To screen the differentially expressed miRNAs (DEMs) and the differentially expressed gene mRNAs (DEGs) in lung adenocarcinoma (LUAD) from the TCGA database and to explore the relationship between miRNAs and the prognosis of lung adenocarcinoma and their biological functions.  The RNA-seq and miRNA-seq data of lung adenocarcinoma samples were downloaded from the TCGA database for analysis, and the R program was used to screen for differentially expressed miRNAs and mRNAs. Then, the molecular functions, biological processes, cellular components, and signaling pathways involved in the occurrence and development of LUAD were analyzed using the functional accumulation analysis software of GSEA. The relationship between the integrated differentially expressed RNAs was analyzed by miRcode, TargetScan, and miRTarbase databases, and the miRNA-mRNA network was constructed.  A total of 516 differentially expressed miRNAs and 5464 differentially expressed mRNAs were identified in LUAD. The GSEA enrichment analysis showed that miRNAs and mRNAs were mainly enriched in extracellular structure organization, external encapsulating structure organization, extracellular matrix organization, and gated channel activity. They were mainly involved in neuroactive ligand-receptor interaction signaling pathway. Some miRNAs and mRNAs in clustering modules were found to be associated with the prognosis of LUAD. Four targeting networks consisting of 22 miRNAs and 531 mRNAs were constructed.  The miRNA and mRNA related to the prognosis of LUAD were screened out, which provided a valuable preliminary basis for the follow-upin-depth clinical research and basic experimental research of LUAD. LUAD is one of the most common malignancies with high morbidity and mortality worldwide . Lung caThe oncogenic process of LUAD is a multistage and multigene process, involving the activation of oncogenes and inactivation of oncogenes . Its treR package for weighted correlation network analysis (WGCNA) is a systems biology approach for describing correlation patterns between genes by microarray samples [GO is a database created by the Gene Ontology Consortium to establish a semantic vocabulary standard for qualifying and describing gene and protein functions that is applicable to a wide range of species  and can  samples .Currently available LUAD markers do not have sufficient predictive power. Most of the current methods are invasive. Therefore, we reanalyzed the TCGA dataset to find new biomarkers that are closely related to the patient TMN stage, survival rate, and other prognosis.In this study, we downloaded raw expression data from LUAD RNA high-throughput sequencing (RNA-HTSeq) from TCGA and analyzed DEGs and DEMs between LUAD patients and normal lung gland tissues. The WGCNA approach based on DEG identified important gene coexpression modules and genes associated with TNM staging. GO and KEGG enrichment analyses were performed to analyze the functional genes and cellular signaling pathways involved. In addition, modular gene networks were constructed, and hub genes associated with TNM staging were identified by Cytoscape software. The relationship between hub genes and survival was analyzed. Four target networks consisting of 22 miRNAs and 531 mRNAs were constructed, which may be related to the development and clinical features of LUAD, laying a theoretical foundation for further research and discovery of new target biomarkers for LUAD.The workflow chart of all experimental contents in this study is shown in https://cancergenome.nih.gov/). After the removal of duplicate and no clinically informative samples, miRNA data sample profiles are as follows: 470 cancer samples and 44 normal samples, while mRNA data sample profiles are as follows: 477 cancer samples and 53 normal samples. RNA-seq data were generated by Illumina HiSeq platform. Read counts were used to indicate gene expression levels [RNA-HTSeq and clinical information data of LUAD patients were downloaded from The Cancer Genome Atlas . At the same time, we calculated the fold of differential expression according to the FPKM value, i.e., fold-change. The screening indicators for this analysis were p-value <0.05, log2FC > 1 or <-1.The  samples . After tGSEA version 2.2.2 software was used for functional enrichment analysis. The miRNAs were divided into high and low expression groups according to their target molecule mRNA expression, and each expression group was enriched by the GSEA software for the Kyoto encyclopedia of genes and genomes (KEGG) signaling pathway analysis. The gene enrichment analysis was performed by default weighted enrichment statistics, and the number of random combinations was set to 1000. Gene sets with false discovery rate (FDR) < 0.25 were considered significantly enriched gene sets in GSEA.N, so that the connection between the genes in the network obeys the scale-free network distribution. This algorithm has more biological significance. The second step is to construct a hierarchical clustering tree through the correlation coefficient between genes. Different branches of the clustering tree represent different gene modules, and different colors represent different modules. Based on the weighted correlation coefficients of genes, the genes are classified according to their expression patterns, and genes with similar patterns are grouped into a module. In this way, genes can be divided into dozens of modules by gene expression patterns, which is a process of extracting and summarizing information.The weighted gene coexpression network analysis is a systems biology method used to describe gene association patterns between different samples, which can be used to identify gene sets with high covariation, and it is based on the interconnectivity of gene sets and the relationship between gene sets and phenotypes. The association identifies candidate biomarker genes or therapeutic targets. WGCNA can be used to identify the cluster modules of highly correlated genes for summarizing such clusters using module signature genes (ME) or hub genes within the modules, correlating modules to each other and to external sample features , and using to calculate module membership metrics. WGCNA is divided into two parts: expression cluster analysis and phenotype association, which mainly include the following four steps: the calculation of correlation coefficient between genes, determination of gene modules, coexpression network, and association between modules and traits. The first step is to calculate the correlation coefficient (Person Coefficient) between any two genes. To measure whether two genes have similar expression patterns, it is generally necessary to set a threshold for screening, and those above the threshold are considered to be similar. In WGCNA analysis, the weighted value of the correlation coefficient is used, i.e., the gene correlation coefficient is taken to the power of nd–7th nt) located at the 5′ end of the miRNA can form Watson-Crick pairing with the 3′ UTR of the target gene. It is the most important factor in the prediction of all miRNA target genes. MiRNA inhibits target gene expression at the post-transcriptional level through partial complementary pairing with the mRNA of the target gene. Studies have shown that miRNAs are involved in various biological processes, including cell proliferation, apoptosis, differentiation metabolism, development, and tumor metastasis, among other biological processes.The so-called seed sequence . MElightcyan was positively correlated with M phase with LUAD . MEsalmon was positively correlated with the total stage of LUAD . As shown in T phase and the total Stage of LUAD . MEred was negatively correlated with the stage with LUAD . MEpink was positively correlated with the total stage of LUAD . MEturquoise was positively correlated with the total stage of LUAD .Since ME can recapitulate gene expression profiles, the correlation between ME and TNM stage was calculated, called module-trait relationship analysis. To identify the module-trait relationships of gene modules, we assigned genes into corresponding modules with reference to the initially constructed modules. The correlation of each module with clinical parameters was calculated using a function of the WGCNA module eigengenes . In addiThe target mRNAs of miRNAs were searched from miRTarBase, miRDB, and TargetSsan databases to construct a targeting network consisting of differentially upregulated and downregulated miRNA-mRNAs, and the analysis was visualized using Cytoscape software.One miRNA in M-phase targets 159 mRNAs , of whicFour targeting networks consisting of 22 miRNAs and 531 mRNAs were constructed.To identify key genes, the DEGs of several modules with significant module-trait relationships were analyzed. Using Cytoscape's cytohubba plugin, key genes were screened out by the degree algorithm. Three key genes were obtained in the MEgrey60 module, which are as follows: CDCA8, KIF23, and CDT1 . The keyp > 0.05, p < 0.05, The key genes of LUAD's overall survival analysis samples were divided into high expression group and low expression group. Compared with the median value of key genes, the expression survival analysis showed that ITGB3, DAPK1, CDH13, and CDH1 had no significant difference in the OS rate with patients (With the popularity of high-resolution CT scans and the improvement of patients' health awareness, the detection rate of pulmonary nodules continues to increase. However, distinguishing benign from malignant pulmonary nodules and identifying different subtypes of NSCLC remain major challenges for clinicians. Some miRNA-mRNA regulatory changes occur at different stages of carcinogenesis. In the early diagnosis of cancer, the detection of new miRNA changes may serve as a new prognostic tumor marker method for LUAD. With advances in high-throughput sequencing technologies, the amount of transcriptome data in public databases has grown exponentially, providing ample data for screening ideal diagnostic biomarkers and evaluating their