Trimethylamine-N-oxide (TMAO) is a circulating biomarker associated with cardiovascular disease (CVD). Production of TMAO is facilitated by gut microbiota and dependent on micronutrients such as choline, betaine, and L-carnitine, present in foods such as red meat and eggs.We sought to predict serum TMAO quartile levels among healthy individuals at increased risk of CVD using clinical data via an ordinal logistic model.P < 0.30 were evaluated further. Second, demographic variables  were included in a multivariable model with each previously identified independent variable controlling for potential confounding. Last, the final model included fixed demographics and candidates from the confounder-adjusted model with P < 0.10.Data from participants (n = 127) enrolled in a longitudinal observational study on CVD were used to build a predictive model for TMAO using ordinal logistic regression with demographic variables and 40 other variables considered related to CVD risk. First, univariate models for each covariate were tested (with serum TMAO quartiles as the dependent variable), and only variables with Eight candidate variables were included in the final model, with only transferrin, high-density lipoprotein cholesterol (HDL-C) and race (white vs. non-white) showing significant associations with TMAO. Participants had 0.16 (Q2), 0.31 (Q3), and 0.20 (Q4) odds of being in a higher TMAO quartile compared with participants in the lowest transferrin quartile. Non-white participants had 2.92 times higher odds of being in the highest TMAO quartile compared to white individuals. Participants in the second quartile of HDL-C had 2.68 times higher odds of being in a higher TMAO quartile compared with participants in the lowest HDL-C quartile.Transferrin demonstrated a significant predictive association with TMAO and may represent a novel potential biomarker of increased CVD risk worthy of further study. These results warrant further examination of iron, metabolism, homeostasis, and gut microbiome to better understand and mitigate known increased CVD risk.The online version contains supplementary material available at 10.1186/s12872-022-02644-3. Heart disease remains the leading cause of death, responsible for more than 655,000 deaths in the U.S. and 17.9 million deaths worldwide each year. Between 2016 and 2017, heart disease resulted in approximately $363 billion in costs of health care services, medicines, and lost productivity due to death in the United States [Apoe−/− mice with L-carnitine leads to aortic atherosclerosis in the absence of adverse alterations in lipoprotein or glucose levels, and TMAO inhibits reverse cholesterol transport [Traditional factors used to predict atherosclerotic cardiovascular disease (ASCVD) risk and reduce costs/outcomes via treatment (e.g. with statins) include blood pressure, glucose, lipids, weight, physical inactivity, stress, smoking, and certain dietary patterns . Conversransport . In addiransport  InterestDietary studies have provided consistent evidence that omnivorous diets are associated with high TMAO levels compared to vegetarian diets. Since TMAO is a metabolite of the precursor nutrients choline and L-carnitine, many studies have investigated the association between diet and TMAO levels, particularly diets high in red meats  or eggs;Thus, TMAO levels demonstrate a relationship with omnivorous diets and CVD risk; however, very few studies have described other CVD-related non-dietary biomarkers associated with TMAO. The objective of this study is to explore potentially novel dietary factors or biomarkers that associate with TMAO levels, among participants at increased risk of incident ASCVD.Data used in this analysis were initially collected from a longitudinal observational study of 127 recruited adults greater than 40 years of age with risk factors for, but without known, ASCVD. Adults without evident ASCVD but with 2 or more risk factors  were prospectively identified primarily by recruitment from ambulatory clinics associated with the Ohio State University Wexner Medical Center. Other participants were enrolled via flyer advertisements, word of mouth, and by visiting other clinics to recruit. No participants had a known history of iron overload or significant blood transfusion. All subjects gave written informed consent to participate in this Institutional Review Board-approved study.All biometric measurements were obtained in person at baseline and at 2 years follow-up. Only baseline data were used for the current study due to the lack of significance in temporality and to maximize precision of the sample of data used for analysis. The leading author of this manuscript had full access to all data in this study and takes responsibility for its integrity and the data analysis.4COOH (5 mM) with 0.1% formic acid) and solvent B (acetonitrile). Using the multi reaction monitoring (MRM) technique on a Thermo TSQ Quantiva (triple quadrupole MS/MS) instrument, the ion pairs of 76.1/58.1 and 85.1/66.1 were used to monitor the concentration of TMAO and the internal standard (IS) d9-TMAO, respectively. All mass spectrometer parameters were tuned to give the optimized MRM signal. For standard curve preparation for calibration, to 160 µL of MeOH, 20 µL of TMAO stock solution  were added together with 20 µL of IS at 1 µg/mL in ACN to yield standard samples . The quality controls (QCs) were at 10, 100, and 500 ng/mL. For human plasma sample preparation, to 50 µL of the sample, 50 µL of IS at 1 µg/mL in ACN and 400 µL of cold MeOH (pre-chilled at − 20 °C) were added. The mixture was vortexed for approximately 3 s, kept at − 20 °C for 30 min, and centrifuged at 13,000 rpm/4 °C for 10 min. A supernatant quantity of 100 µL was transferred to glass vials for LC-MS/MS injection and measurement.Serum concentration of TMAO (the outcome variable) was measured with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Separation for HPLC was performed on a Dionex UltiMate 3000 RSLCnano System with a Waters ACQUITY UPLC BEH amide column at 30 °C with a gradient including solvent A  were abstracted from medical records and collected in person. Biomarkers studied included blood pressure, Modification of Diet in Renal Disease-derived glomerular filtration rate (MDRDGFR), hematocrit, transferrin, ferritin, hepcidin, adiponectin, TMAO, blood total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), oxidized LDL-C, triglycerides, iron, iron saturation, ferritin, and creatinine levels , 16. BasP < 0.30 retained for further testing. Successive stages of testing were based on a stricter threshold (P < 0.20) in multivariable models with age, gender, and race to adjust for potential confounders. In the final model, only covariates approaching statistical significance at P < 0.10 were considered of value for TMAO prediction. All analyses were conducted using Intercooled Stata Version 14.1  software . For the original data set a missing outcome deletion approach was used to deal with the missingness of the outcome data. Therefore, any observations that had missing baseline TMAO data were removed. Continuous measures that were highly skewed including serum TMAO (dependent variable) were recoded into quartiles. Race was operationalized as white versus non-white based on small counts of participants of non-white races. After cleaning and managing the data set, ordinal logistic regression was used to build a predictive model for serum TMAO levels. TMAO levels were stratified into quartiles for testing associations with demographic factors  and 40 clinical variables potentially related to CVD risk. Initially, univariate models were tested and only covariates with A total of 127 participants with reported baseline TMAO serum level data were enrolled  positive associations with TMAO were observed for weight (e.g. 1.01) and HDL-cholesterol , and inverse associations were observed for transferrin  serum iron , iron saturation  and total cholesterol .Table P < 0.10) with TMAO. Participants had 0.16 (Q2), 0.31 (Q3), and 0.20 (Q4) odds of being in a higher TMAO quartile compared with participants in the lowest transferrin quartile (Q1). Non-white participants had 2.92 times higher odds of being in the highest TMAO quartile compared to white participants. Participants in the second quartile of HDL-C had 2.68 times higher odds of being in a higher TMAO quartile compared with participants in the lowest HDL-C quartile.Eight candidate variables with age, gender, and race were included in the confounder-adjusted final model . Of these, only transferrin, race (white vs. non-white), and HDL-C showed significant associations . It is notable that the average BMI and weight was higher than a randomly selected normal population but because obesity/overweight is also a risk factor for CVD  and demoThe observed inverse association of TMAO with transferrin is generally consistent with the reciprocal involvement of these factors in systemic inflammation, vis a vis., TMAO is pro-inflammatory whereas transferrin is anti-inflammatory.Elevated TMAO concentrations have been found to induce inflammatory endothelial injury by inducing the release of inflammatory cytokines, enhancing monocyte adhesion to endothelial cells, and promoting oxidative stress . Recent Important clinical measures of iron absorption and delivery to tissues include serum iron and iron saturation, both of which are related to transferrin. Serum iron measures the amount of iron that is bound to transferrin in the blood, and iron saturation is the percentage of transferrin associated with iron, i.e. the percentage of total iron binding capacity. Notably, in our sample, low serum iron and low iron saturation were significant predictors of high levels of TMAO in univariate models, but these effects were attenuated in the final multivariable model after adjustment for transferrin and other variables. Furthermore, the overall mean iron saturation of our sample of participants was low (20%) suggesting that many participants may have had subclinical iron deficiency anemia or inflammatory anemia (anemia of chronic disease).In our sample of adult participants, all of whom had CVD risk factors, transferrin was the strongest and most consistent predictor of TMAO, e.g., high levels of pro-inflammatory TMAO were markedly reduced (69–84%) in participants with higher levels of anti-inflammatory transferrin Fig. . In contTransferrin also chelates free iron, which is toxic, thereby preventing the formation of inflammatory free radicals and oxidative damage to the endothelium and other tissues . TransfeSeveral previous studies have reported that low transferrin is associated with systemic inflammation and increased risk of CVD. A follow up study of participants in the National Health and Nutrition Examination Survey (NHANES) reported an inverse relationship between transferrin saturation and CVD mortality , and a rSeveral studies have linked transferrin with other chronic conditions strongly associated with CVD such as diabetes and chronic kidney disease –30. As pLower transferrin saturation has been independently associated with higher risk of mortality for CKD patients . InteresThe literature is consistent in the associations between TMAO and transferrin on chronic disease risk and outcomes, which strengthens the biological plausibility of a potential relationship between TMAO and transferrin in our study. What is clear from the discussion above is that TMAO and transferrin each demonstrate associations with various chronic diseases such as CKD, diabetes, and CVD. What remains unclear are the potential mechanistic interactions that TMAO and transferrin might have with one another. One feasible possibility is that decreased transferrin is associated with increased free iron, which favors the presence of certain gut microbiota associated with increased TMAO production , 31. OneNon-white participants had 3 times higher odds of being in the highest quartile of serum TMAO compared to white participants. Similar racial differences in other CVD-related biomarkers have been reported previously, including those related to inflammation, lipids, adipokines, as well as biomarkers of endothelial damage, myocyte injury, and neurohormonal stress . Our finIt is well known that higher total cholesterol and LDL-C are associated with increased risk of CVD, whereas elevated HDL-C is generally considered protective . Recent Most significantly, among participants with risk factors for (but absence of known) ASCVD, transferrin had a significant predictive association with TMAO and may represent a novel potential biomarker of increased CVD risk worthy of further study. The biological mechanistic relationships between TMAO, HDL-C, and transferrin remain unclear. These results warrant further examination of iron, metabolism, homeostasis, and gut microbiome to better understand and mitigate known increased CVD risk.Additional file 1. Dataset used for analysis.


