive for both pTDP-43 and p62 were observed in 67% of cases assigned ALS-TDP type B; and (3) scarce cortical pTDP-43 and p62 aggregates were identified in 15% of cases coined ALS-TDP type SC . Quantitative analyses revealed a significantly greater burden of pTDP-43 GFNI and grains in ALS-TDP type E. Principal component analysis demonstrated significant relationships between GFNIs, grains and ALS-TDP subtypes to support the distinction of subtypes E and B. No significant difference in age at death or disease duration was found between ALS-TDP subgroups to suggest that these subtypes represent earlier or later stages of the same disease process. Instead, a significantly higher ALS-TDP stage, indicating greater topographical spread of pTDP-43, was identified in ALS-TDP type E. Alzheimer’s disease neuropathological change (ABC score ≥ intermediate) and Lewy body disease (Braak stage ≥ IV) was more prevalent in the ALS-TDP type SC cohort, which also demonstrated a significantly lower overall cognitive score.In summary, the present study demonstrates that ALS-TDP does not represent a single homogenous neuropathology. We propose the subclassification of ALS-TDP into three distinct subtypes using standard immuno-stains for pTDP-43 and p62 in the motor cortex, which is routinely sampled and evaluated for diagnostic neuropathological characterisation of ALS. We propose that future studies specify both clinicopathological group and pTDP-43 subtype to advance current understanding of the pathogenesis of clinical phenotypes in pTDP-43 proteinopathies, which will have significant relevance to the development of targeted therapies for this heterogeneous disorder.The online version contains supplementary material available at 10.1186/s40478-023-01670-2. Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disease underscored by diverse yet poorly understood pathomechanisms that contribute to differential therapeutic responses and unsuccessful drug development , 2. ALS C9ORF72 gene [All cases with a pathological diagnosis of ALS-TDP were selected from a neuropathological series collected by the Sydney Brain Bank through regional brain donor programs in Sydney, Australia. The brain donor programs hold approval from the Human Research Ethics Committees of the University of New South Wales and comply with the statement on human experimentation issued by the National Health and Medical Research Council of Australia. Patients were diagnosed using standard clinical diagnostic criteria , 14–18 fF72 gene  which waF72 gene  was idenF72 gene , 23 was C9ORF72-carriers [Pathological classification was based on pTDP-43 aggregates in the motor cortex, which is the earliest cortical region implicated in ALS , 25. TDPcarriers ; and 3) carriers . In addiTen µm thick formalin-fixed, paraffin-embedded tissue sections from each region-of-interest were immunostained with antibodies against phospho-TDP-43 S409/410)  and p62  using a Ventana BenchMark GX autostainer. Both antibodies underwent 30 min antigen retrieval with CC1 reagent followed by a one hour primary antibody incubation and visualization using Optiview DAB IHC Detection Kit. Slides were digitally scanned using the Olympus VS-120 slidescanner at 20 × magnification. Regions of interest measuring 2000 µm × 1000 µm were defined and the percentage area occupied by granulofilamentous pTDP-43 inclusions, pTDP-43 grains and round pTDP-43 neuronal cytoplasmic (NCIs) were quantified using QuPath (version 0.3.2)  , which ep value < 0.05 taken as significant. Group differences were determined using a one-way ANOVA followed by Bonferroni post hoc tests for age, disease duration, postmortem delay, density of different pTDP-43 inclusions, ALS-TDP stage and ACE-III scores, and with chi-square test for gender, site of disease onset, C9ORF72 and co-pathologies. Any relationships between variables were assessed using principal component factor analysis (PCA), which included the density of different pTDP-43 inclusions in the motor cortex, ALS-TDP group, genetic mutation and demographic variables (age and disease duration).Statistical analysis was performed using SPSS (Version 28) with a Granulofilamentous neuronal inclusions (GFNI), abundant grains and oligodendroglial inclusions that were immunopositive for pTDP-43 but not p62 were identified in 18% of cases (n = 11). The morphology and composition of these aggregates resemble that recently reported in FTLD-TDP type E  and as sA predominant pattern of round neuronal cytoplasmic inclusions in superficial and deep cortical layers with relatively few dystrophic neurites were observed on both pTDP-43 and p62-immunostained sections of the motor cortex in 67% of ALS cases (n = 41). The morphology, composition and distribution of these aggregates resemble that seen in the cortical sections of FTLD-TDP