performance. In the present study, we systematically analyzed the transcriptome data of LUAD in the TCGA and GEO databases. Twenty-two miRNAs were identified and validated as possible LUAD-specific prognostic diagnostic biomarkers. Compared with previous studies, the data patient sample size in our study reached more than 1000. Therefore, our advantage lies in a wider range of candidate miRNA-mRNAs. Therefore, it is of great significance for the prognosis and treatment of LUAD to elucidate the occurrence and development mechanism of LUAD and find biomarkers that can be used for early clinical diagnosis and prognosis.N-phase of LUAD. MElightcyan is positively correlated with the M phase of LUAD. MEsalmon is positively correlated with the total stage of LUAD. Among the differentially expressed mRNAs, MEbrown was negatively correlated with the T phase of LUAD. Mered and MEbrown were negatively correlated with the stage phase of LUAD. MEpink and MEturquoise were positively correlated with the total stage of LUAD. It is suggested that these modular signature genes play an important role in the occurrence and development of LUAD.This study employed a systems biology approach, WGCNA, to identify gene modules associated with DEG and TNM staging of LUAD. A total of 1575 DEMs and module-trait relationships were identified. As shown in In addition, GO and KEGG pathway enrichment analysis was performed on the module eigengenes. GO analysis showed that the differentially expressed miRNAs in lung adenocarcinoma were mainly enriched in biological processes and molecular functions. Biological process BP is mainly enriched in extracellular structural tissue, external encapsulated structural tissue, and extracellular matrix tissue. Molecular function MF is mainly enriched in channel activity, passive transmembrane transporter activity, and gated channel activity. The cellular components CC were mainly enriched in the following: transmembrane transporter complexes and extracellular matrix containing collagen. In the KEGG pathway analysis, we found that the signaling pathway was mainly enriched in neuroactive ligand-receptor interactions.Gprotein-coupledreceptor-mediated signaling pathway, has been implicated in the progression of cancers, such as bladder and pancreatic cancer [Previous studies have shown that the biological processes of lung adenocarcinoma and glioma are mainly enriched in extracellular structural tissue, external encapsulated structural tissue, and extracellular matrix tissue , 23. Molc cancer –28. It rc cancer . In our After identifying the GO and KEGG enrichment analysis of LUAD-related module eigengenes, a gene network was constructed based on WGCNA to analyze the correlation between module eigengenes. Meanwhile, core miRNAs were identified using the Cytohubb package, and they are as follows: hsa-miR-6860, hsa-miR-2682-3p, hsa-miR-2682-5p, hsa-miR-503-5p, hsa-miR-503-3p, hubhsa-miR-216b-3p, hsa-miR-216b-5p, hsa-miR-939-5p, hsa-miR-1247-5p, hsa-miR-939-3p, hsa-miR-6726-5p, hsa-miR-6510-5p, hsa-miR- 326, hsa-miR-3615, hsa-miR-6892-3p, hsa-miR-3677-3p, hsa-miR-1247-3p, hsa-miR-99b-3p, hsa-miR-3677-5p, hsa-miR-4664-5p, hsa-miR-4736, and hsa-miR-6892-5p. They may be potential biomarkers to predict the prognosis of LUAD patients. In addition, potential target gene transcriptions were validated in databases, in which some target gene mRNAs were significantly downregulated/upregulated in lung adenocarcinoma tissues. These findings may provide the fundamental evidence for the future identification of potential biomarkers or anticancer targets.in vitro by promoting SALL1 expression [β [Gene modules of these central genes were identified as primarily associated with the clinical metastasis of lung adenocarcinoma. Previous literature suggested that the MIR2682 locus is associated with perineural invasion in head and neck cancer . MiR-268pression . Hsa_cirpression . LncRNADpression . MiRNA-2pression . The ovepression . MicroRNpression . The knopression . MiR-124pression . Tumor-dpression . MiR-361pression . MiR-367ssion [β . Hypoxiassion [β . MiR-473ssion [β . These mIt is worth mentioning that the key roles of these 22 hub miRNAs in LUAD are only predicted based on the WGCNA theory, and if verified, our findings may provide evidence for the study of anticancer targets in LUAD patients.In conclusion, we identified gene modules and hub genes associated with the TNM rank using the WGCNA approach. A total of 516 differentially expressed miRNAs and 5464 differentially expressed mRNAs were identified in LUAD. GSEA enrichment analysis showed that miRNAs and mRNAs were mainly enriched in extracellular structure organization, external encapsulating structure organization, extracellular matrix organization, and gated channel activity, and it is mainly involved in the neuroactive ligand-receptor interaction signaling pathway. It was found that miRNAs and mRNAs in some clustering modules were associated with the prognosis of LUAD, and 4 targeting networks consisting of 22 miRNAs and 531 mRNAs were constructed. Mechanistically, these molecular genes may contribute to the development of changes in lung adenocarcinoma by regulating the pathways.


Microelectrode recording (MER) guided subthalamic nucleus deep brain stimulation (STN-DBS) under local anesthesia (LA) is widely applied in the management of advanced Parkinson's disease (PD). Whereas, awake DBS under LA is painful and burdensome for PD patients. We analyzed the influence of general anesthesia (GA) on intraoperative MER, to assess the feasibility and effectiveness of GA in MER guided STN-DBS.Retrospective analysis was performed on the PD patients, who underwent bilateral MER guided STN-DBS in Wuhan Union Hospital from July 2019 to December 2021. The patients were assigned to LA or GA group according to the anesthetic methods implemented. Multidimensional parameters, including MER signals, electrode implantation accuracy, clinical outcome and adverse events, were analyzed.A total of 40 PD patients were enrolled in this study, including 18 in LA group and 22 in GA group. There were no statistically significant differences in patient demographics and baseline characteristics between two groups. Although, the parameters of MER signal, including frequency, inter-spike interval (ISI) and amplitude, were obviously interfered under GA, the waveforms of MER signals were recognizable and shared similar characteristics with LA group. Both LA and GA could achieve effective electrode implantation accuracy and clinical outcome. They also shared similar adverse events postoperatively.GA is viable and comparable to LA in MER guided STN-DBS for PD, regarding electrode implantation accuracy, clinical outcome and adverse events. Notably, GA is more friendly and acceptable to the patients who are incapable of enduring intraoperative MER under LA. Parkinson's disease (PD) is one of the most common neurodegenerative diseases, characterized by resting tremor, rigidity, bradykinesia, postural instability and gait disturbance . BilaterGenerally, the procedure of electrode implantation is implemented under local anesthesia (LA), allowing for intraoperative microelectrode recording (MER) and test stimulation during DBS surgery , 3. IndeOwning to these concerns of awake DBS under LA, it has been a growing trend to perform STN-DBS under general anesthesia (GA) , 9, 10. PD patients who were referred to our center for bilateral MER guided STN-DBS from July 2019 to December 2021, were included in this study. Exclusion criteria were: (a) previous PD related surgery; (b) medication history, which might have influences on MER signals; (c) MER data lost, incomplete, or with quality problems; (d) lost to follow-up within 6 months. The consort flow diagram was presented as The anesthetic method was determined mainly according to the patients' preferences. Whereas, patients who were incapable of enduring awake DBS, including those with extreme anxiety, reduced cooperation, severe convulsions and difficult breathing, were operated under GA. We also showed reasons for the patients who were operated under GA in 1, Y1, and Z1). The X1, Y1 and Z1 coordinates were defined as the distance from the target site to the midpoint of anterior commissure-posterior commissure (AC-PC) line in medial-lateral (X-), anterior-posterior (Y-) and superior-inferior (Z-) axes respectively. The planned trajectories were kept away from intracranial vessels according to MRI images.All patients are required to take a preoperative 3-T MRI scan and a stereotactic frame-based computed tomography (CT) scan. Then, the CT image was co-registered with MRI image to determine the target site, the trajectory angles  and the target coordinates . Postoperative CT scan was applied to verify the electrode placement and exclude hemorrhage in 24 h.MER signals in the target where DBS electrode was implanted in were selected for analysis. MER signals were band-pass filtered (Butterworth filter) at 300–6000 Hz and sampled at a rate of 24 kHz. Only MER signals that had stable activities significantly over the baseline amplitude of background noise were selected and a threshold was applied to detect the spikes. The spike sorting for a single unit was performed by NeuroExplorer (version 5.311). The MER parameters, including spike frequency, inter-spike interval (ISI), minimum and maximum value of waveform, were recorded for further analysis. The borders of STN were identified according to the changes in firing patterns and the length of STN was recorded for further analysis.2, Y2, and Z2) were defined as the distance from the center of implanted electrode to the midpoint of AC-PC line in X-, Y-, and Z-axes. Vector errors  and Euclidean distance  were employed to assess the distance between the electrode implantation site to the intended target site, yielding the electrode implantation accuracy. The length of STN was also applied to verify the suitable target. Postoperative clinical assessment was performed in 6 months. UPDRS  was applied to assess the motor