RSPECT) using SPECT/CT was calculated for detecting metastatic sentinel lymph nodes. To localize sentinel lymph nodes exactly, we compared the positions of sentinel lymph nodes localized using SPECT/CT with positions localized surgically using an intraoperative γ-probe.  We retrospectively enrolled breast cancer patients who underwent SPECT/CT prior to sentinel lymph node biopsy. Quantification of radiotracer uptake from SPECT/CT data was performed. A radioactivity count threshold . No metastasis was found in lymph nodes with RSPECT ≤ 30% in the >2 SLNs group, and thus, 30% (157/526) of SPECT/CT-identified nodes would avoid unnecessary removal. The positions of sentinel lymph nodes localized by SPECT/CT and γ-probe were identical in 42% (39/93) of patients.  491 patients were included, with a median of 3 sentinel lymph nodes/patient detected by the  Quantitative Tc-99 m SC SPECT/CT imaging has the potential to preoperatively locate sentinel lymph nodes and intraoperatively avoid unnecessary sentinel lymph node biopsy. According to the data on the global cancer burden updated in 2020, the incidence of female breast cancer has surpassed lung cancer and now ranks first in the world . Axillarγ-probe. However, Schuman et al. found that processing the two hottest lymph nodes and suspicious nodes is sufficient for initial axillary staging of breast cancer [γ-probe may not detect the hottest SLN successfully for two main reasons: (i) the node is very close to the injection site, obscured by scatter and (ii) the position of the node is too deep to locate. In such circumstances, single-photon emission computed tomography/computed tomography (SPECT/CT) imaging may have an additional value.SLNs in breast cancer are detected using a combination of radiotracer and blue dye . Affectet cancer . Practicγ-probe in determining the exact anatomy of SLNs. Moreover, in a recent study, Kwak et al., simulating the common practice of removing SLNs with ex vivo count rate ≥10% of the hottest node detected by γ-probe, proposed the concept of the radioactivity count threshold, which was calculated based on SPECT/CT quantification, and found that no metastatic SLN (m-SLN) was detected when using a threshold of 20% [SPECT/CT technology has the potential for SLN localization and quantitative analysis of radioactive lymph nodes. Preoperative SLN SPECT/CT imaging provides accurate anatomical information of SLNs with lymphatic drainage into the axilla and extra-axillary regions, distinguishing false-positive results caused by nuclide contamination and lymphatic tortuosity and reducing the false-negative rate caused by patient obesity. It compensates for the limitation of the d of 20% . Neverth99mTc-SC SPECT/CT quantification in helping surgeons select SLNs that are likely to be metastatic and minimize the number of SLNs removed. In order to help surgeons quickly locate SLNs, we evaluated the accuracy of 99mTc-SC SPECT/CT for preoperatively marking the position of SLNs.This study aimed to investigate the feasibility of a radioactivity count threshold using Procedures performed in this study were approved by the institutional review board and ethics committee of Shandong Cancer Hospital. All consecutive breast cancer patients scheduled for SLNB were collected between December 2020 and June 2021. Inclusion criteria were primary breast cancer (T1-3) and no palpable lymph node at diagnosis, ≤2 suspicious nodes on imaging, or ≤2 positive nodes confirmed by needle biopsy ± clip placement. Patients with pregnancy and inflammatory breast cancer were excluded. Several patients were randomly selected for preoperative SLN localization on skin. Informed consent was obtained from each patient.99mTc-SC  for surgery planned on the same day or twice the dose (0.5 mCi) for surgery arranged the next day. Planar scintigraphy (PS) was acquired after 20 min (within 4 h), after the injection of the radiotracer, with 3-minute static images in the anterior and lateral projections , followed by SPECT/CT  with the patient lying supine as well as arms positioned above the head. Images were processed by OSEM (ordered-subset expectation maximization) reconstruction and attenuation correction.A dual-head SPECT/CT scanner with low-energy high-resolution collimators  was used. Each patient was given a peritumoral intradermal injection of 9.25 MBq (0.25 mCi) ±10% of −3 m, height: 0.24 m) were arranged at an interval of 0.03 m and fixed with tape orderly. The localization tool was attached to the side with cancer of the patient's chest closely before SPECT/CT. The radiologist diagnosed SLNs on SPECT/CT images and selected the hottest SLN for localization. The position of the hottest SLN was mutually determined by the localization tool, and a laser line of SPECT/CT was marked for surgery reference.We have made a simple tool for preoperative SLN localization. Ten metal sticks  was used for detecting radioactive nodes. Lymph nodes stained blue and/or ≥10% of the ex vivo counts of the hottest node were removed. Suspicious lymph nodes palpable by hand were also processed. Harvested nodes were sent for pathological examination. SLN status (hot/blue/suspicious), position, radioactivity counts, and pathological results were recorded.SLNB was operated on the same day as the radiotracer injection or the next day in the morning. A peritumoral intradermal injection of 1% methylene blue  was performed 10–15 min before surgery. An intraoperative γ-probe detected each extracted SLN for 10–15 s, and the final radioactivity count was obtained by pressing “target button.” The hottest radioactive node detected by either modality (SPECT/CT or γ-probe) was designated as 100%, and the following SLN was calculated as a percentage of the hottest node detected by SPECT/CT quantification or by γ-probe, expressing as RSPECT and Rγ-probe.Quantification of radiotracer uptake in the SLNs from SPECT/CT images was processed in the “Volumetrix MI Evolution for Tissue” module of the Xeleris Functional Imaging Workstation (Xeleris Version 4.0). The radiologist identified radioactive lymph nodes and chose semiautomatic segmentation for the region of interest (ROI) on the highest radioactive uptake SPECT layer. Radioactivity counts of SLNs are generated by clicking on the radioactivity concentration of each node. Graphical images describing the location of the ROIs and the generation of radioactivity counts of hot nodes on SPECT/CT are shown in t-test. A P value of <0.05 was considered as statistically significant.Data analysis was processed using SPSS 19.0. Categorical variables of patient characteristics and metastasis incidence of SLNs were analyzed using the chi-square test or univariate Fisher exact test. Numerical variables were tested for normality and applied the Kruskal–Wallis test and two-sample 99mTc-SC SPECT/CT prior to SLNB were enrolled. 1504 SLNs were excised using a combination of a radiotracer and blue dye, with a median of 3 SLNs/patient. SLNs were identified by SPECT/CT in 82% (404/491) of the patients, with a median of two SLNs/patient. Surgeons removed every node identified by SPECT/CT in 87% (350/404) of the patients. In the remaining patients, the same nodal level was sampled, but the number of SLNs removed intraoperatively was not equivalent to the number identified by SPECT/CT. The most common reason for this phenomenon was that no radioactivity was detected by the γ-probe at this location.491 consecutive breast cancer patients who underwent PS and P < 0.001) or by γ-probe (P=0.049). Additionally, patients younger than 50 y had more SLNs identified by SPECT/CT than those older than 50 y . Patients with a history of tumor excisional biopsy tended to have more intraoperative SLNs . Furthermore, we found that SLN metastasis rates were different in patients with different AJCC stages (P < 0.001) and were statistically higher in male breast cancer than female .The clinical characteristics of patients are summarized in P < 0.001). The most common region of lymph node drainage was the axilla, accounting for 94% (832/886) of all nodes. In 9% (37/404) of the patients, 33 and 11 lymph nodes drained to internal mammary and supraclavicular regions, respectively. 15 cases were presented as hot nodes on PS but interpreted as false-positive results due to tracer contamination on SPECT/CT images.PS visualized SLNs in 66% (322/491) patients and SPECT/CT visualized SLNs in 82% (404/491) of the patients, resulting in a 16% improvement in the visualization rate .Lymph nodes were visualized on SPECT/CT in 404 patients, and AS was metastatic in 28% (112/404) of them. Of the remaining patients, 34% (30/87) of the patients with no lymph nodes visualized on SPECT/CT had metastatic lymph nodes. Metastasis incidence of lymph nodes had a significant difference between different numbers of SLNs . Based oRSPECT of m-SLNs in different 10% gradients (RSPECT ≤ 30% was used as a radioactivity count threshold. This would avoid resection of 30% (157/526) of the SPECT/CT-identified nodes, beneficial for 63 (40.9%) patients. A typical case describing the procedure of SLNB combined with SPECT/CT quantification is presented in In the >2 SLNs group, 15% (23/154) of patients were metastatic with 36 m-SLNs . To avoid removing invalid SLNs that were nonmetastatic, we analyzed the radients . Based oγ-probe and SPECT/CT. A total of 29 m-SLNs were pathologically examined in 23 patients. The quantification of radioactive uptake in m-SLNs is listed in γ-probe (66% (19/29)). Four m-SLNs occurred in the second hottest nodes, and one m-SLN occurred in the third hottest node detected by SPECT/CT. Three m-SLNs mismatched with the SLNs presented on SPECT/CT images, but they were observed to have morphological abnormalities. The γ-probe found metastasis on a fifth hottest node. RSPECT of m-SLNs were all over 30%, but no radioactivity count threshold of Rγ-probe could be found to identify the nature of the lymph node. When using the same radioactivity count threshold in the Rγ-probe, we calculated a test sensitivity of 86% and 44.6% (139/312) of intraoperatively detected nodes that would not need to be removed.In 93 patients chosen for localization, radioactivity counts of SLNs were measured both by the γ-probe were consistent in 42% (39/93) of the patients, and all were located in the anterior axillary region. In the remaining patients, the localization markers were located inside and/or below the position detected by γ-probe, with a deviation of 1–4 cm in distance. Particularly, a typical overweight patient, with a body mass index of 37 kg/m2, failed SLNB by using a combination of a radiotracer and blue dye. With the help of anatomic images and position markers localized by SPECT/CT, surgeons successfully removed a SLN in level I of the axilla, which was 6.69 cm away from the skin  and a history of prior excisional biopsy tended to have more SLNs detected, be exposed to unnecessary SLNB, and have more postoperative risks. These patients would be the target group of our findings. The m-SLNs more frequently occurred in male breast cancer patients than female patients (75% vs. 23.6%), which is consistent with previous studies . Althougγ-probe. The remaining locations were all situated in a fixed range, with a deviation of 1–4 cm from the position using γ-probe. We believe that the posture difference between SPECT/CT imaging and surgery may be the main reason for this discrepancy. Due to the limitation of the small SPECT/CT aperture, patients lay supine with arms positioned above the head, whereas during SLNB, patients most often lay supine with arms extending perpendicular to the body. If possible, patients should maintain the same position during both SPECT/CT imaging and surgery. Our preliminary investigation of preoperatively marking of SLNs on skin according to SPECT/CT images is feasible and reliable, which would aid in probe-directed surgery and reduce operator-dependent variation and time, especially in obese patients.Preoperative SLN localization would reduce the scope of surgical resection, important for breast-conserving surgery. In a previous study, radioisotope (Co-57) was often appropriately positioned on the side of the body opposite the camera for reference, exposing patients to extra radiation . We innoOur study has several limitations. First, the detection rate of SPECT/CT alone was low (82%), compared with other studies (84.4%–92%) , 25. The99mTc-SC SPECT/CT quantification of SLNs might be helpful to guide selective radioactive node resection during SLNB and minimize unnecessary lymph node removal by establishing radioactivity count thresholds, especially for patients with prior excisional biopsy and younger than 50 y.


