Background and Objectives: The rate of head and neck cancer (HNC) is expected to increase by 30% by 2030. However, there are many similarities between the symptomatology of a benign and a malign diagnosis; thus, a protocol for conducting a full head and neck examination is of high importance since the absence of adenopathy does not exclude a malignant diagnosis and also a favorable prognosis. Material and methods: The current study presents a retrospective study on 515 adult patients who underwent a biopsy for possible head and neck tumor pathology. Results: The patients identified with cancer were older than the rest of the group, with a higher developing trend in men than in women. However, the top 10 symptomatology patterns were identical in the malign and benign groups, meaning that new HNC may be missed due to the common symptomatology between benign and malign outcomes. Conclusions: The importance of a full ear, nose, and throat (ENT) examination may be of significant relevance for a proper diagnosis that can improve the overall prognosis of a patient with cancer. The absence of routine screening tests and screening guidelines for oral and pharyngeal cancers represents a significant barrier to secondary HNC prevention. Each year, more than 500,000 new diagnoses of head and neck squamous cell carcinoma (HNSCC) are reported worldwide . The incAround 30–40% of pharyngeal cancer patients are diagnosed with early stage disease and have a 5-year survival rate of 70–90% when under treatment. However, the majority of pharyngeal cancers are discovered at advanced stages (over 75%), when medical treatment is not so efficacious, and surgical treatment deteriorates the organs involved in the speech and swallowing processes . CervicaHowever, advanced-stage chemoradiotherapy (CRT) is the primary approach for treating cancers developing in the pharynx. Nevertheless, HPV-positive HNC generally presents a more favorable prognosis when compared to HPV-negative HNC. Still, several studies are investigating the relationship between dose reduction (of both radiation and chemotherapy) and HPV-positive disease efficacy. According to Bonner et al., most patients diagnosed with HNC need multilateral approaches and, thus, multidisciplinary care . A delayTaking into account all the aspects presented above related to HNC diagnosis and prognosis, the present study aims to compare the diagnostic strategies employed by practitioners and the relevance in the relapse rate and survival based on the initial stage of lymphadenopathy (N0/N1) of the patients upon admission to the hospital.The present study represents a retrospective cohort study of 515 patients older than 18 years who underwent a biopsy for a possible head and neck tumor pathology. The study focused on patients’ malignant histopathological diagnosis, including several aspects, such as date of diagnosis, date of death, treatment of head and neck cancer, classification of histological diagnosis at three levels: pharynx, adjuvants, and anti-neoplastic therapies. The patients were carefully selected from Info World-Hospital Manager between January 2014 and December 2018 using the diagnosis-related group codes: malignant tumor; lesion exceeding the oropharynx (C.10.8); unspecified nasopharyngeal malignant tumor (C11.9); malignant tumor of the piriform sinus (C12); unspecified hypopharyngeal malignant tumor (C13.9); tumor of the lip, oral cavity, and pharynx with unpredictable and unknown evolution (D37.0); unspecified tumor with unpredictable and unknown evolution (D48.9) at Timisoara Municipal Emergency Clinical Hospital, Department of Otorhinolaryngology.The data recorded were initial diagnostic tumor pathology with unpredictable evolution, benign, or malignant biopsy related to the diagnosis. However, the focus was on the group of patients with malignant disease, and the following aspects were evaluated: age at diagnosis, gender, rural or urban environment, localization of the tumor . TNM staging was determined retrospectively, where possible, using disease extent and collaborative staging codes for tumor sizes and locations, following the classification protocol developed by the American Joint Committee on Cancer: days of hospitalization, type of hospitalization (emergency or not), type of surgical intervention, type of biopsy, histological diagnosis, clinical presentation, and overall survival (OS). The study examined patients with malignant head and neck tumors. Data collected included date of diagnosis, date of death, treatment details, and histopathological classification. The tumors were classified via anatomical location in the pharynx. Information on adjuvant therapies and anti-cancer treatments administered was also collected.The primary outcome was defined as the time (in months) from diagnosis to death from any cause, whereas for OS, the time was recorded from the moment of diagnosis to cancer-related death.Exclusion criteria consisted of patients under age of 18 years, without histopathological diagnosis or non-concluded biopsy and laryngeal carcinoma. Also, patients with incomplete staging, treatment, or follow-up data were excluded, or with other anatomic site codes .t-test or analysis of variance  were used for normal distributed data and