type B cases and as such, these cases were designated ALS-TDP type B.Scarce pTDP-43 and p62 aggregates were observed in the motor cortex of 15% of cases (n = 9) and were designated ALS-TDP type SC . Double-labelled immunofluorescence confirmed the absence of ubiquitin and p62 immunoreactivity in the pTDP-43 aggregates of ALS-TDP type E cases . Principal component factor analysis was used to assess all relationships between variables across all cases and loading scores > 0.6 was considered significant. Two separate factors accounted for > 57% of variance. Significant relationships were identified between ALS-TDP subtype (0.74 loading), % GFNI (0.86 loading) and % pTDP-43 grains (0.83 loading) , supporting the construct that pTDP-43 GFNI and grains differentiated the ALS-TDP groups. Factor 2 accounted for 22% of the variance and showed that % round pTDP-43 NCIs (0.633 loading) associated with age (-0.63 loading), disease duration (-0.64 loading) and presence of a C9ORF72 mutation (0.69 loading). This is consistent with the shorter disease duration, younger age at death and round NCIs characteristic of type A/B described in C9ORF72 cases [Assessment of motor cortical burden of pTDP-43 morphologies revealed a significantly greater burden of pTDP-43 GFNI and grains in ALS-TDP type E compared to ALS-TDP type B and ALS-TDP type SC cases  > 6.2, 72 cases .Fig. 4QuC9ORF72 expansion was identified between groups (X(2) = 2.41, p = 0.3)  = 14.49, p < 0001, Table C9ORF72-carriers and sporadic cases (age at death (mean ± SD): 59 ± 8 years in C9ORF72 cases, 66 ± 11 years in sporadic cases, p = 0.1; disease duration (mean ± SD): 2 ± 1 years in C9ORF72 cases, 3 ± 2 years in sporadic cases, p = 0.2; PMI (mean ± SD): 24 ± 10 h in C9ORF72 cases, 28 ± 15 h in sporadic cases, p = 0.5). However, a significantly higher ALS-TDP stage was identified in C9ORF72-carriers compared to sporadic ALS cases . ALS-TDP stage was similar in ALS-TDP type E cases with and without a C9ORF72 expansion .Group comparisons revealed no significant differences in age at death, age at disease onset, disease duration or post-mortem interval between groups Table . No signTo determine if the GFNIs characteristic of ALS-TDP type E represent immature pTDP-43 inclusions that have yet to coalesce into the more prevalent ALS-TDP type B morphology which is ubiquitinated, the frontal cortex, which is affected later in pathological progression (and consequently would demonstrate comparatively “earlier” pathology by the end of the disease progression) were assessed. ALS-TDP type B cases with pTDP-43 pathology in the frontal cortices (n = 21) demonstrated similar round neuronal cytoplasmic inclusions with few neurites that were immunopositive for p62 in these “later affected” regions. As expected, all ALS-TDP type E cases demonstrated GFNI and grains in the frontal cortex that were consistent with that seen in the motor cortex of these cases. Regional differences in pathological subtypes were not identified within cases here nor have they been previously reported Fig. . In the C9ORF72 expansion), 46% of the ALS-TDP type B cohort  and 44% of the ALS-TDP type SC cohort , with no significant group differences in the years between assessments and death (Table p < 0.05) .ACE-III scores  were avaThe present study examined and reports, for the first time, three distinct histological subtypes in ALS cases without FTD: (1) In 18% of cases, a predominant pattern of pTDP GFNIs and grains, which were immuno-negative for p62 were identified, and these cases were designated ALS-TDP type E; (2) In 67% of cases, a predominant pattern of round NCIs that were immuno-positive for both pTDP-43 and p62 were observed, and these cases were designated ALS-TDP type B; (3) In 15% of cases, scarce cortical pTDP-43 and p62 aggregates were observed, and these cases were coined ALS-TDP type SC .Quantitative analyses identified a significantly greater burden of pTDP-43 GFNIs and grains in ALS-TDP type E. PCA analysis of pTDP-43 inclusion morphologies revealed significant associations between GFNIs, grains and ALS-TDP subtypes to support that these pathologies segregate with ALS-TDP type E. No significant difference in age of disease onset, death or survival was found between groups to suggest that these subtypes represent earlier or later-stage disease pathologies. Further to this, pTDP-43 morphology and pattern of p62-immunoreactivity in each case was consistent across early- and late-affected cortical regions, indicating that non-ubiquitinated GFNIs are not a predecessor to ubiquitinated NCIs. Instead, a significantly higher ALS-TDP stage was identified in the ALS-TDP type E cohort. Interestingly, a lower overall cognitive score and higher incidence of Alzheimer’s disease neuropathological change (≥ intermediate level) or LBD (Braak