and non-motor symptoms under on-DBS/off-medication condition. Postoperative LEDD was recorded and compared with the baseline. Reduction in LEDD was regarded as a parameter demonstrating the efficacy of STN-DBS. Within 24 h postoperatively, the patients' experiences were assessed using Visual Analog Scale (VAS) and Kolcaba General Comfort Questionnaire (GCQ). The adverse events were also recorded for further analysis.The outcome analysis included electrode implantation accuracy evaluation and postoperative clinical assessment. Postoperative CT image was co-registered with preoperative MRI image to evaluate the electrode implantation accuracy. The coordinates of electrode implantation site . Categorical variables were evaluated by chi-square test and presented as frequency with percentage (%). P-value < 0.05 was considered as statistically significant difference.Statistical analysis was conducted with the use of SPSS software, version 26.0 (IBM Corp). Continuous variables with normal distribution were evaluated by Student's P > 0.05). These results were presented in A total of 40 PD patients who experienced bilateral MER guided STN-DBS in our center, were enrolled in this study. Among these cases, 18 patients who experienced STN-DBS under LA, were assigned to LA group and 22 patients who experienced STN-DBS under GA, were assigned to GA group. The LA and GA groups have similar demographics and baseline characteristics of patients, including sex, age at onset of PD, age at surgery, duration of PD, UPDRS-III (off-medication), H&Y stage (off-medication), MMSE (off-medication) and LEDD (P < 0.05) and an increased ISI . Meanwhile, the amplitudes of STN MER signals in GA group were reduced, with the minimum of waveform increased  and the maximum of waveform decreased . Whereas, the length of STN showed no statistically significant difference between LA and GA groups   .1-X2|, ΔY=|Y1-Y2|, ΔZ=|Z1-Z2|, mm) and Euclidean distance  were employed to assess the distance between the electrode implantation site to the intended target site. As shown in P > 0.05). Therefore, similar electrode implantation accuracy was achieved in LA and GA groups.Vector errors (ΔX=|XP > 0.05). Furthermore, patients in GA group achieved lower VAS scores  and higher GCQ scores  than those in LA group, indicating that patients experienced less pain and more comfort in GA group. Unexpectedly, the duration of surgery was similar between LA and GA groups   was applied to assess the motor and non-motor symptoms under on-DBS/off-medication condition. Actually, significant improvement of motor and non-motor symptoms with UPDRS  scores declined were observed in both LA and GA groups. However, changes in UPDRS  scores at 6 months from baseline were similar between LA and GA groups. Likewise, there were no statistically significant differences in the separate subscores of tremor, rigidity, bradykinesia, postural instability and gait disturbance (PIGD) between LA and GA groups. Postoperative LEDD was also significantly reduced at 6 months and the reduction of LEDD were similar between LA and GA groups  .There were no hardware-related postoperative adverse events occurred in this study. However, both LA and GA groups had one patient experienced non-symptomatic intracerebral hemorrhage. Two patients in GA group were diagnosed as postoperative pulmonary infection. No surgical site infection happened in both groups. In addition, several stimulation-related complications occurred in this study. For example, each group had one patient suffering from mild depression, one patient in LA group and two patients in GA group endured insomnia, and one patient in GA group complained of knee pain postoperatively.Since DBS surgery was introduced by Benabid and his colleagues in 1987, this technique had experienced continuous improvement . CurrentThe effect of general anesthesia on MER is controversial but likely depends on the type and dose of a particular anesthetic agent, underlying disease, and surgical target , 16–18. In this study, propofol and remifentanil were applied to implement GA and BIS value was maintained at 40–60 during MER. Under the well-defined circumstance, the bursting firing pattern of STN was typical , 16. AltIn addition to the properly selected candidates, precise implantation of the electrodes in STN is the most important factor in achieving expected clinical outcomes . HoweverIn both LA and GA groups, PD patients were benefit from STN-DBS. According to the assessment of UPDRS , the improvement of motor and non-motor symptoms after bilateral STN-DBS under GA was comparable with the improvement achieved under LA. Likewise, the separate subscores of tremor, rigidity, bradykinesia and PIGD achieved similar reduction in LA and GA groups. These findings matched the results of previous studies, which compared the clinical outcome of STN-DBS under different anesthetic methods –32. The The postoperative adverse events were categorized as hardware-related, surgery-related, and stimulation-related complications. No hardware-related postoperative adverse events occurred in this study. However, the incidence of intracerebral hemorrhage was 5%, which was consistent with the overall risk of intracerebral hemorrhage reported before , 34. It There are several limitations in this present study. Firstly, the surgery was performed under the guideline of frame-based MER. The applications of these results are limited in MRI guided STN-DBS or frameless STN-DBS surgery. Secondly, the BIS value recommended for GA remains controversial. To determine the optimal BIS value for GA in MER guided STN-DBS, more studies are necessary. Finally, the patients enrolled in this study was limited and the analysis was performed retrospectively.GA is viable and effective in MER guided STN-DBS. Although, the parameters of STN MER signals, including frequency, ISI and amplitude, were obviously interfered under GA, the waveforms of MER signals were recognizable and shared similar characteristics with LA group. GA was also comparable with LA in MER guided STN-DBS, regarding electrode implantation accuracy, clinical outcome and adverse events. Notably, GA is more friendly and acceptable to the patients who are incapable of enduring intraoperative MER under LA.The original contributions presented in the study are included in the article/Written informed consent for surgery was obtained from all patients and the anesthesia methods were determined on the basis of patients' preferences. The demographics and baseline information of patients were acquired from medical records with their approval.PF and KQ conceived of the paper. KQ wrote the initial draft of the paper and generated the figures and tables. JW and JR collected the medical records and data. PZ, YS, WH, and JH performed the MER signal analysis. XJ edited and revised the manuscript. All authors contributed to the article and approved the submitted version.


According to our analysis of the multicenter EORP ELECTRa (European Lead Extraction ConTRolled) Registry, about one-third of the candidates to complete removal of an implantable pacemaker/defibrillator because of device infection, through standard transvenous lead extraction, presents a systemic infection. Systemic infection is associated with a higher incidence of major in-hospital complications overall and a strong trend for procedure-related complications. Moreover, systemic infection is associated with increased procedure-related and non-procedure-related in-hospital mortality. Patients with cardiac implantable devices (CIED)-related infection with systemic vs. local involvement present different characteristics suggesting that in a relevant subgroup of patients the infection can be systemic from the beginning, without progression from CIED pocket.p = 0.0007) and mortality . Sy-CIEDI was an independent predictor of in-hospital death . Patients with Sy-CIEDI more frequently had an initial CIED implant and a higher prevalence of comorbidities, while subjects with Lo-CIEDI had a higher prevalence of previous CIED procedures. Time from signs of CIEDI and TLE was longer for Lo-CIEDI despite a shorter pre-TLE antibiotic treatment. Conclusions: Patients with CIEDI have a worse in-hospital prognosis after TLE, especially for patients with Sy-CIEDI. These results raise the suspicion that in a relevant group of patients CIEDI can be systemic from the beginning without progression from Lo-CIEDI. Future research is needed to characterize this subgroup of patients.Background: Infections of cardiac implantable devices (CIEDI) have poor outcomes despite improvement in lead extraction (TLE) procedures. Methods: To explore the influence of CIEDI on the outcomes of TLE and the differences between patients with systemic (Sy) vs. local (Lo) CIEDI, we performed a sub-analysis of the EORP ELECTRa (European Lead Extraction ConTRolled) Registry. Results: Among 3555 patients enrolled by 73 centers in 19 Countries, the indication for TLE was CIEDI in 1850: 1170 with Lo-CIEDI and 680 with Sy-CIEDI. Patients with CIEDI had a worse in-hospital prognosis in terms of major complications (3.57% vs. 1.71%;  This tr 0.0001) . These f 0.0001) ,4. The cThe ELECTRa registry was a large multicentre prospective registry of consecutive patients undergoing TLE. The registry was conducted by the European Heart Rhythm Association (EHRA). The executive committee in cooperation with the EURObservational Research Program (EORP) provided the study design, protocol, and the scientific leadership of the registry under the responsibility of the EHRA Scientific Initiatives Committee (SIC). EHRA affiliated centres participating in the registry were required to recruit all consecutive patients with an indication for TLE  in their institution. All subjects provided written informed consent prior to the extraction procedure. No specific protocol or recommendations regarding technique were made for the TLE procedure. A detailed description of the study design and of the electronic case report form (e-CRF) has been