Mann–Whitney U-test or Kruskal–Wallis tests were reported . Continuous variable distributions were tested for normality using the Shapiro–Wilk test and for equal variance using the Levenes test. The strength of association between two continuous variables from non-Gaussian populations was assessed using Spearman’s correlation coefficient. A sample size calculation was performed prior to the study, with the aim of achieving a 95% confidence level and at least 80% statistical power. In this study, p < 0.05 was used as the threshold for statistical significance.Data were collected and analyzed with R (version 3.6.3) using Tidyverse, Final-Fit, MCGV, Survival, Stringdist, Janitor, and Hmisc packages. Results were presented as mean and standard deviation for continuous variables with Gaussian distribution, medians and IQR range for continuous variables with no Gaussian distribution. Frequency and percentages were used for categorical variables. To assess the significance of differences between groups, Students’ The current study was evaluated and approved by the Scientific Research Ethics Committee of Victor Babeș University of Medicine and Pharmacy, Timișoara ).A total of 515 of patients were included in this study with a possible pharyngeal tumor who underwent surgical treatment following diagnosis during the study period. The median age at diagnosis was 55.7 years, with a significant difference in gender distribution: 125 females (24.3%) and 390 males (75.7%).p = 0.001). Also, the urban population was higher with 181.0 (51.7%) malign patients versus 169 (48.3%) patients from rural environments.Data showed that patients with cancer were older, with a mean age of 59.7 (9.8) years versus 47.3 (15.6) years in the benign group  of the total number of patients (513) presented with N0, and 54.0 (15.4%) presented with N1 staging from the malign group. A total of 199 (56.9%) had a malign biopsy, while 146 were benign. Among the 350 patients diagnosed with malignancy, 61 (1.4%) patients presented with N3, 54.0 (15.4%) patients were diagnosed with N1, and 36.0 (10.3%) presented with N2. Hospitalization days ranged from 1 to 37 days, with a mean of 4.9, influenced by emergency enrolment. All results obtained are summarized in A total of 38 different symptoms were reported in patients with benign and malignant tumors. Additionally, 10 symptoms were common to both groups, but with different frequencies. In the malignant tumor group, the most common symptoms were dysphagia (40%), sore throat (39%), dysphonia (15%), and reflex otalgia (12%) a.In the group with benign tumors, the most common symptoms were nasal obstruction (15%), oral respiration (11%), and dysphagia (7%) b.However, because benign and malignant symptoms overlap, clinical investigation must be performed thoroughly and a follow-up employed at all times to accurately rule out a possible malignant diagnosis.Pathology reports revealed malignant disease for 323 patients and benign conditions for 192 patients. The most common malignant pathology was squamous cell carcinoma, of the unkeratinized type, found in 200 patients (57.1%). Keratinized squamous cell carcinoma was identified in 68 patients (19.4%). Other malignant pathologies included rare cancers in 27 patients (7.7%) and non-Hodgkin lymphoma in 24 patients (6.9%). Among the benign conditions, reactive lymphoid hyperplasia was the most prevalent, occurring in 76 patients (46.1%). Other frequent benign pathologies were chronic inflammation in 21 patients (12.7%) and chronic hypertrophic tonsillitis in 15 patients (9.1%). The histopathological results are summarized in p = 0.015). In a multivariable analysis, after adjustment for other variables, this association was no longer significant (p = 0.389). The present study showed that patients presented a grading of dysplasia of G2 ((71.9%), G3 (12.3%), G1 (2.3%), and in situ (2.6%), as shown in p = 0.044). In the multivariable analysis, patients with rhinopharyngeal tumors presented a substantially lower risk of death than those with tumors in the hypopharynx, with an OR of 0.14 (p = 0.129). In the multivariable analysis, patients with acute respiratory failure had a higher risk of mortality, with an OR of 1.90 (p = 0.271). With a p-value of 0.048 in the univariable analysis, however, this association was only close to significance.There is a tendency for mortality rate to rise as N staging increases. Specifically, patients with N3 staging had a 2.87-fold higher risk of mortality compared to patients with N0 staging . If a complete medical examination is not fulfilled, certain symptomatology may be missed; therefore, a delay in the indication for biopsy may cause a delay in diagnosis and treatment. The classic symptoms of HNC depend on both the anatomical site of the primary tumor and the etiology of the tumor. Several aspects of the targeted physical examination raise the possibility of cancer, including i) a 1.5 cm nontender neck mass that is anchored to surrounding tissues and has a skin ulcer over it [Romania has one of the lowest life expectancy rates in the EU and, despite improvements since 2000, it is still more than six years below the EU average. Similar to other developing countries in the EU, Romania needs to make progress in a