stage ≥ IV) was identified in the ALS-TDP SC cohort. To date, pathological subclassification of pTDP-43 subtypes is routinely performed in FTLD, as demonstrated by the nomenclature “FTLD-TDP subtypes A-E”. Given the similarities in the pattern of cortical pTDP-43 in two of our ALS subgroups with that identified in the FTLD-TDP subtypes, we have adopted the same alphabetised subclassification and propose that future studies consider specifying clinicopathological group followed by pTDP-43 subtype (eg. FTLD-ALS type E) to advance pathomechanistic understanding of clinical phenotypes in pTDP-43 proteinopathies.Widespread pTDP-positive p62-negative GFNI, grains and oligodendroglial inclusions were first described in seven FTD cases with motor neuronal pTDP-43 and rapid disease durations of one to three years . This paIn contrast to a previous study that assessed the inclusion morphologies in the ~ 30% of ALS cases with pTDP-43 in the hippocampus , this stC9ORF72 expansion was identified in ALS-TDP subtypes B and E but not subtype SC here. Although this may be considered basis for support of a B-E continuum, it is important to note that in FTLD, the C9ORF72 expansion is predominantly associated with subtypes A and B [C9ORF72 expansion has also been associated with FTLD-TDP subtypes A + B and E [C9ORF72-type B cases, indicating greater topographical spread of pTDP-43 to extramotor regions. Perhaps surprisingly, this was not associated with lower overall cognitive scores in either of these cohorts although it is worth noting that cognitive assessments were performed midway in the disease process. Importantly however, ALS-TDP stage is an indicator of regional presence, not severity [The  A and B , 37–39.  A and B , 33, 40. B and E , highligseverity , 41. Intseverity , it is uIn summary, the present study delineates three distinct histological subtypes in ALS-TDP cases according to the presence, morphology and composition of cortical pTDP-43 pathology. These pathological subtypes demonstrate no significant difference in age at death or disease duration to suggest that they capture an earlier or later cross-sectional process in pathological pTDP-43 development. Rather, they are associated with different motor cortical burden of GFNIs and grains, topographical spreads, overall cognitive performances and co-pathologies to suggest the involvement of divergent pathomechanisms in ALS-TDP. Given the similarities between two of these ALS-TDP subtypes with that seen in FTLD-TDP, we have adopted the same alphabetised classification and highlight the need for further research to elucidate whether these similar subtypes identified in different clinical phenotypes share common disease pathways. As such, we propose that future studies specify both clinicopathological phenotype and pTDP-43 subtype, which will be important for the advancement of current understanding of disease pathogeneses and the development of targeted therapies for pTDP-43 proteinopathies.Additional file 1: Supplementary Figure 1.


This study investigates energy harvesting by a deionized (DI) water droplet flow on an epitaxial graphene film on a SiC substrate. We obtain an epitaxial single-crystal graphene film by annealing a 4H-SiC substrate. Energy harvesting of the solution droplet flow on the graphene surface has been investigated by using NaCl or HCl solutions. This study validates the voltage generated from the DI water flow on the epitaxial graphene film. The maximum generated voltage was as high as 100 mV, which was a quite large value compared with the previous reports. Furthermore, we measure the dependence of flow direction on electrode configuration. The generated voltages are independent of the electrode configuration, indicating that the DI water flow direction is not influenced by the voltage generation for the single-crystal epitaxial graphene film. Based on these results, the origin of the voltage generation on the epitaxial graphene film is not only an outcome of the fluctuation of the electrical-double layer, resulting in the breaking of the uniform balance of the surface charges, but also other factors such as the charges in the DI water or frictional electrification. In addition, the buffer layer has no effect on the epitaxial graphene film on the SiC substrate. Graphene is a monolayer crystal with carbon atoms in a honeycomb structure that has been intensively investigated for various applications because of its extraordinary carrier mobility and chemical stability . GraphenOver the past decade, the number of sensor devices connected to the Internet has increased. These “Internet of things (IoT)” devices can be used anywhere, such as monitoring a person with a pacemaker, pastured livestock, and traffic control . As the However, the origin of such energy-harvesting processes remains