Subacute sclerosing panencephalitis (SSPE) is a late-onset and fatal viral disease caused by persistent infection of the central nervous system by measles virus (MeV). We present the case of a 10-year-old child from South Asia affected by SSPE, stabilized with a combination of intrathecal interferon-α2b (INF-α2b) injections and oral inosiplex and how we continued the treatment when inosiplex was commercially stopped worldwide. Paramyxoviridae family. The illness typically begins with fever and pathognomonic enanthem. Complications occur in 10–40% of cases and subacute sclerosing panencephalitis (SSPE) is one of the most dramatic [Measles is an extremely contagious vaccine-preventable disease caused by the measles virus (MeV), belonging to dramatic . With widramatic .SSPE is a progressive neurological disease associated with high mortality within 1–3 years of onset . Patient® [SSPE treatments include antiviral agents such as isoprinosine, interferon-alpha, or ribavirin. These drugs are usually associated with anticonvulsants such as carbamazepine, levetiracetam, and clobazam to control myoclonic jerks. In the future, a fusion inhibitory peptide may be used . Current® ,8. Despi® . However® ,7,9.® 500 mg tablets  were commercially stopped. This illustrates the real public health problem related to a supply disruption or a marketing cessation of a pharmaceutical specialty, its impact on the clinical management of patients, and the essential role of hospital pharmacotechnical units.Our work aims to present the case of a 10-year-old child affected by SSPE who was stabilized with a combination of intrathecal INF-α2b injections and oral inosiplex and how we continue the treatment when ISOPRINOSINE6 units/m2/week was initiated, associated with ISOPRINOSINE® 500 mg tablets  33 mg/kg/8 h. Seizures were treated with usual antiepileptic drugs .The patient was born in Afghanistan, without any complications. He contracted measles at 6 months old without any complications. No vaccination was recorded. Unfortunately, in December 2017, he was hospitalized due to a brutal neurological degradation, with central hypotonia, spastic tetraplegia, fever flares, and generalized tonic-clonic seizures. The diagnosis of SSPE was made in India in June 2018 with an elevated level of anti-measles antibodies in cerebrospinal fluid and serum. Electroencephalography (EEG) finds asymmetric, unorganized, and slowed-down tracings, with rapid periodic bursts. In addition, magnetic resonance imaging (MRI) showed lesions consistent with SSPE. Intrathecal injection of IFN-α2b RELIFERON® (Reliance life sciences) 3.10On his arrival in France in January 2019, the patient presented spastic stiffness and almost no motricity of the four limbs with hyperreflexia on the left side. The Babinsky’s sign was uncertain. He had clonic eye movements and no ocular following. The pupillary reflex was normal.θ range (5–6 Hz), with a left-right asymmetry. High-frequency, pseudo-periodic, fronto-central bursts were seen. Sleep amplified the previously described abnormalities.A brain magnetic resonance imaging (MRIb) showed cortical and subcortical atrophy, with ventricular and peri-cerebral enlargement. There were diffuse signal anomalies of the white matter, with a hyperintensity in the T2 sequence and hypointensity in the T1 sequence. Basal ganglia and corpus callosum were atrophic and hyperintensity was seen in the internal capsule. In the subtentorial region, there was a demyelination aspect of both mesencephalon and metencephalon. .Figure Mycoplasma pneumoniae, Bartonella sp., and Toxoplasma gondii. Serologies for Cytomegalovirus and Epstein–Barr virus showed a high level of IgG.Chemiluminescence assay on the LCR showed a high anti-MeV IgG antibody titre . Serology was also positive for anti-MeV Immunoglobulin G (IgG). Polymerase chain reaction and all serologies were negative for neurotropic viruses, Decision was made by a multidisciplinary committee to continue the treatment with IFN-α2b and inosiplex , combine6 units/m2 per week during a month, then the maintenance dose was continued at 3.106 units/m2 per month. He received only six injections of IFN-α2b because INTRONA® was discontinued in France, and supply became impossible in March 2020.Concerning IFN-α2b, it was initially administered at the dose of 3.10® 500 mg) and stopped the worldwide marketing in 2014 due to low sales volume. After 3 months of research, the unit for medicines under temporary use authorization in Grenoble University Hospital (CHUGA) was able to import ISOPRINOSINE® 500 mg (Sanofi Aventis) from Belgium. The patient received therefore 20 mg/kg/8 h. Due to his clinical situation, the French drug agency authorized the importation of the specialty and Sanofi Aventis France has graciously offered ISOPRINOSINE® for this compassionate use. As of October 31, 2021, all remaining Sanofi Aventis stock expired. Given the lack of any alternative to provide for this patient, it was considered to make a magistral preparation. We looked for a Good Manufacturing Practices (GMP) supplier of this active pharmaceutical ingredient. In Europe, only one supplier was identified and with the precious help of MedicaPharma (Netherlands), we prepared 500 mg hard capsules of inosiplex in the preparation unit of CHUGA. The patient continued the treatment on an outpatient basis from November 18, 2021 .The paediatric unit requested inosiplex in June 2019. Unfortunately, only SANOFI AVENTIS marketed, in Europe, a speciality containing inosiplex (ISOPRINOSINEDuring the 18 days of treatment’s interruption, the patient's clinical condition deteriorated with an increase in the frequency of seizures. Caregivers reported that his condition had gradually improved since the reintroduction of inosiplex.Today, the patient's health condition is stabilized. Moreover, his neurological disorders, in particular dysautonomia, have not increased and no new tonic-clonic seizures were reported.Inosiplex preparation has contributed to stabilize his health condition until today. It was the only way to provide a safe response to the patient in this situation. The main challenge was to supply raw material for pharmaceutical use at a GMP grade, as inosiplex has a very limited indication and low frequency of use in Western countries. Thus, to our knowledge, CHUGA is the only hospital in France to provide this treatment by inosiplex. This case illustrates the problem of pursuing a treatment when a speciality is no longer marketed, reinforcing the interest in magistral preparation and the role of hospital pharmacies in the current context where stock outs are more and more frequent.Click here for additional data file.


Musa spp.). However, several key genes and their regulation mechanism of controlling plant height during banana development are unclear. In the present study, the popular cultivar ‘Brazilian banana’ (‘BX’) and its dwarf mutant (‘RK’) were selected to identify plant height-related genes by comparing the phenotypic and transcriptomic data. Banana seedlings with 3–4 leaves were planted in the greenhouse and field. We found that the third and fourth weeks are the key period of plant height development of the selected cultivars. A total of 4563 and 10507 differentially expressed genes (DEGs) were identified in the third and fourth weeks, respectively. Twenty modules were produced by the weighted gene co-expression network analysis (WGCNA). Eight modules were positively correlated with the plant height, and twelve other modules were negatively correlated. Combining with the analysis of DEGs and WGCNA, 13 genes in the signaling pathway of gibberellic acid (GA) and 7 genes in the signaling pathway of indole acetic acid (IAA) were identified. Hub genes related to plant height development were obtained in light of the significantly different expression levels (|log2FC| ≥ 1) at the critical stages. Moreover, GA3 treatment significantly induced the transcription expressions of the selected candidate genes, suggesting that GA signaling could play a key role in plant height development of banana. It provides an important gene resource for the regulation mechanism of banana plant development and assisted breeding of ideal plant architecture.Plant height is an important and valuable agronomic trait associated with yield and resistance to abiotic and biotic stresses. Dwarfism has positive effects on plant development and field management, especially for tall monocotyledon banana ( Musa spp.) are the most important staple food and fruit crops in tropical and subtropical regions [Bananas  genes control plant height and grain size, further influencing production in rice [GmLHY genes not only reduces plant height, but also shortens the internodes in soybean [Numerous studies prove that dwarfism is an important plant architecture trait in crop breeding. It is undeniable that the dwarf plant has characteristics of lodging resistance, high planting density and convenient management . It playse yield . The knond yield . MiR529a in rice . Mutagen soybean . TPS) involved in the early step of GA biosynthesis cause plant dwarfism in maize and Arabidopsis [P450-mediated GA biosynthesis has a negative impact on plant height development in maize and rice [It is well known that hormones play a key role in regulating cell division, elongation and differentiation of the apical meristem and vascular bending tissue ,25,26,27bidopsis ,43,44. Iand rice . Additioand rice . Excavatand rice . HoweverIn this study, the critical period points of plant height differences in ‘BX’ and its mutant were determined. The ‘RK’ is a natural dwarf mutant of ‘BX’, with a stable phenotype in the field for many years. Based on the transcriptomic analysis and weighted gene co-expression network analysis (WGCNA), differentially expressed genes (DEGs) were identified between the selected five developmental stages. The important gene modules and hub genes related to plant height were predicted. Real-time PCR was performed to evaluate the expressions of important DEGs after treatment with GA3. This study provides a gene resource for further assaying the regulatory network of banana plant height.Banana variety ‘RK’ is a natural mutation of ‘BX’. In the field, the plant height showed a stable significant difference at different years A,B. The The cDNA libraries of ‘BX’ and ‘RK’ pseudostem samples during different growth stages were prepared and sequenced using the Illumina 6000 platform. After filtering low-quality reads and removing adapter, a total of 207.22 Gb of clean data was obtained. The average clean data of each sample reached 4.23 Gb. The percentage of GC was among 47.59–53.76%. Q30 ranged from 93.21% to 95.88% , indicatCompared to the transcriptome data of ‘RK’, there were 2593, 4999, 3294, 1997 and 1523 down-regulated genes, and 1970, 5508, 3223, 2205 and 2156 up-regulated genes from the third week to the seventh week in ‘BX’, respectively . Especiap < 0.05), respectively. There were 1656, 226 and 406 GO entries in ‘BX’, and 1831, 278 and 422 GO entries in ‘RK’ for the Biological Process, Molecular Function, and Cellular Component categories, respectively. The most highly enriched GO terms belonged to the GA-mediated signaling pathway  in ‘BX’ , and the other four genes were not annotated B. In theTo further reveal transcriptome changes in different growth stages, WGCNA was used to analyze the transcription levels of ‘BX’ and ‘RK’ at different growth stages. After filtering, a total of 17922 genes were selected for WGCNA analysis. A total of 20 modules with different colors were identified, and grey represented no assignment to any module. Significant differences were found between different modules A,B. ExprThe modules of bisque4 and salmon were positively related to plant height, and contained 1549 and 1560 genes, respectively. Combining with DEG analysis, six genes from GA-mediated signaling pathway  and two genes from IAA biosynthesis (ko00901) were obtained from the bisque2 module A. Two geMYB transcription factors, one HLH, one GID1C and one ARF, were identified. The GID1C was also found from up-regulated DEGs in ‘BX’ between the third week and the fourth