number of sectors related to public health . Usually over it ; ii) the over it ,20. The  over it . HoweverNodal levels require the most attention during treatment planning and post-treatment surveillance periods. Our hope is that patients, surgeons, and radiation therapists will use these data to decide whether to electively treat the neck in the N0-N1 setting and, if so, where the treatment should be focused. Therefore, a benefit– risk assessment can be made to determine whether the neck should be treated or observed as part of initial cancer therapy. These data demonstrate the feasibility of FDG-PET/CT in guiding the personalization of neck management for cN0-staged HNC , althougTNM/AJCC staging plays an essential role in tumor treatment planning and prognostic assessment. The rate of lymph node and distant metastases of pharyngeal cancer is increased; the rate of lymph node metastasis at first presentation is high with a distribution of N3 17.4%, N2 10.3%, and N1 15.4%, the rate of possible metastatic lymph node presence at first presentation of patients is 33%. The presence of a single metastatic lymph node (LN) commits patients to an advanced-stage disease category and has been shown to confer up to a 50% decrease in overall survival (OS). The control rate for primary and cervical lymph node metastases has gradually improved in patients with squamous cell carcinoma (SCC), showing improved treatments. Therefore, identifying the high-risk patient presenting distant metastasis represents a key factor to ensure effective treatment as soon as possible. The incidence of uncontrolled primary tumor and cervical lymph node metastases may lead to distant metastases during the development of these recurrent tumors ,28.p < 0.001), while we had to perform emergency tracheotomy in 6.3% of cases and total laryngectomy in 0.9% of cases.Even after treatment, 30–60% of patients diagnosed at an advanced stage with successful remission will develop recurrent locoregional cancer or second primary cancer. Therefore, the number of affected lymph nodes of a cancer patient may be a better measure of prognosis ,29. Due Histologically, the progression to mild, moderate, and severe carcinoma in situ and, finally, invasive HNC carcinoma follows an ordered series of steps, beginning with epithelial cell hyperplasia, followed by dysplasia .Depending on the location of the primary tumor, patients with HNC may experience specific symptoms, such as oral dysfunction and swallowing or speech problems, during treatment, which may often improve 6 months post-treatment ,32. The Currently, there is no standard or routine screening test to diagnose oral and pharyngeal cancers, and no screening guidelines have been provided for the early detection of oral and pharyngeal leukoplakia and erythroplakia lesions or cancers in the general population. There is also the problem of the absence of a cancer program separate from the general medical history, especially for cancer patients from private institutions and which should handle the treatment, history, stage of a patient, and tertiary prevention. Nevertheless, for better assessment of the patient, all institutions providing healthcare in the area of cancer must work together to develop a well-organized regimen for better outcomes and easy follow-up of the patient. Clinicians gathering past medical histories should also ask patients about tobacco use and alcohol consumption in order to assess whether smoking cessation intervention and/or counseling (for tobacco) is needed. Future research is needed to understand the risk factors and impact of financial toxicity on HNC patients, as well as the effectiveness of interventions, such as financial navigators, policies addressing drug price transparency, and value-based insurance design, in alleviating the cancer-related financial burden. What we must strive to understand when assessing the influence of financial burden on HNC care is the patient-level impact of perceived financial harm on clinically meaningful outcomes.Though the many strengths of this study, our findings are representative of patients treated in a single healthcare system with similar treatment-related costs and relatively homogenous demographics, which may not be representative or generalizable to all HNC patients. Furthermore, despite our high participation rate, future studies should be multi-institutional in nature, including analyses of additional financial coping mechanisms, and should also consider larger sample sizes to increase the generalizability of findings to allow subgroup analysis in regression modeling. In addition, it should be noted that comorbidities and ethnicity were not available and may represent unidentified sources of confounding.In conclusion, this study highlights the importance of identifying risk factors associated with malignancy and toxicity in patients with head and neck cancer. Early diagnosis and access to multidisciplinary teams are crucial to improving survival rates and health-related QoL. Financial limitation remains a major concern for patients with HNC, and further research is needed to understand the impact and efficacy of interventions. The absence of routine screening tests and screening guidelines for oral and pharyngeal cancers represents a significant barrier to the secondary prevention of HNC. Despite the strengths of this study, larger multi-institutional studies are needed to increase generalizability and account for potential confounders, such as comorbidities and ethnicity. Overall, our findings highlight the need for a well-organized and coordinated approach to HNC care that addresses the various challenges and barriers faced by patients and healthcare providers.