previously described . The ELEFrom 1 November 2012 to 31 May 2014, 73 EHRA affiliated centres from 19 countries participated in the ELECTRa registry. A total of 3555 consecutive patients were enrolled, of whom 3510 98.7%) underwent TLE. Among this cohort of patients 1850/3510 (52.7%) underwent TLE for CIEDI, either local (Lo-CIEDI) or systemic (Sy-CIEDI) ,7,8. For.7% underThe primary endpoint of the current analysis was to describe the characteristics associated with CIEDI in candidates to TLE and to evidence the differences between patients with CIEDI Lo-CIEDI vs. Sy-CIEDI in terms of baseline characteristics, indications, and outcomes for TLE and in-hospital mortality. The main secondary endpoint was the identification of predictors of mortality before discharge considering the presence of Lo-/Sy-CIEDI.p < 0.05 in univariate, except those with more than 20% of missing data) and the type of CIEDI (Lo- Vs. Sy-)  using SAS statistical software version 9.4 .Results were summarized by absence/presence and type of CIEDI (Lo-CIEDI vs. Sy-CIEDI). Continuous variables were reported as mean ± standard deviation (SD) or as median and inter-quartile range (IQR). Among-group comparisons were made using a non-parametric test (Mann-Whitney test). Categorical variables were reported as counts and percentages . Among-group comparisons were made using a Chi-square test or the Fisher’s exact test (if any expected cell count was less than five). A stepwise multiple Cox regression was used to determine the predictors of in-hospital all-cause mortality and major complications including death selecting into the models all the respective candidate variables (variables with ublished ). A signp < 0.0001) and more frequently males  with a higher prevalence of comorbidities such as hypertension , diabetes , chronic kidney disease  , Chronic obstructive pulmonary disease . On the contrary, despite no difference in the prevalence of reduced left ventricular ejection fraction (CIEDI 31.49% vs. 32.99%), history of chronic heart failure was more prevalent in subjects undergoing TLE for non-infective indications .From 1 November 2012 to 31 May 2014, 73 European centres from 19 countries participated in the study. A total of 3555 consecutive patients were enrolled in the study: 1850/3555 (52.04%) underwent TLE for CIEDI and 1634/3555 (45.96%) for other non-infective indications, mainly to add/substitute a CIED lead. The remaining patients were not included in the analysis since 44 did not underwent TLE and in 26 attributions to a specific subgroup was not possible despite post-hoc CRF revision. Patients with CIEDI were older  and with less prevalent obesity  and presented more comorbidities such as: reduced left ventricular ejection fraction , history of chronic heart failure , diabetes mellitus  and chronic kidney disease . Focusing on the 1850/3555 (52.04%) who underwent TLE for CIEDI: 1170/1850 (63.24%) with Lo-CIEDI and 680/1850 (36.76%) with Sy-CIEDI. p < 0.0001). However, devices for cardiac resynchronization therapy (either defibrillator or pacemaker) were more frequent in patients with CIEDI . Patients with non-infective indications underwent TLE after their first implant  and with the lesser prevalence of previous CIED-related complications  which more frequently implicated a previous malfunction , while patients with CIEDI presented a previous infection as the principal cause of previous complication .Unsurprisingly the higher prevalence of chronic heart failure in patients with non-infective indications of TLE was associated with a higher frequency of defibrillators among the implanted devices  with similar prevalence of biventricular ICD  and the number of leads to be extracted . Notably, patients with Sy-CIEDI more often developed CIEDI after their initial CIED implant compared to patients with Lo-CIEDI . However, patients with Lo-CIEDI had a more complex CIED history with a half more previous CIED complications  and more revisions of the CIED system . Finally, no difference was present in terms of elective box exchange as the last procedure before CIEDI  or prevalence of previous upgrades .Compared to Lo-CIEDI, Sy-CIEDI patients presented only minor differences in the implanted hardware at the time of TLE , with a p = 0.0049). More interestingly, the median time from the first manifestation of infection to enrollment was longer in the Lo-CIEDI group , coupled with a slightly shorter pre-TLE antibiotic treatment . Finally, there was also a trend toward a longer time from the last implanted lead to the first sign of CIEDI in Lo-CIEDI patients .Obviously, patients with Sy-CIEDI presented higher WBC counts, average C reactive protein concentration, and prevalence of positive blood cultures and masses  at echocardiography which are confirming the presence of systemic involvement of the infectious process . Notablyp = 0.0007) and mortality  when compared to patients without CIEDI  in patients with Sy-CIEDI 29/680 (4.26%) vs. 13/1170 (1.11%) in patients with Lo-CIEDI  and non procedure-related mortality   . The ind 0.0001) .p < 0.0001), which was just below significance when the analysis was limited to TLE-related events   . The ind 0.0527) . p = 0.03). Certainly, our results can be currently considered only as hypothesis generating but if the suspicion that in a relevant subgroup of patients Sy-CIEDI and Lo-CIEDI are not two steps of the same process while they are two different diseases this will heavily impact on CIEDI management, from prophylaxis until post-TLE management [p = 0.020) while they were more frequently at their first implant , without difference in terms of implanted hardware. Moreover, they presented more frequently ghosts after TLE  despite a trend of a lower lead indwelling time and no difference in terms of anticoagulant use. All these findings suggest that in a range between one-fifth to one quarter of CIEDI patients another possible mechanism can be involved, maybe as previously suggested Sy-CIEDI could develop independently from Lo-CIEDI through direct seeding during intermittent bacteremia upon leads with higher abrasion [The ELECTRa registry was the first large prospective European independent registry on TLE . The prinagement ,9. This nagement . In thisabrasion ,21. In tabrasion , evaluatabrasion . Despiteabrasion .The ELECTRa findings are subject to the limitations inherent to observational studies, including the possibility of unknown confounders and bias in management strategy. To ensure data integrity source data and database quality control was performed by dedicated data monitors to ensure that all consecutive patients were included in participating centres. The participation in the ELECTRa registry was based on a voluntary basis: and complication rates may therefore be underestimated since there are centres, physicians, and surgeons performing lead extraction that did not participate in the Registry. Although there was participation from all of the major centres/countries performing extraction the patients recruited may not represent the daily practice of lead extraction in all countries where a high proportion of lead extraction is undertaken by non-EHRA affiliated centres. Similarly, patients with an indication for TLE who were referred for open surgical extraction were excluded from the study. The purpose of ELECTRa was to offer a multicentre prospective overview of TLE safety and efficacy in Europe. Albeit it was concluded in 2014 the approach to lead extraction still reflects current practice. Predictors of outcomes were identified and discussed although the exact cause-effect relationships remain speculative. At the same time, ELECTRa was not a registry focusing on CIEDI but on TLE with the possible bias of leaving unexplored patients who were not candidates to TLE. For this reason, it did not consider diagnostic procedures like PET/CT scans . HoweverCIEDI is the most frequent indication for TLE in current clinical practice in Europe and it is associated with worse in-hospital outcomes. In particular patients with Sy-CIEDI have an increased risk of procedure-related and overall in-hospital mortality. Patients with Sy-CIEDI have more multiple comorbidities. Several clinical and instrumental factors suggest that in a relevant subgroup of patients, Sy-CIEDI at the time of TLE was not a progression from Lo-CIEDI. This hypothesis if confirmed by ongoing studies will have relevant impact on the pre- and post-procedure management of candidates for TLE and CIEDI prophylaxis.


The COVID-19 pandemic was met with strict containment measures. We hypothesized that societal infection control measures would impact the number of hospital admissions for respiratory tract infections, as well as, the spectrum of pathogens detected in patients with suspected community acquired pneumonia (CAP).This study is based on aggregated surveillance data from electronic health records of patients admitted to the hospitals in Bergen Hospital Trust from January 2017 through June 2021, as well as, two prospective studies of patients with suspected CAP conducted prior to and during the COVID-19 pandemic . In the prospective cohorts, microbiological detections were ascertained by comprehensive PCR-testing in lower respiratory tract specimens. Mann–Whitney’s U test was used to analyse continuous variables. Fisher’s exact test was used for analysing categorical data. The number of admissions before and during the outbreak of SARS-CoV-2 was compared using two-sample t-tests on logarithmic transformed values.Haemophilus influenzae was the most frequent bacterial detection in both cohorts, followed by Streptococcus pneumoniae in the pre-COVID and Staphylococcus aureus in the COVID cohort.Admissions for respiratory tract infections declined after the outbreak of SARS-CoV-2 (p < 0.001). The pre-COVID and the COVID cohorts comprised 96 and 80 patients, respectively. The proportion of viruses detected in the COVID cohort was significantly lower compared with the pre-COVID cohort , and the proportion of bacterial- and viral co-detections