unclear. Various mechanisms for nanocarbon-based energy-harvesting devices based on solution flow have been proposed, such as the momentum transfer by phonon , breakin2 bonds without defects and dislocations. Furthermore, protein and amino acid adsorption on the epitaxial graphene film generally shows electron doping , respectively. The carrier-type change is typical of H2 intercalated epitaxial graphene films [Typical Raman spectra of the epitaxial graphene film before and after Her layer  and not ubstrate . The forne films . These r2 intercalation for the parallel and perpendicular electrode configurations were 63 and 46 mV, respectively, which were similar to those before H2 intercalation, indicating that the buffer layer under the epitaxial graphene film was not influenced by energy harvesting from the solution flow.A summary of the voltage generated by 50 measurements using DI water flow is shown in The dependence of the generated voltage on the droplet amount in the epitaxial graphene film with the buffer layer is shown in As shown in The origins of energy harvesting by solution droplets flowing on the graphene film differ as mentioned in the introduction. A key interpretation is the significance of the existence of anions and cations as well as the broken uniform balance of surface charges in graphene by the electrical double layer in the moving solution droplet . TherefoBecause epitaxial graphene films on the SiC substrate used in this study were single crystals with large areas with no polymer residue, ideal results were expected for graphene-based devices. Therefore, the energy harvested by the solution flow on the graphene surface must not be anions or cations in the solution. Hence, the electrical double layer is inessential in this case.The generated voltages depended on the number of DI water droplets as shown in This study investigated energy-harvesting characteristics of single-crystal epitaxial graphene films synthesized on SiC substrates using DI water flow. A voltage as high as 100 mV was generated by the DI water flow, and the generated voltage was independent of the electrode configurations parallel and perpendicular to the DI water flow as well as the existence of the buffer layer under the epitaxial graphene film. The average electrical power of energy harvesting by the DI water flow exceeded 1 µW. The epitaxial graphene film-based energy-harvesting devices using solution flow can be used for electrical power supply to IoT devices in the presence of flowing water, such as river or seashore, with no electrode configuration in the direction of water flow.


In a discovery cohort, we found significantly higher mortality in patients with IPF who had elevated platelet counts within the normal range. However, our validation cohort did not confirm this observation, despite significantly increased platelets, neutrophils, active TGFβ1, and CCL5, a chemokine produced by inflammatory cells, in the blood, lung, and bronchoalveolar lavage (BAL) of patients with IPF. In vivo, we showed that despite platelets being readily detected within the lungs of bleomycin-treated mice, neither the degree of pulmonary inflammation nor fibrosis was significantly different between TGFβ1fl/fl.PF4-Cre and control mice. Our results demonstrate for the first time that platelet-derived TGFβ1 does not significantly mediate inflammation or fibrosis in a PF animal model. Furthermore, our human studies revealed blood platelet counts do not consistently predict mortality in IPF but other platelet-derived mediators, such as C-C chemokine ligand 5 (CCL5), may promote neutrophil recruitment and human IPF.Transforming growth factor-β1 (TGFβ1) is the key profibrotic cytokine in idiopathic pulmonary fibrosis (IPF), but the primary source of this cytokine in this disease is unknown. Platelets have abundant stores of TGFβ1, although the role of these cells in IPF is ill-defined. In this study, we investigated whether platelets, and specifically platelet-derived TGFβ1, mediate IPF disease progression. Patients with IPF and non-IPF patients were recruited to determine platelet reactivity, and separate cohorts of patients with IPF were followed for mortality. To study whether platelet-derived TGFβ1 modulates pulmonary fibrosis (PF), mice with a targeted deletion of TGFβ1 in megakaryocytes and platelets  is unclear. We identified that patients with IPF have significantly more platelets, neutrophils, and active TGFβ in their airways than control patients. Using an animal model of IPF, we demonstrated that platelet-derived TGFβ does not significantly drive lung fibrosis or inflammation. Our findings offer a better understanding of platelets in both human and animal studies of IPF. Interstitial lung diseases (ILDs) consist of over 200 inflammatory and fibrotic diseases affecting