Osteosarcoma is the most prevalent malignant bone tumor. In this study, we identified metastasis-related genes (MRG) that are differentially expressed between primary and metastatic osteosarcoma and employed them to create metastasis-related risk tags (MRSs) for the overall survival of osteosarcoma patients. Using consistent cluster analysis, patients with osteosarcoma in the TARGET database were divided into subgroups with different metastatic scoring patterns. The clinicopathological traits, survival rates, tumor microenvironment traits, immune-related scores, and therapeutic responses of these patients varied. Additionally, we constructed MRS-based risk characteristics and nomographs and developed an MRG Score to improve patient characteristics. Thorough evaluations demonstrated that prognostic models and metastasis scores can distinguish high-risk patients from low-risk individuals, offering excellent predictive value. Finally, western blotting was used to confirm the expression of five identified MRG markers, which are crucial for osteosarcoma invasion and migration in terms of mechanism. Our findings represent a novel and practical predictive biomarker for osteosarcoma. Osteosarcoma (OS) is the most common primary malignant bone cancer. It mostly affects children and teenagers, predominantly males, with an annual incidence rate of one to four cases per million people. OS is highly malignant and tends to progress rapidly . OS treaImmunotherapy is a novel therapeutic approach that has shown promising results in the treatment of various diseases, including hepatocellular carcinoma and breast cancer , 7. The The development of high-throughput next-generation sequencing and gene microarrays has made it possible to analyze gene expression patterns using bioinformatics to investigate possible causes of the disease and prospective treatment targets. Some studies have used bioinformatics to screen biomarkers in patients with OS. To identify the putative pathogenic genes for OS, Cao et al.  thoroughIn this study, we screened 15 metastasis-related signals (MRS) associated with prognosis. Specifically, we investigated the interaction between genes and pharmacology by focusing on IL10RA and TLR7, which are known to suppress the production of TLR7 and have been shown to significantly enhance the migration, invasion, and adhesion of OS cells . HoweverIn the present study, we identified 28 metastasis-related genes (MRGs) of OS based on the target database and constructed a risk tag composed of five MRGs. Additionally, we constructed a nomogram model based on metastasis-related signatures (MRSs) to predict clinical OS. Further research was conducted to determine the connection between MRSs and immune-infiltrating cells. Investigating functional medications and potential pharmaceuticals linked to the metastatic mechanism of OS may aid in the exploration of new therapy options. This was confirmed in the OS GSE39055 and GSE21257 cohorts, and the functions of the MRSs were fully validated. We examined the effects of MRSs on the proliferation, migration, and invasion of OS cells using a functional phenotypic method and investigated the mechanism of action of MRSs in OS metastasis. Our study advances our knowledge of OS metastasis and provides new and efficient therapeutic options.Based on metastatic and non-metastatic patients, we identified 28 DGEs. We found 16 upregulated and 12 downregulated genes . The expTo explore the relationship between MGRs and the prognosis of OS, we performed a survival analysis of each MGR in patients with OS from the TARGET database. High levels of SEMA5A, HILPDA, WIF1, ALPL, CXCL8, LOX, and SEC61G were significantly associated with poor overall survival in OS, whereas ASPA, RBM24, GPR174, NLGN1, SLFN13, IGF2, and PDLIM3 showed the opposite trend . The intFirst, based on the 28 MGRs, we conducted univariate Cox and LASSO region analyses to identify five risk genes , 3B. TheAccording to Kaplan-Meier analysis, the OS of the low-risk group was considerably superior to that of the high-risk group . The ROCAdditionally, more individuals had metastatic disease in the high-risk group than in the low-risk group . UnivariThe association between the risk score and the number of immune cells was assessed using the CIBERSORT method. The low-risk group exhibited greater immune cell infiltration than the high-risk group . AdditioBased on the mRNA expression levels of the 28 MGRs, OS samples in the TARGET database were clustered using unsupervised non-negative matrix decomposition. The best k value was determined using molecular clustering and comprehensive correlation analysis. We examined the k values between 2 and 9. For clustering and overall survival analysis, k = 3 was the most appropriate of all possible values , 5B. CluGene clusters