week. The most highly enriched GO terms were cell cycle  and intracellular steroid hormone receptor signaling pathway . Cytoscape was used to analyze the network in the white module, and a total of 17 hub genes were identified. These genes showed significant differences at different growth stages from the third week to the fifth week in ‘BX’ and ‘RK’ , respiratory chain complex , NADH dehydrogenase complex , ribose phosphate metabolic process  and ATP metabolic process . A total of 21 hub genes were obtained, including 3 ATP synthases  in the pseudostem of ‘BX’ treated with GA3 after one hour ’ by chemical and physical mutagenesis, respectively [In the early research, new dwarf and semi-dwarf varieties were obtained mainly through hybridization of tall and dwarf varieties in crops such as wheat, maize and rice ,51,52. Hectively . A new dectively . HoweverGID1. The functions of the other four genes were unknown. It suggested that the difference in plant height between ‘BX’ and ‘RK’ may be caused by different gene expressions of GA signal transduction. Previous studies also showed that mutations involved in GA biosynthesis and metabolic pathways led to dwarf phenotypes in plants [GID1) and DELLA proteins play an important role [GID1, binding and activating downstream DELLA proteins [DELLA binds to the E3 ubiquitin ligase SLEEPY1 (SLY1) and triggers its degradation by the 26S proteasome pathway [SPLs), GAMYB, SUPPRESSOR OF CONSTANS 1 (SOC1), and phytochrome-interacting factors (PIFs), and the downregulation of SPINDLY (SPY) [GID1 was identified as the hub gene from the positively module. In addition, downstream DELLA genes showed significant differences between ‘BX’ and ‘RK’. All of the GID1 homologous genes were significantly up-regulated after GA3 treatment, suggesting these genes were involved in GA signal transduction. Our results further supported that GA signal transduction is critical for plant height.Plant height is a quantitative trait that is controlled by multiple genes . In thisn plants . Thus fan plants . In the ant role . Bioactiproteins . Then, t pathway . After dLY (SPY) ,61,62. SDELLA, including AUXIN RESPONSE FACTOR (ARF) family and AUXIN INDUCIBLE/INDOLE-3-ACETIC ACID INDUCIBLE (AUX/IAA) [ARF was found in the white module positively associated with plant height, and GID1 was also involved in this module  ,63,64. Is module . TherefoMusa spp. AAA group, ‘BX’) and its mutant (Reke 3) were selected as materials. The seedlings from vegetative propagation were planted in the experimental field and greenhouse of the Chinese Academy of Tropical Agricultural sciences in Haikou, Hainan, China. The plant height of seedlings was measured and recorded once a week from planting.The banana cultivar Brazilian . Genomic DNA contamination was removed with RNase-free DNase I. RNA degradation and contamination were assessed using 1.2% agarose gel electrophoresis. The integrity and concentration of RNA were detected using a NanoDrop One . A total amount of 3 μg RNA was used to construct each cDNA library. A total of 48 RNA libraries from eight growth stages were constructed. All libraries were sequenced on the Illumina 6000 platform by the BioMarker Technologies Company .https://banana-genome-hub.southgreen.fr/ accessed on 6 December 2022). Hisat2 (v2.1.0) and QualiMap (v.2.2.1) were used to analyze the genome coverage and mapping rate. The gene expression levels were analyzed by the htseq-count (v1.99.2), and then converted the gene count to the FPKM (expected number of fragments per kilobase of transcript sequence per million base pairs sequenced). DEGs between samples of ‘BX’ and ‘RK’ at different growth stages were identified using the DESeq2 R package (v1.32.0) according to the standard of FC ≥ 2, FDR < 0.01 and p < 0.05.The Q20, Q30 and GC content were calculated using the FastQC (v0.11.9) software. The adapter, ploy-N and low-quality reads were filtered according to the quality control by the fastp (v0.20.0) software. The clean reads were aligned with the banana reference genome v2 download from the Banana Genome Hub . The protein function was annotated, and the GO and KEGG of DEGs were analyzed using the clusterfiler in R package.GO enrichment of DEGs was analyzed using the eggNOG-mapper (v1.0.3) against the reference database  of gene connection strength by average hierarchical clustering. The network modules used the dynamic tree cut algorithm with the minimum cluster size of 30 and the merging threshold function of 0.80 for identification. To link the physiological measurement with the network, the module eigengenes were associated with the physiological data. The GO and KEGG enrichment of each module was analyzed using clusterfiler in R package. The hub genes of modules highly related to phenotype were identified using cytoscape (v3.8.2). The hub genes were identified through 12 algorithms of cytoHubba (v0.1).To further validate the reliability of candidate genes, GA3 was used for spraying the whole banana plant in the greenhouse of the Chinese Academy of Tropical Agricultural sciences. The concentration of GA3 was set as 20 mg/L, with sterile water as the control. The whole plant of banana seedlings was sprayed two weeks after planting. The banana pseudostem tissue was collected at 0 h, 1 h, 6 h, 12 h and 24 h after treatment, respectively. At least three plants from each group were collected randomly and were stored at −80 °C.https://www.ncbi.nlm.nih.gov/tools/primer-blast; accessed on 17 May 2022). Diluted cDNA was amplified using TB Green premix EX Taq II. PCR was performed on a LightCycler96 Real-Time System . Each sample was performed with three independent biological replicates. The actin gene was used as an internal control [For the quantitative RT-PCR analysis, total RNA was extracted using the RNA extraction kit, and a reverse transcription kit  was used for the first complementary DNA (cDNA) synthesis. The RT-PCR primers of candidate genes  were des control .t-test within SPSS 22.0 . Statistically significant differences were indicated with p < 0.05 (*).For statistical analysis, at least three samples were taken to calculate the average ±SD. Statistical analysis was performed using Student’s In this study, the key period of plant height development of ‘BX’ and its dwarf mutant ‘RK’ were identified via phenotypic analysis. A total of 13 candidate genes in the signaling pathway of GA, and 7 candidate genes in the signaling pathway of IAA were found with WGCNA and transcriptome analysis in the critical period. Especially, the GA-related genes were induced significantly after GA3 treatment. Those genes showed higher expression levels in ‘BX’ than ‘RK’ at the fourth week, and were consistent with the transcriptome analysis.


Unrelated cord blood transplantation (CBT) is an alternative curative option for adult patients with acute myeloid leukemia (AML) who need allogeneic hematopoietic cell transplantation (HCT) but lack an HLA-matched related or unrelated donor. However, large-scale data are lacking on CBT outcomes for unselected adult AML. To investigate the trends of survival and engraftment after CBT over the past 22 years, we retrospectively evaluated the data of patients with AML in Japan according to the time period of CBT (1998–2007 vs 2008–2013 vs 2014–2019). A total of 5504 patients who received single-unit CBT as first allogeneic HCT for AML were included. Overall survival (OS) at 2 years significantly improved over time. The improved OS among patients in ≥ complete remission (CR)3 and active disease at CBT was mainly due to a reduction of relapse-related mortality, whereas among patients in first or second CR at CBT, this was due mainly to a reduction of non-relapse mortality. The trends of neutrophil engraftment also improved over time. This experience demonstrated that the survival and engraftment rate after CBT for this group has improved over the past 22 years. The availability of cryopreserved cord blood could allow patients without appropriate donors to receive allogeneic hematopoietic cell transplantation (HCT). This is because cord blood transplantation (CBT) is acceptable for tolerance to human leukocyte antigen (HLA) mismatch and is rapidly available –3. IndeeThe major limitations of CBT for adult patients are higher rates of engraftment failure and mortality, particularly in non-relapse mortality (NRM). Indeed, patients with advanced disease frequently received CBT, which might be associated with poor outcomes. Nevertheless, our recent study showed that early mortality and engraftment failure have improved after CBT as the first allogeneic HCT for 9678 adults over the past 20 years in Japan . This wan = 95) or related CBT (n = 1) were excluded from this study. We also excluded patients that lacked data about survival status (n = 12), and those with a previous history of allogeneic HCT (n = 1670). Finally, 5504 patients were eligible for this study. The primary outcome was overall survival (OS). Secondary outcomes were relapse-related mortality, NRM, neutrophil and platelet engraftment, and acute and chronic graft-versus-host disease (GVHD). This study was approved by the adult AML working group of the JSTCT and by the institutional review board of the Institute of Medical Science, The University of Tokyo (2021-30-0729).This retrospective study was conducted by the Adult AML Working Group of the Japanese Society for Transplantation and Cellular Therapy (JSTCT). The clinical data were collected by the Transplant Registry Unified Management Program (TRUMP) of the Japanese Data Center for Hematopoietic Cell Transplantation (JDCHCT) and the JSTCT , 20. Pat9/L or the platelet count was higher than 20 × 109/L without platelet transfusion, respectively. The grading of acute and chronic GVHD was defined using standard criteria  vs reduced-intensity conditioning [RIC]), use of total body irradiation [TBI] (non-TBI vs TBI), GVHD prophylaxis (methotrexate [MTX]-based vs. other than MTX-based), and time period of CBT (1998–2007 vs 2008–2013 vs 2014–2019). Missing data of HCT-CI and sex incompatibility were accounted for as separate categories. Cryopreserved cord blood TNC and CD34+ cell counts were divided according to an approximately median value. Results are expressed as the hazard ratio (HR) and 95% confidence interval (CI).To adjust for differences in baseline characteristics, all possible confounding variables were considered for the multivariate analysis. These variables included age (<55 vs ≥55 years), HCT-CI (0–2 vs ≥3 vs unknown), cytogenetic risk (other than adverse vs adverse), prior history of myelodysplastic syndrome (MDS)/myeloproliferative neoplasm (MPN) (yes vs no), disease status at CBT (early phase vs. advance phase), cryopreserved cord blood total nucleated cell (TNC) count  patients in CR1 and CR2 at CBT (early phase at CBT); and (2) patients in advanced disease status at CBT (advanced phase at CBT).