the lung interstitium , with idTransforming growth factor-β1 (TGFβ1) has a plethora of biological roles ranging from inflammation through fibrosis  and wountgfb are associated with IPF disease progression  of patients with IPF , and mougression . Fibroblgression . Howeverproposed –14. IdenPlatelets are key mediators in immunity and tissue remodeling and represent an abundant source of TGFβ1, which can be released rapidly on cell activation . The rolThe role of platelets in chronic respiratory conditions such as IPF is now actively being pursued . It has Platelets and neutrophils act in a synergistic manner to cross the endothelium during inflammation in vitro and in vivo . Along wDespite the growing body of evidence demonstrating the increased presence of activated platelets in clinical and animal studies of IPF, it is still unclear how platelets drive fibrotic disease progression and whether platelets represent a major source of profibrotic TGFβ1 in IPF. Therefore, in this study we sought to define whether platelets, and more specifically, platelet-derived TGFβ1, mediate IPF disease progression using both clinical patient samples and an animal model of pulmonary fibrosis.fl/fl.PF4-Cre mice were obtained from Prof. Richard Hynes , BALF or lung biopsies from male and female healthy controls, patients with IPF or non-ILD, were collected with written informed consent and research ethics committee approval (IRAS study I.D. 29531). For survival analysis, male and female patients with IPF from the University College London Hospital (UCLH) or Exeter ILD Services were followed prospectively for mortality based on blood platelet counts as described in Supplemental Methods (ethics study number REC reference 18/L0/0937). Blood platelet counts from a cohort of male and female patients with IPF or healthy controls were obtained from the UK Biobank (applications 9072 and 44046). Patient demographics are shown in n = 6–12). BAL cells were stained with anti-CD61 antibody  or mouse IgG1 isotype control , and percentage of CD61+ platelets in BALF (n = 3–6) was quantified on a BD FACSVerse flow cytometer  and analyzed with FlowJo v10 software .Patient BALF and plasma were collected and processed as described in Supplemental Methods. Cell counts were performed on cytospun BALF (n = 4–5), plasma (n = 10–37), and BALF (n = 7–16) using ELISA kits . Active TGFβ1 was quantified by a Mink lung epithelial cell (MLEC) bioassay , and total matrix metalloproteinase-7 (MMP7) were quantified in PRP (bioassay  as descrn = 4) were added to the top of 8 μm PET trans-wells  and chemoattractants: 100 nM N-formylmethionine-leucyl-phenylalanine (fMLP) (Sigma-Aldrich), 1 ng/mL TGFβ1 (R&D systems), 10 ng/mL leukotriene B4 (LTB4)  or PRP added to the bottom. After 1 h at 37°C, cells that had migrated toward the chemoattractant in the bottom chamber were counted on a BD FACSVerse flow cytometer.Human or murine neutrophils and PRP were isolated as described in Supplemental Methods. In brief, untreated, 1 μM SB-525334 , 10 μM Galunisertib  ALK5 inhibitors or DMSO vehicle pretreated neutrophils  were randomized into treatment or control groups. The treatment groups were given 25 or 50 IU bleomycin  in a volume of 50 μL 0.9% saline by oropharyngeal instillation. The control group was given 50 μL 0.9% saline by oropharyngeal instillation. Mice were monitored for disease severity and culled at 6, 21, and 28 days post instillation. Individual mice were coded for each experiment to allow for blinded result assessments and data analysis.Male and female TGFβ1BALF was obtained from murine lungs by three washes with 1 mL PBS. BALF was centrifuged and cell pellet was processed for cytospin cell counts. Murine lungs were homogenized in PBS before red blood cell lysis. Cells were blocked with Fc block  and stained with anti-Ly6G-PE , anti-CD11b-APC , and anti-CD11c-APC-H7  antibodies. Cells were analyzed on a BD FACSVerse flow cytometer and FlowJo software.Dehydrated murine lungs were scanned on a SkyScan-1072 micro-CT scanner and images were reconstructed using SkyScan NRecon software . The degree of fibrosis in the lungs was assessed using InForm image analysis software  as previously described . Total lMurine or human lungs were processed for histological analysis as described in Supplemental Methods. Immunohistochemistry (IHC) was performed for murine  or human CD61 . Sections were scanned on a Nanozoomer Digital Slide Scanner and analyzed using NDP.view software . Quantification of collagen deposition in alveolar walls based on modified Martius Scarlet Blue (MSB) staining was performed using Orbit Image analysis software .U test, unpaired t test, one- or two-way ANOVA with Holm–Sidak post hoc testing were performed with Prism v9 software . Cox proportional hazard modeling, with multivariate