A and B were created based on the DEGs between the risk categories . AccordiThe MRGs score and the distribution and survival of the gene and MRGs clusters are represented in the Sanggi diagram . In addiWe used two external queues to evaluate the reliability and stability of five MRS predictions. The risk score for each patient on these lines was determined using the same method. The patients were then divided into high- and low-risk groups according to the previously set median of the training queue. The survival rates of the two patient groups , 8D and We used qRT-PCR and western blotting to study the expression of HILPDA, LOX, NLGN1, SEMA5A, and WIF1 in MG63 cells. Compared to hFOB1.19, HILPDA, LOX, SEMA5A, and WIF1 showed increased mRNA and protein expression in MG63 cells, whereas NLGN1 showed the opposite trend , 9B.The scratch test was used to analyze the migration of MG63 cells. Compared to the NC group, the migration area of the si-HILPDA, si-LOX, si-SEMA5A, si-WIF1, and oe-NLGN1 groups decreased significantly, indicating that the low expression of HILPDA, LOX, SEMA5A, WIF1, and other genes and the overexpression of NLGN1 effectively inhibited the migration ability of MG63 cells .We used a Transwell assay to evaluate the invasive ability of MG63 cells. Compared to the NC group, the number of MG63 cells passing through the chamber in the si-HELPDA, si-LOX, si-SEMA5A, si-WIF1, and oe-NLGN1 groups decreased significantly, indicating that the low expression of HILPDA, LOX, SEMA5A, WIF1, and other genes and the overexpression of NLGN1 can effectively inhibit the invasiveness of MG63 cells .OS is a bone tumor known for its high metastatic potential, with the lungs being the most common site of metastasis , 14. At We used the TARGET and GEO databases to explore the characteristics of osteosarcoma (OS) and the tumor immune microenvironment (TIME), with a focus on OS metastasis and patient prognosis. Despite recent research on OS metastasis, there are currently few biomarkers available for early prediction. Therefore, it is crucial to gain a thorough understanding of the molecular process of metastasis and to discover efficient, sensitive, and specific molecular markers. For this study, we gathered information from the aforementioned databases. GO analysis revealed the wide heterogeneity of OS metastatic capacity, highlighting the need for further investigation of the molecular determinants of metastasis. To assist medical professionals in creating more successful immunotherapy regimens, we propose a metastasis score system to assess individual metastases and better understand the TME.Firstly, we discussed the impact of 28 MRGs on the survival of osteosarcoma (OS) patients. To analyze the mRNA expression profiles of these MRGs, we employed unsupervised clustering approaches. Our findings demonstrated that the survival outcomes of OS patients varied significantly between the two groups, based on the mRNA expression profile of MRGs. Furthermore, the role of immune-related characteristics in the development and management of cancer, as well as their significance in clinical outcomes and survival, cannot be overlooked. Our comprehensive immune analysis showed remarkable consistency across various analyses, including risk models, metastasis score, gene cluster, and MRGs cluster. The findings indicated that TME infiltration was higher in the groups with good prognosis. However, research on the immune correlation of metastasis score remains limited. Hence, further studies are required to shed light on this aspect of the disease.Secondly, we conducted corresponding experiments to validate our analysis. The results of qRT-PCR and western blot revealed that the mRNA and protein levels of HILPDA, LOX, SEMA5A, and WIF1 were increased in MG63 cells, whereas NLGN1 exhibited the opposite pattern. Moreover, the scratch test and Transwell test findings showed that overexpression of NLGN1 or knockdown of HILPDA, LOX, SEMA5A, or WIF1 effectively inhibited the migration and invasion of MG63 cells. By unraveling the process of OS proliferation and metastasis, it becomes possible to identify biomarkers for early diagnosis and assess the incidence and spread of the disease. Despite its significance, this study has some limitations that should be acknowledged. Firstly, we did not investigate the effect of metastasis score on osteosarcoma. Moreover, there is a need for further research to comprehensively understand the relationship between metastasis score and the immune microenvironment. Therefore, we urge for more in-depth studies on metastasis score in future osteosarcoma research. It is essential to identify the limitations of any study to provide a comprehensive understanding of the research and to lay the groundwork for future studies.The OS database was obtained from the TARGET and GEO databases. The TARGET database contained 88 tumor samples, GSE21257 contained 53 samples, and GSE39055 contained 37 samples. The TARGET database was used for the training set, and GSE21257 and GSE39055 datasets were used for the verification set.2 (fold change) >1 or Log2 (fold change) < −1“. We investigated the upregulated genes and conducted a functional enrichment analysis of the data to further validate the possible