was less than half in the COVID cohort compared with the pre-COVID cohort . The proportion of bacteria detected was similar (p = 0.162), however, a difference in the bacterial spectrum was observed in the two cohorts. During the first year of the COVID-19 pandemic, the number of admissions with pneumonia and the microbiological detections in patients with suspected CAP, differed from the preceding year. This suggests that infection control measures related to COVID-19 restrictions have an overall and specific impact on respiratory tract infections, beyond reducing the spread of SARS-CoV-2.The online version contains supplementary material available at 10.1186/s12879-022-07732-5. Streptococcus pneumoniae and influenza virus . The proportion of detected bacteria remained stable between the two cohorts; 71% (79/112) in the COVID cohort vs 62% (108/174) in the pre-COVID cohort .Figure Haemophilus influenzae was the most frequent bacterial detection in both cohorts . Streptococcus pneumoniae (25%) followed H. influenzae in the pre-COVID cohort, while Staphylococcus aureus increased significantly and was the second most common detection in the COVID cohort (23%).The overview of the microbiological findings is listed in Table Streptococcus pneumoniae was detected more frequently in combination with a viral pathogen in the pre-COVID cohort compared with the COVID cohort , moreover influenza A virus was detected in 54% (13/24) of patients with S. pneumoniae (pre-COVID cohort). In the COVID cohort, S. aureus was detected more frequently in patients with SARS-CoV-2 compared to patients without SARS-CoV-2 .The Norwegian COVID-19 restrictions were comprehensive and intrusive, but comparable to other European countries . During The role of viruses in CAP have been increasingly recognized in the last decades. The introduction of PCR-based methods capable of rapidly and accurately detecting viral pathogens, has led to a great increase of viral detections in CAP patients, either alone or in combination with bacteria , 18–20. S. pneumoniae, H. influenzae or a combination of these. A large study analysing surveillance data from 26 countries and territories across six continents on invasive disease due to S. pneumoniae, H. influenzae and Neisseria meningitidis before and during the outbreak of SARS-CoV-2, showed a substantial and sustained reduction of hospital reported invasive disease for these pathogens [We observed a shift in the microbial patterns of detected bacteria in the COVID cohort, including, fewer bacterial- and viral co-detections. Notably, there was a reduced proportion of patients with detected athogens . AlthougS. pneumoniae and H. influenzae after the outbreak of SARS-CoV-2. This can be interpreted in support of the hypothesis that person-to-person transmission of microbes is an important cause of LRTIs for both viruses and bacteria. However, the proportion of detected S. aureus, which also has the potential for person to person spread, increased in our COVID cohort. It is known that S. aureus tend to colonize the upper respiratory tract of adults more frequently than S. pneumoniae and H. influenzae and this might explain why S. aureus did not decrease in the same manner [S. aureus is strongly associated with infection and clinical studies consistently describe a significantly greater risk of bacteremia among carriers [S. aureus invasive infections, than they do for other pathogens.The development of LRTIs, including bacterial CAP, is complex and still not completely understood. Our data indicate a reduction of detected bacterial pathogens with a known potential to transmit by respiratory droplets, like e manner –29. Nasacarriers . SocietaS. aureus was found more frequently in patients with detected SARS-CoV-2 compared with patients without . An interaction between viral and bacterial respiratory pathogens in CAP has been discussed for many years [S. pneumoniae both as a colonizer and as a pathogen [S. pneumoniae were found in combination with a viral pathogen, most frequently with influenza virus.Many respiratory tract pathogens colonize the upper respiratory tract, especially among the children and elderly, without causing an infection –29, 31. ny years , 32–37. ny years . Severalpathogen , 35, 37.In relation, data from the Norwegian Cause of Death Registry, show a decrease in age-adjusted death rate of LRTIs, including CAP, in 2020 compared with 2010–2019 . This inH. influenzae have decreased [The major strength of our work is that microbiological data were collected from two prospective studies with an attention to detail and accuracy of lower respiratory tract sampling. Lower respiratory tract samples were collected from all patients and tested with a comprehensive multiplex molecular panel in addition to standard methods in most patients. The study has limitations; the inclusion of CAP patients at a single hospital in Norway, a limited sample size and enrolment of patients restricted to fixed hours during weekdays. We excluded patients that were unable to provide a lower respiratory tract sample, e.g. due to confusion, severe hypoxemia, need of assisted ventilation, or a non-productive cough; symptoms which are often found in patients with COVID-19. In addition, in Norway, patients with mild COVID-19 were often treated outside the hospitals at designated COVID-19 wards. Finally, concerning surveillance data from electronic health records, ICD-10 diagnoses are registered by the treating physician and the primary diagnosis may have been influenced by the pandemic. Nevertheless, national surveillance data show that influenza, invasive pneumococcal disease, and systemic disease caused by ecreased .In conclusion, admissions with pneumonia and microbiological detections in patients with suspected CAP during the first year of the COVID-19 pandemic, differed from the previous year, suggesting that infection control measures related to COVID-19 restrictions are effective beyond reducing the spread of SARS-CoV-2. The number of detected viruses declined, and accordingly, the proportion of patients with bacterial- and viral co-detections decreased. Furthermore, we observed a change in both the proportion and pattern of certain bacterial detections, implying that presence of viruses may facilitate colonization and infection by certain types of bacteria and play an important role in the etiopathogenesis of CAP.Additional file 1. BioFire® FilmArray® Pneumonia panel plus (FAP plus) targets.


Lung cancers are the leading cause of cancer-related deaths worldwide. Studies have shown that non-small cell lung cancer (NSCLC), which constitutes the majority of lung cancers, is significantly more responsive to early-stage interventions. However, the early stages are often asymptomatic, and current diagnostic methods are limited in their precision and safety. The cell-free RNAs (cfRNAs) circulating in plasma (liquid biopsies) offer a non-invasive detection of spatial and temporal changes occurring in primary tumors since the early stages. To address gaps in the current cfRNA knowledge base, we conducted a pilot study for the comprehensive analysis of transcriptome-wide changes in plasma cfRNA in NSCLC patients. Total cfRNA was extracted from archived plasma collected from NSCLC patients (N = 12), cancer-free former smokers (N = 12), and non-smoking healthy volunteers (N = 12). Plasma cfRNA expression levels were quantified by using a tagmentation-based library preparation and sequencing. The comparisons of cfRNA expression levels between patients and the two control groups revealed a total of 2357 differentially expressed cfRNAs enriched in 123 pathways. Of these, 251 transcripts were previously reported in primary NSCLCs. A small subset of genes (N = 5) was validated in an independent sample (N = 50) using qRT-PCR. Our study provides a framework for developing blood-based assays for the early detection of NSCLC and warrants further validation. Lung cancers are the leading cause of cancer-related deaths in both men and women in the U.S. and worldwide. Non-small cell lung cancer (NSCLC) constitutes approximately 84% of all lung cancer cases and consists of two main histological subtypes: adenocarcinoma (AC) and squamous cell carcinoma (SCC) . The maiBecause lung cancers are often asymptomatic in early stages, most patients are diagnosed at advanced stages, resulting in only about 15–20% of patients surviving five years after diagnosis . Early-sRecently, the concept of liquid biopsies has garnered excitement among the scientific community for its potential to provide real-time information on spatial and temporal changes in tumor markers in an easily obtained peripheral blood sample . SeveralStudy design: In this pilot study, we first compared the expression levels of plasma cfRNA obtained from SCC and AC patients  and cancer-free former smokers . As all patients in the case group were also heavy smokers, we included a second control group of non-smoking healthy individuals  to exclude differentially expressed cfRNAs associated with smoking, rather than pathological processes underlying NSCLC. Each participant provided whole blood samples as part of an umbrella protocol approved by the Institutional Review Board of the University of Maryland Baltimore [UMB IRB protocol ID: HP-00040666] and the Veterans Affairs Maryland Health Care System. All participants provided written informed consent to participate in the research conducted at the University of Maryland Medical Center and the Baltimore VA Medical Center. Diagnosis of lung cancer was established by the pathological examination of tissues obtained via surgery or biopsy. Histological diagnoses were made on bronchoscopic biopsy specimens and thoracotomy according to the World Health Organization (WHO) categories. The NSCLC stage classification was based on the WHO classification and the International Association for the Study of Lung Cancer staging system. The smokers consisted of former smokers who had a minimum smoking history of 30-pack years and quit within