+ cell dose, MAC regimens, use of non-TBI regimens, and GVHD prophylaxis without MTX. The data for PS, HCT-CI, and anti-HLA antibody were mostly unavailable during the former time periods (1998–2007). Among 1096 patients who had anti-HLA antibody, 56 (5%) patients had donor-specific anti-HLA antibody (DSA).A total of 5504 patients who received single-unit CBT for AML were included Table . Among tIn the subset analysis, 2289, and 3091 patients with early phase at CBT, and advanced phase at CBT were analyzed, respectively (Table P < 0.001 by log-rank test)  and 2014–2019  , HCT-CI ≥ 3 , adverse cytogenetic risk , advanced phase at CBT , cryopreserved cord blood CD34+ cell count ≥0.8 × 105/kg , female donor to male recipient , and unknown status of sex incompatibility  were also significantly associated with overall mortality . In the univariate analysis, the 2-year OS significantly improved over time  Fig. . In the 01) Fig. . In relaity Fig. .Fig. 1OvP < 0.001 for early phase at CBT; and P < 0.001 for advanced phase at CBT)  in two cohorts  but not with early phase at CBT   Fig. .Fig. 2OvP < 0.001 by Gray’s test)  but not 2008–2013  , history of MDS/MPN , advanced phase at CBT , HLA disparities ≥2 mismatch , female donor to male recipient , unknown status of sex incompatibility , RIC regimens , and GVHD prophylaxis without MTX  were also significantly associated with relapse-related mortality . The 2-year relapse-related mortality was 30.8% for 1998–2007, 32.5% for 2008–2013, and 27.5% for 2014–2019 (st) Fig. . In the 10) Fig. . In relaity Fig. .P = 0.029), but not those with early phase at CBT (P = 0.393)   Fig. .Fig. 3ReP < 0.001)  but not 2008–2013  , HCT-CI ≥ 3 , unknown status of HCT-CI , adverse cytogenetic risk , history of MDS/MPN , HLA disparities ≥2 mismatch , and GVHD prophylaxis without MTX  were also significantly associated with NRM  for the entire cohort. The 2-year NRM was 31.3% for 1998–2007, 26.9% for 2008–2013, and 25.2% for 2014–2019 (01) Fig. . In the 10) Fig. . In relaNRM Fig. .P < 0.001) and advanced phase at CBT (P = 0.010)   Fig. A, B. In 09) Fig. .Fig. 4NoFor the entire cohort, the cumulative incidences of neutrophil and platelet engraftment were 80.0%  at 42 days and 65.1%  at 100 days after CBT, respectively. Improvements of neutrophil and platelet engraftment were observed in the periods 2008–2013 and 2014–2019 compared with 1998–2007 in the univariate and multivariate analyses Fig. .P < 0.001 for early phase at CBT; P < 0.001; and for advanced phase at CBT)  in all three cohorts  and those with advanced phase at CBT   Fig. A, B. In T Fig. A,rts Fig. , and in 38) Fig. .Fig. 5NeP = 0.003), the univariate and multivariate analyses showed that acute and chronic GVHD was not significantly different across the time periods  and 11.4%  at 100 days after CBT, respectively Fig. A, S2B. Tods Fig. .P = 0.047) (data not shown).Among two distinct cohorts, the multivariate analysis showed that acute and chronic GVHD was not significantly different across the time periods, except that grade III–IV acute GVHD was significantly lower in the middle time period (2008–2013) compared with 1998–2007 only among patients with early phase at CBT  and the middle (2008–2013) time periods in our cohort. However, improvements in survival were observed in each patient cohort based on disease status. This suggests that an initial poor prognosis of CBT for adults is independent of advanced disease status at CBT. Furthermore, although the proportion of patients with adverse cytogenetics increased over time throughout all cohorts, relapse-related mortality was significantly improved in the recent time period in patients with advanced phase at CBT but was not improved in patients with early phase at CBT. Although a reduction in the risk of relapse could depend on both the intensity of conditioning regimen , 29 and Our previous study demonstrated a significant improvement of early NRM after CBT as the first allogeneic HCT for adults aged between 16 and 70 years with various hematological diseases over the past 20 years . Our cur+ cell dose in the cryopreserved cord blood unit [The most important limitation of CBT for adults is that the frequency of primary graft failure is higher after CBT compared with allogeneic HCT from adult donors . Althougood unit . This isood unit , 34. IndOur study had several limitations. First, data for the mutation profile of AML were insufficient in our registry data, which could strongly affect the outcomes after CBT for AML . Second,In summary, our registry-based study under real-world settings demonstrated that the survival and engraftment rate after CBT for adult patients with AML has improved over the past 22 years. The causes are likely to be multifactorial including the recent progress of cord blood unit selection, conditioning regimen, and improvements of supportive care. However, mortality after CBT still has room for further improvement. Therefore, our real-world experience can support the next major approaches to reduce mortality after CBT for adult patients with AML.Supplementary Figure 1Supplementary Figure 2Supplementary Figure 3


S. aureus (VISA) remain unclear. In this study, we systematically identified 3,260 Ksucc sites in 799 proteins and 7,935 Kac sites across 1,710 proteins in the VISA strain XN108. Functional analyses revealed that both Ksucc and Kac sites were highly enriched in several critical metabolic pathways, including ribosomal metabolism, tricarboxylic acid cycle, and glycolysis. Furthermore, a remarkable cross talk between Ksucc and Kac modifications was observed that almost 75% of the succinylated sites were also frequently acetylated. In addition, we identified SaCobB, a Sirtuin 2-like lysine deacetylase, as a bifunctional enzyme with both deacetylation and desuccinylation activities in S. aureus. We demonstrated the first lysine succinylome and acetylome in a VISA and identified SaCobB, a functional enzyme taking part in the regulation of Ksucc and Kac in S. aureus. Our findings provide valuable information for further study on the regulatory mechanisms of PTMs in S. aureus.Protein posttranslational modifications (PTMs) play important roles in regulating numerous biological functions of prokaryotic and eukaryotic organisms. Lysine succinylation (Ksucc) and acetylation (Kac) are two important PTMs that have been identified in various bacterial species. However, the biological functions of Ksucc and Kac in vancomycin-intermediate IMPORTANCE Lysine succinylation (Ksucc) and acetylation (Kac) are two important protein posttranslational modifications (PTMs) that regulate numerous biological functions in prokaryotes and eukaryotes. However, the functions of Ksucc and Kac in Staphylococcus aureus are seldom described. Understanding of Ksucc and Kac modifications in S. aureus will facilitate the development of new strategies to control infections. Herein, we quantified both Ksucc and Kac in a vancomycin-intermediate S. aureus (VISA) strain XN108, analyzed the interaction between these two PTMs, and identified SaCobB as a bifunctional enzyme with both deacetylation and desuccinylation activities. This study is the first description of dual PTMs, Ksucc and Kac profiles, in the VISA. The findings could provide valuable information for the following researches on the regulatory roles of PTMs in S. aureus. The classic central dogma states that DNA, as the main carrier of genetic information, is the major determinant of life science. Recently, increasing evidence has emphasized the importance of proteins, since they are the actual executors of most biological activities , 2. Gene12–Escherichia coli in 2011  is one of the most well studied PTMs and extensively exists in eukaryotes and prokaryotes . Acetyla in 2011 . Then, Kcapsulae , Vibrio olyticus , Porphyrdrophila , and Mycrculosis , have berculosis . As two rocesses . NeverthStaphylococcus aureus is an important human pathogen that distributes widely in hospitals and in communities  strain isolated in China .The total protein sample of andem MS . All of A total of 3,260 succinylation sites on 799 proteins were identified in XN108 wild-type strain (details listed in Data Set S1). Among the identified Ksucc proteins, about 90% were succinylated at 1 or 2 sites, the remaining proteins were succinylated at no less than 3 sites, including 12 proteins containing more than 20 Ksucc sites . To veriMycobacterium tuberculosis and Pseudomonas aeruginosa  . Among truginosa , 30, and seeding . The aceolyticus  and KacTolyticus . In addiThe Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of all identified succinylated proteins were performed to understand the roles of Ksucc in VISA XN108. Bubble plots were constructed to visualize the proteins containing succinylation sites . GO annoE. coli  in S. aureus XN108. Compared with SaCobB, AID40891.1 lacks only three amino acids at its N terminus. Therefore, we also designed AID40891.1 as SaCobB in this study. Next, we wondered whether SaCobB (AID40891.1) possesses only deacetylase activity or has both deacetylase and desuccinylase activities in S. aureus, like CobB in E. coli.In the process of protein acylation and deacylation, enzymes are needed to catalyze the transfer of acyl groups to different amino acid residues. A study has reported that the Sir2-like enzyme CobB shows comparable deacetylase and desuccinylase bifunction in E. coli , whereasectively . Specifielicolor . In 2019us HG001 . By homoin vivo, the XN108-ΔcobB mutant was constructed . Western blot was performed to detect the PTM levels of XN108-WT and XN108-ΔcobB. As shown in cobB strain were slightly enhanced compared with that of XN108-WT, indicating that SaCobB might function as both a desuccinylase and a deacetylase in vivo.To determine the desuccinylase and deacetylase activity of SaCobB cobB and XN108-WT were performed using antibody-based affinity enrichment, followed by liquid chromatography-mass spectrometry (LC-MS/MS) analysis  among three biological replicates of the XN108-WT and XN108-ΔcobB mutant displayed a high correlation , suggesting a reliable quantification analysis. In addition, a standard of localization probability >0.75 was used to filter the identification data. For succinylome analysis, as many as 2,936 succinylation sites on 741 proteins were quantified and normalized to the proteome data (Data Set S4). With a quantification ratio of >1.5  as upregulation threshold and <1/1.5  as downregulation threshold, 101 Ksucc sites across 85 succinylated proteins were found to be regulated by SaCobB. Among which, 98 sites on 83 proteins were hypersuccinylated, and only 3 sites on 2 proteins were hyposuccinylated after cobB gene deletion as exhibited by volcano plot , especially in purine nucleotide biosynthetic process and ribonucleoside-related biosynthetic process . Based on molecular function classification, SaCobB-affected proteins were mainly associated with peptidase activity . GO classification showed that SaCobB-regulated Kac-modified proteins were enriched in metabolic and cellular processes, including tRNA aminoacylation for protein translation, catalytic proteins, and cytoplasmic proteins . KEGG analysis indicated that the proteins regulated by SaCobB were mainly involved in central metabolism and biosynthetic processes  and were particularly involved in pathways associated with carbon metabolism, pyruvate metabolism, and ribosome and microbial metabolism in diverse environments. Interestingly, we found that proteins associated with peptidoglycan synthesis have a high tendency to undergo succinylation or acetylation modification . Furthermore, several proteins associated with oxidative stress and bacterial survival and a few transcriptional regulators regulated by SaCobB were observed . Overall, these results indicate that the proteins, of which the Ksucc and Kac modifications were regulated by SaCobB, are highly involved in central process of S. aureus metabolism and biosynthesis.GO annotation and KEGG analysis of SaCobB-regulated Ksucc and Kac proteins were performed to make clear the regulatory role of SaCobB in cobB compared with those in the XN108-WT were subjected to protein-protein interaction (PPI) analysis based on the STRING database. Interactions identified with high confidence (confidence score >0.7) were mapped with STRING and visualized with Cytoscape software. Four highly interconnected networks containing SaCobB-regulated succinylated or acetylated proteins were indicated  succinylation and β-lactam susceptibility by observing that Atl was the most succinylated protein in their study . All S. aureus strains were cultured in tryptic soy broth . E. coli strains DH5α were obtained commercially and cultured in Luria-Bertani (LB) medium . Unless otherwise indicated, all strains were cultured at 37°C, or at 30°C when carrying temperature-sensitive plasmids. The anti-succinyllysine and anti-acetylation antibodies were purchased from Jingjie PTM BioLab, Inc., , and the goat anti-mouse IgG-horseradish peroxidase (HRP) antibody was purchased from Abmart Inc. (China).The VISA strain XN108 was originally isolated from a burn patient of Southwest hospital and was used as backbone strain. The laboratory cobB mutant was constructed by homologous recombination method as previously described , resuspended with lysis buffer , and then lysed by ultrasonication. After that, the lysate was centrifuged at 12,000 × g at 4°C for 10 