analysis using mortality data of patients with IPF, was conducted using OriginLab statistical software. Unless stated otherwise, means ± SE are shown in all graphs with a P < 0.05 considered significant.All experiments were conducted with at least three technical replicates. For neutrophil experiments, three to four biological replicates were used to minimize donor variability. All statistical analyses with the Gehan–Breslow–Wilcoxon test, linear regression, Mann–Whitney 9/L) and lowest in those with the lowest platelet counts . In addition, data from the UK Biobank revealed that patients with IPF had significantly higher blood platelet blood counts than healthy controls (P < 0.01).We initially sought to investigate the role of platelets in IPF disease progression. A discovery cohort of consecutive, unselected patients with IPF  from UCLCox proportional hazard modeling with multivariate analysis was conducted to further investigate any potential correlations with mortality in the UCLH and Exeter patient cohorts. In both the UCLH and Exeter cohorts, higher percentage in forced vital capacity (FVC) was found to be significantly protective (probability for hazard ratio was 0.005 for UCLH cohort and 0.0001 for Exeter cohort). In addition, age at diagnosis was found to offer protection in the Exeter cohort, but not in the UCLH patient cohort . Cox proportional hazard modeling based on sex or platelet counts did not show any significant correlation with mortality in either patient cohort. These findings suggest that although blood platelet counts do not consistently predict IPF disease progression or survival, patients with IPF have significantly more platelets in their blood than healthy controls.P = 0.044). In addition, we found no correlation in the levels of total or active TGFβ1 with blood platelet counts from patients with IPF    was usedcontrols A, confirfl/fl.PF4-Cre mice had regained initial weight loss with no significant difference between bleomycin-treated littermate or TGFβ1fl/fl.PF4-Cre mice . HPLC analysis for hydroxyproline (a major component of collagen) content in whole lungs (Following 21 days after bleomycin (50 IU) treatment, littermate and TGFβ1Cre mice B. No difCre mice B. To detCre mice  revealedCre mice C. A trenobserved D and E ble lungs F confirmfl/fl.PF4-Cre animals did not differ , the percentage weight loss between littermate and TGFβ1t differ A. Micro-t differ B and C. t differ D. No difanalyzed E. This s+ platelets and platelet aggregates were found within IPF lung, particularly in the blood vessels , indicating that platelet-derived TGFβ1 does not affect neutrophil counts at baseline in naïve mice. During the peak of inflammation at day 6, littermate and TGFβ1fl/fl.PF4-Cre mice treated with bleomycin lost significantly more weight than saline-treated mice . This suggests that although platelet-derived TGFβ1 influences neutrophil migration in vitro, it does not significantly drive cellular recruitment into the lung or airways in vivo.To address whether platelet-derived TGFβ1 can influence cellular recruitment during pulmonary inflammation in vivo, littermate or TGFβ1ted mice A. Signifted mice B–E. HoweCre mice C–E. FlowCre mice F–I. BleoCre mice I, with eCre mice I. To ver+ platelets in BALF  , on IPF Recent evidence has proposed the lung as a major site for platelet biogenesis in mice with a distinct megakaryocyte subset, residing within the lung interstitium . In addifl/fl.PF4-Cre lungs; however, the degree of fibrosis was equivalent in both treatment groups, suggesting that platelet-derived TGFβ1 is redundant in this animal model of PF. Of importance, it should be noted that although bleomycin remains the gold standard animal model for PF research, it does not fully recapitulate the human disease. The degree of lung fibrosis in mice is dependent on bleomycin dose  or APB5 (a PDGFR-β blocking antibody) attenuates bleomycin-induced PF , a European Commission FP7 award PIEF GA-2012–326928 to C. Rebeyrol, and a GW4 BioMed MRC Doctoral Training Partnership award to A. Duckworth.No conflicts of interest, financial or otherwise, are declared by the authors.D.L.W.C., C.J.S., and J.C.P. conceived and designed research; D.L.W.C., T.A.M., J.S., C.R., H.S.G., H.L.B., M.H., E.K.D., R.J.J., A.A.K., A.D., M.L., and C.J.S. performed experiments; D.L.W.C., T.A.M., J.S., E.K.D., and A.D. analyzed data; D.L.W.C., T.A.M., J.S., E.K.D., C.J.S., and J.C.P. interpreted results of experiments; D.L.W.C. and T.A.M. prepared figures; D.L.W.C. and T.A.M. drafted manuscript; D.L.W.C., T.A.M., J.S., C.R., H.S.G., H.L.B., M.H., E.K.D., R.J.J., A.A.K., A.D., M.L., C.J.S., and J.C.P. edited and revised manuscript; D.L.W.C., T.A.M., J.S., C.R., H.S.G., H.L.B., M.H., E.K.D., R.J.J., A.A.K., A.D., M.L., C.J.S., and J.C.P. approved final version of manuscript.