the past 15 years. The exclusion criteria were similar to Leng et al. 2017 . The demg for 30 min at 4 °C to remove any cellular components in the plasma. The supernatant was extracted and centrifuged again at 13,000× g for 30 min at 4 °C and stored at −80 °C until the day of cfRNA extractions. The quality control procedures for the plasma sample preparations were similar to our earlier study [® Serum/Plasma Advanced Kit (Qiagen) according to the manufacturer’s guidelines and was tested for RNA integrity using an Agilent bioanalyzer system. The libraries were prepared using a tagmentation-based method consisting of a two-step probe-assisted exome enrichment for cfRNA detection  [Sample preparation and sequencing: The archived plasma samples  . An IlluEnsembl repository) using HISAT2, a fast splice-aware aligner for mapping next-generation sequencing reads [p-values were generated using the Wald test implemented in DESeq2 and then corrected for multiple hypothesis testing using the Benjamini–Hochberg correction method [Sequencing data analyses: The raw sequence reads generated for each sample were analyzed using the CAVERN analysis pipeline . Read qung reads . The reang reads  and the n method . The sigRNA Spike-In Kit, For RT was added to the plasma samples according to the manufacturer’s protocol  prior to the extraction of cfRNA to control for cfRNA isolation across the samples. The extracted total cfRNA samples were then split into equal volumes for cDNA synthesis and the subsequent mRNA quantification and detection of the three miRNA spike-ins in parallel. We used miRCURY LNA RT and miRCURY LNA SYBR Green PCR kits (Qiagen) for the reverse transcription and qPCR of spike-in miRNAs and the ® Reverse TranscriptionQuantiTect and QuantiTect SYBR Green RT-PCR kits (Qiagen) for the reverse transcription and qPCR of the selected protein-coding genes. All qPCR reactions were performed in triplicates with 1:10 cDNA dilutions in a Bio-Rad CFX real-time PCR detection system , according to the protocols associated with each kit. As stable endogenous reference genes for quantifying circulating mRNA in plasma samples have not been established in the literature and normalizing to a global mean of all expressed mRNA was not applicable to the analyses of five genes, we opted not to use a reference gene in this pilot study. We also explored the possibility of using GAPDH—the commonly used endogenous reference gene for cellular mRNA—and did not detect any amplification. Therefore, we adopted a method of, first, assessing the between-sample variability using three spike-ins to identify outlier samples and then performing qRT-PCR for the five selected genes, excluding outliers. Two-tailed t-tests using GraphPad Prism software  were performed for statistical comparisons.Quantitative RT-PCR (qRT-PCR) for validation of a subset of cfRNA: Based on the findings from the sequencing data analyses, we selected five differentially expressed protein-coding genes, as listed in cfRNA processing and quality control: cfRNA was extracted from all 36 samples at mean concentrations of 0.111 ng/uL in cases, 0.085 ng/uL in control_smokers, and 0.151 ng/uL in control_healthy. The RNA integrity numbers (RIN) ranged from 1 to 5.3. All samples had sequence reads that mapped >80% to the reference sequence and mapped to the exonic regions. Total Gene Abundance ranged from approximately 10 to 70 million. Of these genes, 0.5–10% were Hb coding genes, 0.5–20% mitochondrial genes, <0.03% ribosomal RNA (rRNA) genes, and up to 4% were other non-coding RNA (ncRNA) genes. Amongst the protein-coding genes, the most abundant were actin, myosin, platelet-specific genes, and pseudogenes.Identification of differentially expressed cfRNAs between cases and controls: The differential expression of cfRNA was analyzed after excluding Hb, mitochondrial, and rRNA transcripts. As shown in Statistical power analysis: The post hoc power analysis revealed that the samples of 12 cases and 24 controls afforded a 78.5% power to detect differentially expressed genes with a 2-fold effect size using a 5% false discovery rate.Exploratory subgroup analyses: We performed two subgroup analyses exploring the differentially expressed cfRNAs between (1) subtypes of cases, AC vs. SCC, and (2) based on NSCLC stages, stages I vs. II, compared with both control groups, irrespective of their statistical significance in the combined case group. Literature review to identify DEGs previously reported in primary NSCLC biopsies: We performed an exhaustive review of all the published studies listed on the National Center for Biotechnology Information (NCBI)’s database for gene-specific information, using gene IDs for each of the 2357 identified DEGs. Studies reporting DEGs in primary NSCLC biopsies were identified and are referenced in p = 0.037), and SRXN1 showed a trend towards downregulation in cases (p = 0.056) compared with the combined control group. PDE4DIP showed a trend towards downregulation in cases compared only with the healthy non-smokers (p = 0.079). The other two genes, RAB38 and BLID, did not show statistically significant expressed cfRNA levels between cases and the controls.Quantitative RT-PCR (qRT-PCR) for validation of replicated cfRNA of protein-coding genes: While all the listed genes in p = 0.0041) and 24 cfRNAs in cases vs. control_smokers group (adjusted p = 5.98 × 10−5). However, at an individual gene level, only two cfRNAs (MIR874 and MIR551B) in GO:0010629 were common to the two groups, both in terms of direction and type. The cases vs. control_smokers and cases vs. control_healthy comparisons did not share any significantly enriched pathways. Eighty-five pathways were commonly enriched in cases and cancer-free smokers when each group was compared with the control_healthy group. Details of the 37 pathways that were uniquely enriched in cases compared with both control groups include general mechanisms underlying cancer biology and are presented in Gene ontology (GO) enrichment analysis of differentially expressed cfRNA: The unbiased pathway analysis with cfRNA for the differentially expressed genes included in each category of Twenty-five of thirty-seven uniquely enriched pathways in cases were compared against the non-smoking control group, of which twenty were in the GO domain of biological process (BP) and five in the domain of molecular function (MF). For the BP domain the significant terms were: GO:0001501, GO:0007186, GO:0007200, GO:0007399, GO:0008154, GO:0009888, GO:0009953, GO:0010454, GO:0032501, GO:0042221, GO:0042246, GO:0042692, GO:0043403, GO:0043503, GO:0045165, GO:0051272, GO:0051493, GO:1902903, GO:1904888, and GO:2001046. For the MF domain, the significant terms were: GO:0005125, GO:0005198, GO:0019958, GO:0030545, and GO:0048018. The remaining 12 of the 37 pathways uniquely enriched in cases were compared against the cancer-free smokers. These were in the BP (N = 7), MF (N = 2), and cellular component  domains. For the BP domain, the significant terms were: GO:0010608, GO:0016441, GO:0016458, GO:0031047, GO:0035194, GO:0035195, and GO:0040029. For the MF domain, the significant terms were: GO:0003729 and GO:1903231. For the CC domain, the significant terms were: GO:0016442, GO:0031332, and GO:1990904.Various subtypes of circulating cfRNA have been tested in plasma for the early-stage detection of NSCLC. Building upon these studies, we performed a comprehensive analysis of circulating plasma cfRNA using next-generation sequencing technologies to expand the repertoire of non-invasively measurable NSCLC signatures. We identified 2357 cfRNAs enriched in 123 pathways in those with a diagnosis of NSCLC compared with the control groups consisting of cancer-free smokers and non-smokers. Nearly half of the detected cfRNAs were transcripts of protein-coding genes, and 251 of the 2357 cfRNAs (10.65%) conformed to previously reported differentially expressed genes found in primary tumor biopsies from NSCLC patients. A majority (174 of 251) of these replicated transcripts were protein-coding genes, while the rest were previously reported miRNAs and other non-coding RNAs. In fact, two of the snoRNAs—SNORD115-41 and SNORD12—were previously reported in NSCLC tissue biopsies by our group .Importantly, our pilot study used a workflow that can be easily adopted to develop a clinical assay for profiling cfRNA using plasma volumes smaller than those that have been reported elsewhere . The arcAlthough identifying biomarker signatures associated with NSCLC was not the primary objective of this proof-of-concept pilot study that sought to test the potential of an NGS-based method for the comprehensive detection of circulating cfRNA in plasma, we further evaluated the cfRNA of the 251 genes to explore potential candidates for future NSCLC-associated biomarker development studies. We first searched for cfRNAs that were differentially expressed in the plasma samples from NSCLC patients (regardless of the subtypes) compared with both smokers with benign PNs and non-smokers. Our results indicated fifteen genes that included six protein-coding, six miRNA, and three other non-coding genes. Twelve of the fifteen genes had low inter-patient variabilities  for cfRNA expression. These included five cf-mRNAs , four cf-miRNAs , and all three of the other non-coding genes . Notably, the cfRNA for the two snoRNAs, genes SNORD115-41 and SNORD12, which we have previously reported , were noOur pilot study has several limitations. First, biological factors such as gender and age have been shown to play a major role in the development and prognosis of lung cancers . For exaIn summary, we have presented transcriptome-wide cfRNA profiling using small volumes of plasma, providing a framework for developing a non-invasive (blood-based) assay for the potential early detection, diagnosis, and monitoring of NSCLC to facilitate high rates of patients able to receive curative surgical resections. Further studies are required for the evaluation of our methodology and its clinical application.