min to remove the remaining debris. Finally, the supernatants were collected, and the protein concentration was determined by bicinchoninic acid (BCA) kit .Three biological repeats of cobB protein samples were used for trypsin digestion. First, protein samples of different strains were adjusted to the same volume by adding lysis buffer. Then, one volume of cold acetone was added into protein samples, followed by mixing on vortex. The mixture was added with another four volumes of cold acetone and kept at −20°C for 2 h for protein precipitation. After centrifugation at 4,500 × g for 5 min, the supernatant was discarded, and the remaining precipitates were washed twice with cold acetone. After that, the air-dried precipitations were dissolved in 200 mM tetraethyl ammonium bromide (TEAB) for sonication and further added with trypsin for digestion overnight (the ratio of trypsin to protein was 1:50). The protein solution was reduced with 5 mM dithiothreitol (DTT) for 30 min at 56°C and alkylated with 11 mM iodoacetamide (IAA) for 15 min in darkness at room temperature.Equal amounts of XN108 and XN108-Δ2O). The resin-bound peptides were eluted three times by using 0.1% trifluoroacetic acid (TFA) and vacuum-dried. Finally, the obtained peptides were desalted with C18 ZipTips (Millipore) according to the manufacturer’s instructions and were analyzed by LC-MS/MS.The peptides were dissolved in IP buffer , and the supernatants were transferred to the prewashed succinylated resin  or acetylated resin  at 4°C overnight with gentle shaking. The resins were washed four times with IP buffer and twice with double-distilled water (ddHThe peptides were dissolved in liquid chromatography solvent A (0.1% formic acid and 2% acetonitrile) and a gradient of solvent B (0.1% formic acid and 100% acetonitrile) for separation by using NanoElute  with ReproSil-Pur Basic C18 column . The gradient of solvent B was set as following: increased from 6 to 22% for 0 to 44 min, from 22 to 35% for 44 to 56 min, 35 to 80% for 56 to 58 min, and at 80% for 58 to 60 min. All increments were performed at a constant flow rate of 450.00 nL/min on a NanoElute system. The peptides were separated by NanoElute and were ionized by injecting into the capillary ion source. Then, the peptide segments were analyzed by TIMS-TOF Pro mass spectrometry . The electrospray voltage was set at 2.0 kV. The parent ion of the peptide segments and their secondary fragments were detected and analyzed by using high resolution time-of-flight (TOF). The MS scan range was 100 to 1,700 for full scan, and the parallel cumulative serial fragmentation (PASEF) mode was used for data acquisition. Secondary MS scan with the charge of the parent ions in the range of 0 to 5 were collected in PASEF mode , followed by 10 MS/MS scans with 30-s dynamic exclusion.S. aureus XN108  database concatenated with a reverse decoy database. Trypsin/P was specified as cleavage enzyme allowing up to four missing cleavages, seven modifications per peptide, and five charges. The mass tolerance for precursor ions was set as 10 ppm in the first search and 20 ppm in the main search, and the mass tolerance for fragment ions was set as 0.02 Da. Carbamidomethylation on cysteine was set as a fixed modification. Acetylation modification on the protein N terminus, oxidation on methionine, and succinylation and acetylation on lysine were specified as variable modifications. Label-free quantification (LFQ) was used for a quantitative method. The false discovery rate (FDR) thresholds for protein, peptide, and modification sites were set to 1%.The resulting MS/MS data were processed using Maxquant 1.6.6.0. Tandem mass spectra were searched against the S. aureus strains of interest were cultured in TSB medium overnight. Bacterial cells were harvested by centrifugation and lysed by ultrasonication, and then the supernatant was collected after centrifugation. The remaining cell lysate was incubated and immunoprecipitated with rabbit anti-MurA polyclonal antibody (produced in our laboratory) and 50 μL of SPA-coated beads  at 4°C overnight with shaking. The serum of an unimmunized rabbit served as isotype control. In the next day, the immune complexes were washed with sterilized PBS five times, and the pellets were resuspended in 1× SDS sample buffer followed by boiling for 5 min. Then, the precipitated proteins were collected by centrifugation and a Western blot by using mouse anti-succinyllysine MAb to confirm the succinylation of MurA protein.The IP test was performed to pull down the MurA protein, which was detected to be succinylated in our MS studies. Briefly, the (i) Annotation methods. GO annotation proteome was derived from the UniProt-GOA database (http://www.ebi.ac.uk/GOA/) and was used to identify succinylated or acetylated proteins, first converting identified protein ID to the UniProt ID and then mapping to GO IDs by protein ID. If some identified proteins were not annotated by UniProt-GOA database, the InterProScan software would be used to annotate the protein’s GO function based on the protein sequence alignment method. Then, all identified proteins were classified by GO annotation into three categories: biological process, cellular component, and molecular function. The domain functional description of identified proteins was annotated by using InterProScan software based on a protein sequence alignment method, and the InterPro domain database (http://www.ebi.ac.uk/interpro/) was used. The KEGG database was used to annotate protein pathway. The KEGG online service tool KAAS was used to annotated protein’s KEGG database description, followed by mapping the annotation results on the KEGG pathway database using KEGG mapper. The subcellular localization prediction software WoLF PSORT was used, and the subcellular structures of prokaryotes were assigned using CELLO software. Motif-x and MoMo were used for analysis of the amino acids, which are overrepresented or underrepresented at the −10 to +10 position adjacent to a succinylation site (or an acetylation site).(ii) Functional enrichment. For each GO annotation category, a two-tailed Fisher’s exact test was employed to test the enrichment of the differentially expressed protein against all identified proteins. The GO with a corrected P value of <0.05 is considered significant. KEGG database was used to identify enriched pathways by a two-tailed Fisher’s exact test to test the enrichment of the differentially expressed protein against all identified proteins. The pathway with a corrected P value of <0.05 was considered significant.(iii) Enrichment-based clustering. All of the categories obtained after enrichment along with their P values were collated, and then those categories that were enriched in at least one of the clusters with P values <0.05 were filtered. This filtered P-value matrix was transformed by the function x = −log10 . Finally, these x values were z-transformed for each functional category. These z scores were then clustered by one-way hierarchical clustering (Euclidean distance and average linkage clustering) in the Genesis.(iv) Protein-protein interaction network. PPI networks were analyzed using the STRING database, version 11.0. Only interactions between the proteins belonging to the searched data set were selected, thereby excluding external candidates. STRING defines a metric called “confidence score” to define interaction confidence. All interactions that had a confidence score of ≥0.7 (high confidence) were fetched, and the interaction work from STRING was achieved and visualized with Cytoscape software.I) were centralized and transformed into relative quantitative values (U) of modified peptides in each sample. The formula is listed as follows: ijR = ijI/mean (jI) . Second, the ratio of the average value between the two samples is calculated. The ratio is used as the final quantitation. For normalization to succinylated (or acetylated) peptides, the naked intensities of succinylated peptides (or acetylated peptides) were first measured and then were divided by the corresponding protein intensities. If both proteomics and PTM profiling were conducted on the same cohort, the relative quantitative value of the modified peptide is divided by the relative quantitative value of corresponding protein to remove the influence from protein expression of modifications. Finally, the significance P value of the difference modification is calculated.The raw LC-MS data sets were first searched against database and converted into matrices containing reporter intensity of peptides across samples. The relative quantitative value of each modified peptide was then calculated based on intensity information by the following steps. First, the intensities of modified peptides , and then P value was calculated by the two-sample two-tailed t test method. Sites with XN108-ΔcobB/XN108-WT ratios of >1.5 and P values of <0.05 were considered upregulated succinylation (or acetylation). Sites with XN108-ΔcobB/XN108-WT ratios of <1/1.5 and P values of <0.05 were considered downregulated succinylation (or acetylation).The relative quantitative values of each sample were set as logEqual amounts of protein samples from different bacteria strains were separated by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were transferred to polyvinylidene fluoride (PVDF) membranes. Anti-succinyllysine mouse MAb (PTM Bio) or anti-acetyllysine mouse MAb (PTM Bio) at a dilution of 1:1,000 was used as the first antibody, respectively. Secondary antibody conjugated to horseradish peroxidase  was diluted by 1:5,000. The images were visualized with a chemiluminescent detection imager .t test was used to treat samples between two groups, and one-way analysis of variance (ANOVA) was used for testing multiple groups. Each experiment was carried out at least three times. The results are presented as mean ± standard deviations (SDs), and a P value of less than 0.05 was considered statistically significant .Statistical analysis of results was carried out using GraphPad Prism 5. Unpaired two-tailed Student’s PXD033341.The raw data and annotated MS spectra are available in the proteomics repository PRIDE with ProteomeXchange data set accession number of


Objectives Due to the constant battle regarding the controversial topic of orthodontic extraction, this study aims to assess the changes in vertical dimensions of patients treated with premolar extractions compared with nonextraction orthodontic patients.Materials and Methods A sample of 60 borderline patients were recruited and divided into extraction and nonextraction groups. Eleven pretreatment cephalometric measurements were recorded using WebCeph and patients were followed-up until the completion of treatment.Statistical Analysis Intragroup and intergroup comparisons were made using pairedt-test and two-sample independentt-test, respectively. The joint significance of differences was measured using F-tests.Results The intragroup comparison revealed that in the extraction group, the vertical dimension was significantly increased posttreatment for four cephalometric measurements, that is, mandibular plane angle (p < 0.05), palatal plane angle (p < 0.05), Frankfort mandibular plane angle (p < 0.05), and y-axis (p < 0.05). In the comparison of the posttreatment values of both groups, the mean differences of the posttreatment values for sella nasion (SN)-gonion (Go)-gnathion (Gn) angle (p = 0.008), the total anterior (p = 0.050), and lower anterior facial heights  were significantly higher. At the same time, the Jarabak ratio was significantly (p = 0.006) lower in the extraction group than in the nonextraction group.Conclusion The increase in vertical dimension is significantly higher in the extraction group than in the nonextraction group which indicates a significant impact of orthodontic extraction on the vertical dimensions. Orthodontists and general dental practitioners have spent decades battling orthodontic extractions. Although there are several strategies to manage orthodontic patients, there are two significant schools of thought regarding orthodontic management based on using extraction as a part of treatment