Experimental studies proposed a direct effect of follicle-stimulating hormone (FSH) on the skeletal metabolism, but results of human studies mainly conducted in females are controversial. The present study aims to investigate the possible role of FSH excess in male bone health, by comparing for the first time primary and central hypogonadism.119 men were enrolled in this cross-sectional observational study at the time of the first diagnosis of hypogonadism. All participants had spontaneous pubertal development. Regarding patients with hypergonadotropic hypogonadism (Hyper-H), Klinefelter syndrome (KS) patients were distinguished from the other forms (non-KS-Hyper-H) based on the onset of FSH elevation. Bone mineral density (BMD) at both lumbar spine (LS) and femoral neck (FN), as well as the prevalence of morphometric vertebral fractures (VFx), were assessed.Across the whole cohort, higher LS and FN BMD were associated with older age at diagnosis and higher body mass index (BMI), respectively. After adjusting for potential confounders , non-KS-Hyper-H patients showed significantly lower LS BMD and tended to show lower FN BMD values, as compared to those with hypogonadotropic hypogonadism (Hypo-H). In KS men, LS BMD was significantly lower than in those with non-KS-Hyper-H. No significant differences in the prevalence of VFx were found between the groups.These findings suggest a potential negative effect of FSH excess on the male bone mass, especially at spine. The duration of high FSH levels may also contribute to these findings. It consists of a hormone-specific β subunit non-covalently associated with the α subunit that is also part of other glycoprotein hormones . FSH acts (GPCR) . The bins (GPCR) . Gonads s (GPCR) .in vitro and, when injected into ovariectomized mice, to attenuate bone loss  –14. A poWith regards to males, the existing literature is sparse and conflicting. On the one hand, according to previous studies involving osteoporotic men , patientHowever, all these studies present some limitations, particularly relating to selection and/or confounding bias, as well as short duration and lack of baseline assessment. Therefore, on balance, the putative role of FSH as a direct modulator of skeletal physiology remains matter of debate. Intriguingly, men with primary versus central hypogonadism, both presenting with low T but with differing FSH values, might represent a novel attractive study model in this context.The present study aims to investigate the possible association of FSH excess with male osteoporosis, by assessing BMD and prevalence of fragility fractures in men with primary hypogonadism (high FSH levels) compared to those with central hypogonadism .One hundred and nineteen men, consecutively referred to the Hypogonadism Clinic both of Istituto Auxologico Italiano  and Newcastle upon Tyne Hospitals (NHS Foundation Trust) for clinical suspicion - later confirmed - of hypogonadism, were enrolled in this cross-sectional observational study. Diagnosis of hypogonadism was based on the finding of consistently low T levels .The project was approved by the Institutional Review Board of IAI (05C622_2016). Recruitment lasted 18 months. Subjects fulfilling one or more of the following criteria were excluded: I) absent puberty; II) current or previous treatment with testosterone and/or gonadotropins, antiresorptive and/or anabolic agents, or any other medication impacting on bone metabolism ; III) comorbidities that may affect skeletal health, such as hyperthyroidism, hyperparathyroidism, rheumatoid arthritis, malabsorption, alcohol abuse, chronic kidney or liver disease. The flow-chart used to select participants is depicted in Causes of hypogonadism and clinical characteristics of the whole cohort are summarized in All the participants were studied at the time of the first diagnosis of hypogonadism. Relevant patient data were prospectively assembled as part of routine clinical practice based on the delivery of good clinical care.Personal and family history was investigated, with focus on previous fragility fractures as well as any conditions or drugs that may affect bone health. Regarding smoking habits, a daily consumption of more than 5 cigarettes was considered as a risk threshold .Weight and height were measured, and body mass index (BMI) was calculated. Waist circumference (WC), arm span (AS), Tanner stage, testicular volume, blood pressure (BP) and heart rate (HR) were assessed, too.t-score) and age-matched (z-score) reference healthy population.Bone mineral density at both lumbar spine  and femoral neck (FN) was measured using dual-energy X-ray absorptiometry , and expressed as standard deviation (SD) units in relation to the young  was evaluated by performing vertebral morphometry on conventional thoraco-lumbar-sacral spine radiographs . The semiquantitative visual assessment previously described by Genant et al.  was emplBesides, serum calcium (Ca), albumin, phosphorus (P), alkaline phosphatase (ALP), glucose and lipid profile were determined using standard techniques. 25-hydroxy-vitamin D levels (25OHD) levels were measured by means of Elecsys Roche assay. Albumin-adjusted calcium (aCa) levels were obtained. Free testosterone (cfT) levels also were calculated from total testosterone (tT), albumin and sex hormone-binding globulin (SHBG) using the Vermeulen equation .BonesGone project, these data were extracted from case records and collated in fully anonymised format prior to statistical analysis. Written consent was obtained from each patient or subject after full explanation of the purpose and nature of all procedures used, unless these formed part of everyday clinical care.As per the Statistical analysis was performed using SPSS version 27.0 statistical package . The normality of distribution for continuous variables was tested using Kolmogorov–Smirnov test. Data were expressed as median (range) for non-normally distributed continuous variables or as mean ± SD for normally distributed variables, whereas as absolute and relative frequencies for categorical variables.post hoc analysis was performed to compare the continuous variables among KS, non-KS-Hyper-H and Hypo-H  was employed to assess the independent associations between BMD and the variables that were found to be different among groups and those known to be relevant for bone health.Continuous variables were compared between Hyper-H and Hypo-H  and overweight/obesity prevalence  with no significant differences in age, prevalence of type 2 diabetes (T2D) and smoking habits , and cfT  as well as aCa levels  tended to be higher as compared with Hypo-H group, while the BMD values and the prevalence of fragility fractures were comparable between the groups.However, after adjusting for age at diagnosis, BMI, cfT and 25OHD levels by means of GLM analysis, LS BMD was significantly lower , and FN BMD tended to be lower  in Hyper-H patients than in Hypo-H ones.The same analysis showed that in the whole cohort LS BMD was directly associated with age at diagnosis  but not with BMI, cfT and 25OHD levels, whereas FN BMD was associated with BMI  but not with age at diagnosis, cfT and 25OHD levels. The bivariate associations between LS BMD and age at diagnosis, and between FN BMD and BMI are depicted in In order to evaluate the possible influence of the duration of FSH excess, we compared KS patients with non-KS-Hyper-H patients  and prevalence of overweight/obesity  than Hypo-H patients. After adjusting for age at diagnosis, BMI, cfT, 25OHD levels and smoking habits, LS BMD was significantly lower , and FN BMD tended to be lower  in non-KS-Hyper-H than in Hypo-H men.We also compared the clinical characteristics between non-KS-Hyper-H and Hypo-H ones  and overweight/obesity prevalence , but not with smoking habits, cfT and 25OHD levels, whilst FN BMD tended to be associated with age at diagnosis  but not with overweight/obesity prevalence, smoking habits, cfT and 25OHD levels.This is the first study comparing men with primary and central hypogonadism to better delineate the role of FSH in the male bone health. Notably, such findings indicate a potential negative effect of FSH excess on the male bone mass, especially at spine, either in the whole cohort or within the subset of hypogonadal patients with adult-onset FSH elevation. The duration of high FSH levels may also play a part in this setting, as revealed by the comparison between KS and non-KS-Hyper-H men.Indeed, several findings support the FSH fostering role on bone loss in males. For instance, in KS patients neither T levels nor polymorphisms in the androgen receptor (AR) gene - consisting of a variable number of CAG repeats which modulate its sensitivity - are associated with osteoporosis , thus poIn our project, after adjusting for potential confounders, Hyper-H patients showed lower LS BMD, and tended to show lower FN BMD levels, as compared to Hypo-H patients . Since we enrolled only patients naïve of any treatment or condition possibly interfering with BMD, these findings give a strong support to the negative effect of FSH excess on the male bone mass. Trabecular bone seems to be affected to a greater extent, in keeping with what is observed in perimenopausal women and, more generally, in high-turnover osteoporosis. In fact, FSH elevation may partly explain the decline in bone density that occurs during the early menopausal transition when oestrogen levels are still well-preserved , 35.Furthermore, in KS patients LS BMD values were lower than in the other Hyper-H ones. Notably, this may reflect the effect of long-lasting FSH excess (starting from puberty) on trabecular bone. Indeed, it is worth noting that hypogonadism can just partly justify bone fragility in KS , 37, andBy carrying out the GLM analysis in the whole cohort, we found LS BMD and FN BMD to be independently and directly associated with age at diagnosis and BMI, respectively . Under sAs expected , Hypo-H Our project also presents some limitations. Firstly, the final sample size of patients was limited but balanced by the rigorous design of the study that excluded many hypogonadal patients  can be found below: 10.5281/zenodo.6472089.The studies involving human participants were reviewed and approved by the Ethics Committee of Istituto Auxologico Italiano. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.IC, MB, and LG contributed to conception and design of the study. LG organized the database. LG, MB, RQ, LP, and BC contributed to recruit patients. SC helped to collect data. IC and LG performed the statistical analysis. LG wrote the first draft and sections of the manuscript. IC, MB, RQ, and LP contributed to manuscript revision. All authors read and approved the submitted version.This study was partially supported by Ricerca Corrente funds from IRCCS Istituto Auxologico Italiano: (grant numbers: O5C202_2012 and 05C622_2016).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cardiac magnetic resonance (CMR) imaging is a well-set diagnostic technique for assessment of valvular heart diseases and is gaining ground in current clinical practice. It provides high-quality images without the administration of ionizing radiation and occasionally without the need of contrast agents. It offers the unique possibility of a comprehensive stand-alone assessment of the heart including biventricular function, left ventricle remodeling, myocardial fibrosis, and associated valvulopathies. CMR is the recognized reference for the quantification of ventricular volumes, mass, and function. A particular strength is the ability to quantify flow, especially with new techniques which allow accurate measurement of stenosis and regurgitation. Furthermore, tissue mapping enables the visualization and quantification of structural changes in the myocardium. In this way, CMR has the potential to yield important prognostic information predicting those patients who will progress to surgery and impact outcomes. In this review, the fundamentals of CMR in assessment of aortic valve diseases (AVD) are described, together with its strengths and weaknesses. This state-of-the-art review provides an updated overview of CMR potentials in all AVD issues, including valve anatomy, flow quantification, ventricular volumes and function, and tissue characterization. Aortic valve disease (AVD) affects approximately 0.9% of the general population , with a Although echocardiography still represents the first-line technique to assess the aortic valve, cardiovascular magnetic resonance (CMR) imaging is emerging as a method able to provide a comprehensive evaluation of many aspects of aortic valvulopathy.CMR is a non-invasive, multiplanar, and high-spatial-resolution imaging technique. It offers a robust alternative for assessing the severity of aortic stenosis (AS) , is supeThe aim of this review is to provide an updated overview of CMR in AVD. In this paper, we will first review the CMR sequences commonly used to assess patients with aortic valvulopathy. Then, we will discuss the role of CMR in AS and AR. Next, we will focus on the application of CMR in patients requiring transcatheter aortic valve prosthesis implantation (TAVI).The steady-state free precession (SSFP) pulse sequence is commonly used for the assessment of valve morphology and function ,7. ImageCine sequences enable visualization of post-stenotic and regurgitant blood flow. Qualitative analysis relies on the evaluation of signal void artifact, which results from intravoxel spin dephasing due to turbulent flow. Gradient echo imaging (GRE) , a formeQuantitative analysis of flow velocity can be performed using through-plane phase contrast (PC) velocity mapping . PC pulsCMR gives the possibility to characterize the anatomy of the thoracic aorta; aortic root dilatation is associated with functional AR, whereas post-stenotic remodeling occurs in AS, especially in bicuspid aortic valves. In patients scheduled for aortic valve intervention, it is fundamental to provide aortic measurements. One of the most used tools is contrast-enhanced MR angiography (CEMRA), which is usually non-ECG-triggered . Thus, iA three-dimensional (3D) self-navigated free-breathing high-resolution whole-heart CMR sequence with either end-systolic or diastolic gating  grants aFour-dimensional (4D) flow or time-resolved 3D phase-contrast CMR is applied for the synchronous evaluation of morphometry and flow parameters along the thoracic aorta. It holds the possibility to measure non-laminar flow in any direction in space during the cardiac cycle . VelocitCMR is the gold-standard imaging technique to evaluate LV volume, mass, and function . AccuratLate gadolinium contrast-enhanced (LGE) imaging is deemed to be the reference standard to quantify myocardial replacement fibrosis and scar. Standard LGE sequences obtained 10 to 15 min after contrast agent injection. Furthermore, inversion recovery gradient echo sequences are used, consisting of an inversion pulse to suppress normal myocardium and a T1-weighted GRE acquisition. In regions with higher gadolinium concentration, T1 time is shorter than in adjacent areas and shows high signal intensity on LGE images. Normal myocardial tissue will appear darker compared with the bright signal of the damaged myocardium where gadolinium washout is delayed . AnotherAS is the most common valvulopathy in developed countries with a prevalence constantly increasing due to rising life expectancy ,32. TranEven though less utilized in clinical practice, CMR offers several advantages in patients affected by AS, allowing a non-invasive, multiplanar, radiation-free, and high-resolution assessment of valvular anatomy and severity of stenosis, coupled with a thorough functional evaluation. Furthermore, compared to other modalities, CMR offers the unique asset of myocardial tissue characterization. In patient candidates for TAVI, as described later, CMR represents an alternative tool to CT for procedural planning in subjects with contraindications to contrast agents.In patients with non-diagnostic TTE due to poor acoustic windows, CMR can be used for aortic valve anatomy assessment and to determine the degree of stenosis. Assessment of AS severity by CMR utilizes two parameters: planimetry of the valve area A and peaThe latter, different from Doppler echocardiography, is time-consuming and tends to underestimate the transvalvular gradients secondary to intravoxel dephasing errors in presence of high-velocity flows . On the LV remodeling in the setting of AS starts as a compensatory process to maintain wall stress, but often it progresses to a maladaptive response characterized by myocyte hypertrophy, interstitial fibrosis, and apoptosis. Although LV reverse remodeling occurs after aortic valve replacement (AVR), the intervention is often performed late after irreversible maladaptive LV remodeling and fibrosis . Dweck aMyocardial fibrosis is a hallmark of severe AS and has an important prognostic role. Three main patterns have been described: endocardial fibrosis, microscars (mainly in the subendomyocardium), and diffuse interstitial fibrosis .Although the gold standard for assessing myocardial fibrosis is histology on endomyocardial biopsy, CMR is able to non-invasively assess the presence of both focal and diffuse fibrosis. Focal non-ischemic fibrosis, often identified as an area of mid-wall LGE D, is freSimilarly, diffuse fibrosis, identified with native T1 and ECV, is a relevant risk marker in patients with AS. Lee et al. showed that high native T1 value on non-contrast T1 mapping CMR is an independent predictor of adverse outcome in patients with significant AS . More reRecent guidelines suggest that CMR is indicated for AR evaluation when echocardiographic images are suboptimal, echo parameters are discordant, disagreement subsists between clinical assessment and echocardiographic grading, inadequate echocardiographic measurements of LV volumes and systolic function are obtained in patients with moderate/severe AR, and insufficient aorta evaluation by echocardiography is achieved in patients with bicuspid aortic valve .CMR assessment of AR is advisable due to the high degree of accuracy for measurement of LV volumes and function as well as aortic regurgitant volumes ,54. ThisCMR assessment of AR initiates from the visual inspection of the aortic valve, aortic root, LV, and LV outflow tract structure and function with SSFP A. Valve A preliminary evaluation of the severity of AR can be achieved by visualization of the signal void of the regurgitant jet on cine imaging A. A narrHowever, the most commonly used method to quantify AR is through-plane PC imaging A, which Regurgitant fraction and regurgitant volume are independent predictors of outcome in patients with AR. A regurgitant fraction of >33% and a regurgitant volume threshold of >42 mL have been shown to predict the likelihood of requiring surgery (mean follow-up of 2.6 years). No patients with a regurgitation fraction <26% progressed to surgery . MoreoveThe presence of holodiastolic retrograde flow (HRF) in the descending aorta can also be assessed easily by PC imaging. HRF on CMR was strongly and independently associated with heart failure, hospitalization, and cardiovascular death .Four-dimensional flow MRI is an emerging tool for the assessment of AR B. The adAccurate LV volumes with CMR are fundamental to clinically assess the impact of AR D. In a mR = 0.70, p < 0.001) [p = 0.001), implying the presence of expanded interstitial fibrosis [R = 0.79, p = 0.011) [p < 0.03 for both), despite a recovery in LV ejection fraction and a shrinkage of LV dimensions [Pressure and volume overload exerted on the LV by AR induce interstitial fibrosis, characterized by increased fibronectin and glucosamine deposit with altered collagen organization . Replace< 0.001) . Malahfj< 0.001) . Sparrowfibrosis . In anot= 0.011) . CMR-FT = 0.011) . Moreovemensions . In addiCT is the gold standard imaging tool to assess the feasibility of TAVI. CT provides accurate annular sizing, determination of risk of annular injury and coronary occlusion, and co-planar fluoroscopic angle pre-procedural prediction. Further benefits of cardiac CT have also been demonstrated in the follow-up of TAVI for assessment of post-procedural complications, including identification of leaflet thickening ,72.However, CT requires contrast agent administration that may be high-risk in allergic patients and in subjects with chronic renal insufficiency, who make up a vast percentage of patients undergoing TAVI. The use of ionizing radiation in CT was not considered particularly relevant for the population involved in the intervention in the past; however, now, it represents a non-negligible issue with the extended indications to younger subjects with lower perioperative risk .CMR may be a valid alternative to CT providing high-quality 3D multislice images without the administration of contrast agents and ionizing radiation. It offers the unique possibility of a one-stop-shop approach to not only assess the anatomical feasibility of TAVI, but to also provide a thorough analysis of the heart, including biventricular function, LV remodeling, myocardial fibrosis, and associated valvulopathies. Moreover, compared to CT, heart rate control is not a major concern because of the superior temporal resolution of CMR. Drawbacks of the use of CMR for TAVI include a longer study time, a greater patient collaboration, and underestimation of calcifications given that calcified tissue produces little signal.A complete evaluation with CMR of the aortic root, including assessment of annular size, aortic leaflet dimensions, and height of coronary artery ostia, is attainable and accurate when compared to cardiac CT ,74 Figu.In order to perform these measurements, a CMR protocol pre-TAVI should include two long axis cine images of the aortic root and a stack of cine images acquired orthogonally to the above two planes, covering the entire aortic root.Magnetic resonance imaging (MRI) can also be used to assess TAVI peripheral access route using CEMRA or, in patients allergic to contrast agents, a 3D-SSFP navigator-echo and ECG-gated  sequence for the thoracic aorta while a non-contrast-enhanced MR angiography can be used for aorto-iliac evaluation . HoweverThe identification of myocardial tissue characterization abnormalities with CMR allows prognostic stratification of patients before TAVI. Indeed, the presence of LGE in patients undergoing TAVI predicts higher cardiovascular disease related mortality .Moreover, CMR is a powerful tool for the screening of association between amyloidosis and AS, which occurs in one of eight patients evaluated for TAVI . Indeed,The recent study by Nitsche et al. demonstrated that although patients with AS and cardiac amyloidosis were older and had worse clinical presentation , they had similar outcomes to those with lone AS .However, there was a trend for higher mortality at 1 year in AS cardiac amyloidosis versus lone AS and other relevant clinical outcomes, including re-hospitalization for heart failure, functional class, and quality of life, which were not considered. Moreover, the study was limited to a 3-year follow-up, whereas cardiac amyloidosis may have an impact on longer-term outcomes .Although the diagnosis of cardiac amyloidosis in symptomatic patients with severe AS should not preclude the consideration for TAVI, its identification with CMR is of importance as it may lead to consideration for pharmacological treatment .In the post-intervention phase and follow-up, CMR can be valuable for the assessment of para-valvular aortic regurgitation, a condition associated with long-term mortality after TAVI . TTE is In the future, real-time CMR (RT-CMR) may be considered for guiding TAVI. Owing to an unlimited scan plane orientation and an unsurpassed soft-tissue contrast with simultaneous device visualization, RT-CMR could allow safe device navigation and offer optimal orientation for precise axial positioning. Non-contrast, radiation-free, and RT-CMR-guided TAVI has been successfully implanted in animals using dedicated conditional equipment , paving