plan:In contrast, the nonsupportive school of thought has a more conservative approach. Their evidence comes from numerous studies that consider premolar extractions a fundamental cause of temporomandibular joint (TMJ) disorders.Although multiple studies have been performed on this controversial topic of orthodontics extraction, no consensus has been established. Our study differs based on strict inclusion criteria. Additionally, to reduce observer error, we incorporated artificial intelligence (AI) software to compare pre- and postcephalometric radiographs. This study aims to assess the changes in the vertical dimension of patients treated with premolar extractions and compare them with the nonextraction group to assess the impact of orthodontic extractions on the vertical dimension of patients.This prospective cohort study was conducted at Armed Forces Institute of Dentistry (AFID), Pakistan, to evaluate the impact of orthodontic treatment with or without premolar extraction on the skeletal vertical dimension. The study was approved by the Institutional Review Board of AFID  and was conducted from April 2019 to August 2021.n = 22) and 63.3% females (n = 38). The mean age of our sample was 16.63 ± 2.7 years. The inclusion criteria for the study had both male and female patients having a complete set of permanent teeth except third molars. An A point, Nasion, B point (ANB) of −1 to 6 degrees, angle's class I or II malocclusion, crowding 5 to 9 mm, and overjet of 4 to 6 mm were also part of the inclusion criteria. Any patient with dentofacial deformity, transverse discrepancy, temporomandibular joint disorders (TMDs), or any previous history of orthodontic/orthognathic treatment was excluded from the study. Patients with impacted, missing, or decayed teeth were also excluded.The sample included 36.7% males .The cephalometric points, lines, and measurements used in this study to evaluate the vertical dimension of patients' pre- and postorthodontic treatment are shown inThe sample size calculations were done using PS software version 3.1.6, assuming that the difference in the response of matched pairs (pre- and posttreatment) is usually distributed with a standard deviation (SD) of 2.75 degrees. If the true difference in the mean response of matched pairs is 1.7 degrees, we will need to study 30 pairs of patients to be able to reject the null hypothesis setting the significance level at 0.05 with a probability (power) of 0.9.t-tests were used to assess the differences between the extraction and nonextraction groups' pre- and posttreatment cephalometric measurements based on the various parameters of vertical dimension.The normal distribution of the sample was assessed using QQ plots for various parameters of vertical dimension. The data were normally distributed, and, therefore, Student'st-test, these values were compared to see if the differences were statistically significant.For intragroup comparison, the mean and SD of pretreatment values and the posttreatment values of the 11 cephalometric parameters were recorded for each group (extraction and nonextraction). The difference between these pre- and posttreatment values was calculated and, then, using a pairedt-test. The joint significance of differences was measured using F-tests. All analyses were performed by the computer program SPSS, version 26.For the intergroup comparison, the pretreatment values were deducted from the posttreatment values for each of the 11 cephalometric variables to assess the alterations produced by orthodontic treatment. The changes in these cephalometric variables resulting from orthodontic treatment were compared between two groups (extraction vs. nonextraction) for statistical differences using a two-sample independentn = 60) had 30 patients in each extraction group and nonextraction group. The overall sample comprised of 36.7% males (n = 22) and 63.3% females (n = 38). The extraction group had a similar distribution with 33% males and 67% females, whereas the nonextraction group had slightly more males (40%). The overall mean age of participants was 16.63 ± 2.72 years, with the majority  within the age range of 15 to 18 years. About 82% of the sample belonged to a high socioeconomic status (SES).The final study sample (n = 39) of the sample belonged to skeletal class I, 30% to class II, and only 5% to class III. About 63% (n = 19) of the participants from the extraction group and 67% (n = 20) from the nonextraction groups had a skeletal class-I relationship. About 70% of patients (n = 42) had a dental class-I occlusal relationship, whereas 30% had class II. Of those from the extraction group, 73% (n = 22) had a dental class-I relationship, while approximately 67% (n = 20) from the nonextraction group had a dental class-I relationship , palatal plane angle , Frankfort mandibular-plane angle , and y-axis (p = 0.015). These values show a significant impact of orthodontic extraction on the vertical dimension of patients and reject the null hypothesis. The negative values for the mean differences (pretreatment − posttreatment) show a rise in the vertical dimensions postorthodontic treatment. However, in the nonextraction group, none of the 11 variables had a significant mean difference at ap-value of <0.05. The negative values of the mean difference indicated an increase in the vertical dimension but these differences were not statistically significant , the anterior (p = 0.050) and lower anterior facial heights , and the Jarabak ratio (p = 0.006). The mean difference between the two groups for these four variables was 4.31, 5.54, 6.42, and −3.42, respectively -gonion (Go)-gnathion (Gn) angle  until the completion of their treatment.p < 0.05. The variables that showed a significant increase in the extraction group included MPA (p = 0.012), PPA (p = 0.009), FMA (p = 0.011), and y-axis (p = 0.015). Six more variables , AFH, LAFH, Jarabak's ratio, gonial angle, and SN-GoGn angle) showed an increase in vertical dimension posttreatment but the results were not statistically significant. These results were in agreement with StaggersThe intragroup comparison results had negative mean difference values , indicating a rise in the vertical proportions after the completion of treatment. Out of the 11 cephalometric parameters evaluated for vertical change, 4 were significantly higher in the extraction group posttreatment, whereas none of them was significantly high in the nonextraction group atOn the other hand, Kirschneck et alp < 0.05. Some variables showed an increase in the vertical dimension , while others decreased . These results were similar to Beit et alIn the nonextraction group, the mean difference of cephalometric measures showed mixed results but was insignificant atp = 0.008), the anterior (p = 0.050), and LAFHs (p = 0.011), and Jarabak's ratio (p = 0.006). The mean difference in the posttreatment values of these four variables between the two groups were 4.31, 5.54, 6.42, and −3.42, respectively. Even though most of the studiesRegarding the intergroup comparison of posttreatment cephalometric values, the extraction group showed significantly high vertical dimensions for four variables compared with the nonextraction group . These p = 0.01, and JaThe study had several strengths, such as prospective study design, adequate sample size, and uniform distribution of the sample into two groups based on age, gender, SES, and treatment plan (extraction vs. nonextraction). We used a software that was based on AI to automatically locate landmarks and carry out analysis. This reduced observer error and was cost and time effective. AI-based cephalometric analysis is rapidly gaining popularity among orthodontists and is gaining precedence over manual tracing. It also enables accurate diagnosis and treatment planning.Our study found only 4 out of 11 variables significantly high in postorthodontic treatment involving extractions. However, no variable was significant in cases that were treated without extraction. Four variables were significantly different when comparing posttreatment values of extraction versus nonextraction groups. Therefore, based on our study's findings, it seems reasonable to conclude that the increase in vertical is due to a lack of attention given to orthodontic mechanics. If caution is taken to control the extrusion of molars after premolar extraction, there will most likely be no significant change in the vertical dimension of orthodontic patients. It is also recommended to be more cautious in patients with hyperdivergent facial profiles.


Staphylococcus aureus (S. aureus). We retrieved adults with S. aureus CAP or HCAP diagnosed by blood or pleural effusion culture in 2.6 years, and compared with those of Streptococcus pneumoniae (S. pneumoniae) CAP or HCAP diagnosed by blood or respiratory culture, or urine antigen. We found 18 patients with CAP and 9 HCAP due to S. aureus , and 48 patients with CAP and 15 HCAP due to S pneumoniae . Diabetes mellitus , hemodialysis , skin lesions , cavitary nodules  and pleural effusions  were more common in staphylococcal than pneumococcal group. Three patients with staphylococcal pneumonia had acute myocardial infarction. Pneumonia severity index  and 30-day mortality  were higher in staphylococcal group. Multivariate analysis showed underlying disease , risk class 4/5, altered mentality, shock and bilateral pneumonia were risk factors for 30-day mortality.This study aims at identifying characteristics, risk factors and mortality of community-acquired (CAP) and health-care-associated pneumonia (HCAP) by  Staphylococcus aureus (S. aureus) has been reported since 2005, following the influenza season of 2003–2004 and 2006–2007 in the United States (USA)3. Among the initially reported cases many occurred in young adults without underlying diseases but with preceding influenza, and most were caused by methicillin-resistant S. aureus (MRSA). S. aureus was later reported to be an important cause of health-care-associated pneumonia (HCAP)4.Severe communityacquired pneumonia (CAP) caused by S. aureus varied in studies from different regions. The average age of patients with staphylococcal CAP was 45, 49, and 60 years in studies from Brazil5, Canada4, USA6, respectively. The mortality of S. aureus CAP was 0%, 13% and 46%, in reports from Canada, USA, and Australia, respectively8. Preceding influenza was a risk factor for primary staphylococcal pneumonia in an earlier series2, but not in a prospective study published later that identified chronic hemodialysis as a risk factor for MRSA CAP6. Therefore, clinical characteristics and risk factors for staphylococcal CAP/HCAP may differ among regions. To help first-line physicians diagnose this potentially fatal pneumonia, we conducted this study to investigate characteristics and risk factors of S. aureus CAP and HCAP in New Taipei City  and compared them with those of CAP/HCAP caused by Streptococcus pneumoniae (S. pneumoniae).However, clinical features of CAP or HCAP caused by n = 18) or HCAP (n = 9). All had S. aureus isolated from blood  sputum, 2 (7%) nasopharynx and 7 (26%) non-respiratory sites including wound and urine. All the 27 patients (except for one who was found comatose on roadside and sent to our ER) had respiratory symptoms at presentation, suggesting primary staphylococcal pneumonia. The one with initial coma had lobar pneumonia in the right lower and patchy consolidation in right upper lobe, liver cirrhosis, prolonged prothrombin time and cerebral hemorrhage. Thus all 27 patients were diagnosed to have primary staphylococcal pneumonia. Image study found that chest radiographs and computed tomography  of the 27 patients all showed patchy or lobar consolidation with or without nodules, compatible with primary staphylococcal pneumonia with or without secondary septic emboli.During the period of 2 years and 7 months, we identified 27 adult patients with staphylococcal CAP (n = 48) or HCAP (n = 15) during the same period. Sixty one (97%) has blood culture performed at ER or within 48 h after admission. Only four (6%) had positive blood culture of S. pneumoniae (Table n = 2) or positive pneumococcal antigen alone (n = 57). All the 63 patients had respiratory symptoms at presentation, suggesting primary pneumococcal pneumonia. Chest radiographs and CT (performed in 16 or 25%) of the 63 patients all showed lobar or patchy consolidation with or without mottling shadows, compatible with primary pneumococcal pneumonia.We identified 63 adult patients with pneumococcal CAP (p = 0.435). MRSA accounted for 13 (48%) of staphylococcal pneumonia. Underlying diseases were present in 26 (96%) and 21 (67%) patients with staphylococcal and pneumococcal pneumonia, respectively (p = 0.007). Diabetes mellitus (DM) and chronic hemodialysis were more common in the staphylococcal than pneumococcal group . Among patients with staphylococcal CAP, two abused intravenous drugs.Table p < 0.001). All those with skin lesions were diabetic.The two groups had similar clinical manifestations. The presenting oxygen saturation was lower in the staphylococcal group. The most obvious differences in physical signs were skin lesions, including cellulitis, scabies, wound infection, herpes zoster, pressure sores, carbuncles or intravenous devices, at presentation in 11 of the staphylococcal, but none of the pneumococcal group  and multiple cavitary nodules . Patients with staphylococcal pneumonia had significantly higher 30-day mortality than those with pneumococcal pneumonia . Patients with methicillin-susceptible S. aureus (MSSA) and those with MRSA pneumonia had similar PSI  and 30-day mortality . Among 30-day survivors, the average admission duration was 24.2 and 12.8 days for those with staphylococcal and pneumococcal pneumonia, respectively (p = 0.001).Average PSI was significantly higher in the staphylococcal group. Totally 85% and 63% of the staphylococcal and pneumococcal group, respectively, presented with a PSI risk class of ≥ 4 (Table p = 0.567). By contrast, empirical antimicrobials used for patients with pneumococcal pneumonia were cefoperazone sulbactam, fluoroquinolone, piperacillin tazobactam and amoxicillin clavulanate, without vancomycin, teicoplanin, or any combinations including them. Seven (11%) did not receive antimicrobials at ED, but all were discharged uneventfully after 5–18 days.Empirical antimicrobials used at ED for patients with staphylococcal pneumonia were mostly β-lactam-β-lactamase inhibitor (BLBLI) combinations, including piperacillin tazobactam and cefoperazone sulbactam, second-generation cephalosporin, and combination of antimicrobials that often included vancomycin or teicoplanin (Table S. aureus or S. pneumoniae) using multivariate regression analysis. First we discovered that all patients without co-morbidity (n = 22) survived. Therefore, THE presence of comorbidity is the most crucial prognostic factor for mortality , skin lesions, and effective antimicrobial use within 3 days. We identified five additional risk factors for 30-day mortality: cancer, cirrhosis, altered mental status, shock, and bilateral lung lesions  in our study was similar to that reported in a prospective study from USA (8.1%)6, indicating that staphylococcal pneumonia can occur with or without preceding influenza.Our study found that staphylococcal CAP and HCAP occurred throughout the year in our community, with a prevalence lower than pneumococcal pneumonia. Influenza virus has been identified as a potential predisposing factor for staphylococcal CAP3. The age range of our staphylococcal patients also differed from that of the pneumococcal group, in which 10% were aged < 40 years and healthy. The absence of patients with no underlying disease in the staphylococcal group suggested that certain condition predisposed hosts to S. aureus pneumonia. DM, chronic hemodialysis and skin lesions were more common in the staphylococcal group, similar to previous studies from Brazil, USA and Australia7. Patients with these underlying diseases had higher risk of staphylococcal pneumonia because: (1) diabetic or chronic hemodialysis patients are prone to S. aureus colonization in nose and skin, which increases the risk of S. aureus infection10. (2) Skin sores, scabies and intravascular lines  were risk factors for invasive staphylococcal infection7. Three of the patients with staphylococcal pneumonia and DM  had scabies at presentation. Scabies is associated with overcrowding and poor hygiene11. In Taiwan it is associated with bed-ridden status, nursing home residence and catheters12. Because scabies can be treated with scabicides and by cleaning bedding and clothing, early diagnosis of scabies may reduce the risk of staphylococcal pneumonia.We reviewed only adults in this study. Our patients with staphylococcal pneumonia were middle-aged or old, mostly with underlying diseases, in contrast to a previous study in which half of the patients with staphylococcal CAP were < 18 year-old and healthy13. The association of rapidly progressive cavitating pneumonia with a preceding influenza due to Panton–Valentine leukocidin (PVL) produced by MRSA or MSSA has been reported14. We did not perform PVL assays and could not verify this association and our patients were elderly with negative influenza antigen or PCR. Nevertheless, the mortality was equally high in our study.Bilateral cavitary nodules (37%) and pleural effusions (48%) were more common among our patients with staphylococcal pneumonia, similar to previous studiesStaphylococcus aureus septicemia has been known to induce platelet and coagulation activation, fibrinolysis, thrombosis and DIC15, but AMI has not been previously reported to be associated with staphylococcal pneumonia. Whether AMI is related to DIC with thromboembolism in the coronary system remains unclear, given that all the three patients had risk factors for coronary artery disease, including smoking, DM, and CKD.Three of fatal patients with staphylococcal pneumonia had AMI. Laboratory data suggested DIC in two. 6. The mortality was even higher (57%) among those without effective antimicrobials in 48 h. For the pneumococcal group, the 30-day mortality was 9.5%, similar to data from a USA (12.5%)16 and an Australian study (11.1%)7, and the seven patients in pneumococcal group without antimicrobial treatment at ED survived. The observation suggests higher virulence of S. aureus than S. pneumoniae, and poorer host immunity of patients who contracted staphylococcal pneumonia. Another factor that may contribute to the lower PSI score and mortality in the pneumococcal group was the percentage of bacteremia, which was 100% (27/27) for the staphylococcal and 6% for the pneumococcal group. Fifty-nine (94%) of the 63 patients in the pneumococcal group was diagnosed by positive urinary antigen; among them 57 had negative blood culture. The low rate of positive culture in patients with pneumococcal pneumonia may be due to the tendency of S. pneumoniae to autolyze when reaching the stationary phase of growth and antibiotic treatment prior to specimen collection17. Nevertheless, previous studies revealed that patients with pneumococcal pneumonia and positive pneumococcal bacteremia had higher18 or similar19 30-day mortality than those without bacteremia. In our study patients with pneumococcal pneumonia and bacteremia had 30-day mortality rate (0/4) similar to non-bacteremic patients , and on multivariate analysis bacteremia was not a significant risk factor. We need to perform a prospective, larger study to see if bacteremia contributes to mortality.The average PSI score and 30-day mortality of patients in the staphylococcal group were higher than the pneumococcal group, similar to previous reportS. aureus20. In a recent study of S. aureus CAP (age 29–67 years), MRSA and PVL were risk factors of mortality21. For pneumococcal pneumonia, age, smoking, alcohol abuse, solid tumor, liver and renal diseases were predictors of mortality22.Multivariate analysis showed risk factors for 30-day mortality were underlying diseases, risk class 4/5, cancer, cirrhosis, presenting with altered mental status, shock and bilateral pneumonia. A previous study found hemoptysis, erythroderma and leukopenia were associated with mortality in young patients (median age 14.5 years) with CAP due to PVL-producing Our study has limitations: (1) Because of the retrospective nature, not all patients underwent testing for coexisting pathogens. Whether influenza was associated with staphylococcal or pneumococcal pneumonia was unclear. (2) We did not examine PVL that may help explain the virulence and complications associated with staphylococcal pneumonia. (3) Case number was small.In conclusion, Staphylococcal CAP/HCAP in our community mainly affected elderly patients with DM or CKD. Many had skin lesions or intravenous catheters. AMI was a lethal complication. 30-day mortality in the staphylococcal group (41%) was higher than the pneumococcal group (9.5%), especially among those without effective antimicrobials in 48 h (57%). Risk factors for 30-day mortality for all patients with staphylococcal or pneumococcal pneumonia were underlying disease, risk class 4/5, cancer, cirrhosis, altered mentality, shock, and bilateral pneumonia.S. aureus as the causative organism of pneumonia if it was isolated from blood or pleural fluid23. From the electronic database of our microbiology laboratory, we retrieved adult patients (> 17 years-old) with S. aureus isolated from blood or pleural fluid between October 2017 and April 2020, and reviewed their electronic records. We then identified all cases that fulfilled criteria for CAP or HCAP. Patients who presented to our emergency (ED) or outpatient department with at least 2 of the following criteria were defined as having CAP: fever, chest pain, cough, shortness of breath, in addition to having radiographic pneumonia, and without any criteria for HCAP24. Patients were determined to have HCAP if they had been hospitalized within 90 days; lived in nursing home; or had received intravenous antibiotics, chemotherapy, wound care, or hemodialysis in health-care facility within 30 days25. We calculated their pneumonia severity index (PSI) score and risk of death within 30 days26.This study was conducted in Fu Jen Catholic University hospital, a primary-care hospital opened in October 2017 in New Taipei city with 460 beds currently. The study was approved by the ethics committee of Fu Jen Catholic University Hospital (FJUH108004), and the requirement of the informed consent was waived for the study. We defined S pneumoniae as the causative organism of pneumonia if it was isolated from blood or pleural fluid, or if pneumococcal antigen was detected in urine23. We compared their clinical data and images with those of patients with staphylococcal CAP or HCAP.For comparison, we also retrieved adult patients with diagnosis of pneumococcal CAP or HCAP during the same period. We defined S. pneumoniae CAP. Blood culture was performed using Virtuo BacT/ALERT system . Staining and culturing of samples for common bacteria/fungi or Mycobacterium tuberculosis, urinary antigen tests for S. pneumoniae and Legionella pneumophila , nasopharyngeal swab for influenza antigen, influenza polymerase chain reaction (PCR)  or SARS-CoV-2 real-time PCR  were performed if deemed necessary by the attending physician. Bacterial identification and antimicrobial susceptibility were performed using VITEK 2 system .All patients had blood culture sampled at ED or within 48 h of admission, except for two patients with t-test. Percentages of cases with certain characteristics were compared using chi-square test. Associations between 30-day mortality and various factors were analyzed using chi-square test and multivariate logistic regression model. A two-sided p < 0.05 was considered significant. All analyses were performed using SAS .Quantitative data were expressed as mean ± standard deviation, and differences between groups were analyzed by We conducted the trial in accordance with good clinical practice guidelines and the Declaration of Helsinki. The study was approved by the Institutional Review Board of the Fu Jen Catholic University Hospital, New Taipei City, Taiwan (FJUH108004). The informed consent was waived because of the retrospective nature of this study.Supplementary Information.