functions of the potential targets. The “ClusterProfiler” package in R was used to examine the GO function of prospective mRNA and enhance the KEGG pathway in order to better comprehend the carcinogenesis of the target gene.The differential expression of mRNA was examined using the “limma” software package of R (version: 3.4.1), which was based on the initial count of the TARGET dataset and the associated clinical data. The screening of threshold mRNA differential expression was specified as “adjusted p = 0.05 and LogConsensus unsupervised clustering analysis was carried out using the R package “CONSENSUSClusterPlus” based on the expression profiles of 28 MRGs. OS patients in the TARGET database were divided into three molecular subgroups of MRGs: clusters A, B, and C.We evaluated the correlation among OS clustering, clinicopathological features, and survival outcomes to assess the clinical importance of various clustering approaches. The Kaplan-Meier technique was used to compare the overall survival rates.The weighting coefficient was determined using least absolute contraction and selection operator (LASSO) Cox regression analysis, and the risk model was created in R using the glmnet package. Based on the best critical value of the risk score, patients were separated into high- and low-risk groups, and the Kaplan-Meier technique was used to determine the survival rate of the patients. To define the ROC curve and obtain the AUC for survival analysis, use the “Survival ROC” tool in R. Finally, a Norman Chart was updated to include clinical and independent prognostic variables.To find DEGs between various risk score groups, R’s “limma” package is utilized. Genes were deemed to be expressed differently when their adjusted p-values were 0.05, and | log2 (FC) | was greater than 1.0. Finally, three DEGs were predicted to be prognostic genes and analyzed. The “clusterProfiler”, “enrichplot”, and “ggplot2” packages of R were used for the enrichment analysis of MRS-related differential genes in OS. The screening criteria were p<0.01 and q<0.05.Based on the three prognostic DGEs, we calculated the MRG scores for each patient with OS using PCA. Subsequently, the MRGs gene score was established using PCA, and principal components 1 and 2 were extracted as feature scores. The score formula was as follows: The MRGs score = Σ (PC1i+PC2i).The tumor immune estimation resource (TIMER) was utilized to evaluate the level of immune cell infiltration between different clusters using the Estimation of Stromal and Immune cells in malignant tumor tissues using Expression data (ESTIMATE) method. Single-sample gene set enrichment analysis (ssGSEA) was performed to determine the relative abundance and activity levels of each immune cell type in OS, followed by CIBERSORT analysis to assess the fraction of invading immune cells in OS patients with specific expression patterns, in order to explore differences between immune cell subtypes.The immune checkpoint inhibitor response to a single sample or subtype was predicted using the Tumor Immune Dysfunction and Exclusion (TIDE) algorithm . The medBiomics Biotech created and manufactured si-HILPDA, si-LOX, si-SEMA5A, si-WIF, and their respective negative controls (NCs) . Their sequences were listed in Total RNA from hFOB1.19 and MG63 cells should be extracted using TRIzol (Invitrogen), and 2ug of total RNA should be reverse-transcribed into cDNA using the TransScript One Step gDNA Removal and cDNA Synthesis SuperMix kit . The ABI Prism 7500 rapid real-time PCR equipment and TransStart Green qPCR SuperMix kit  were used to measure the expression levels of HILPDA, LOX, NLGN1, SEMA5A, and WIF1 . Actin was used as the internal reference for mRNA at the same time. Shanghai Sangong Biotechnology Co., Ltd. created all the primers . The primer sequences used in the experiments are listed in Radioimmunoprecipitation (RIPA) lysis buffer was used to fully lyse the cells, and the resulting protein samples were collected, separated by electrophoresis, and transferred onto PVDF membrane . The PVDF membrane was then sealed with a fast-sealing solution and incubated with primary antibodies against HILPDA , LOX , NLGN1, SEMA5A , WIF1 , and GAPDH at 4° C. The corresponding secondary antibodies were incubated at room temperature for 1.5 h. Finally, the protein bands were visualized using an ECL kit.The scratch test was used to analyze the migratory ability of MG63 cells. After the cells were digested, the seeds were spread onto a well plate. After the cells were digested, the seeds were spread onto a well plate. When the degree of cell fusion reached 95%, a sterile plastic tip was used to scratch the cells. After PBS washing (1-2 times), the cells were cultured in serum-free medium for 24 h. The test results were analyzed by Image J, and the migration areas of the cells in each group were compared.The invasive ability of the MG63 cells was analyzed using a cross-pore chamber. According to 1 × 106 cells/ml, 200ul ratio was plated in a Transwell chamber . The same cells or 500ul MEM medium containing 10% fetal bovine serum were used in the inferior chamber and incubated for 24 h. The cells were fixed with 4% paraformaldehyde for 30 min, and 0.5% crystal violet dye was used for 20–30 min. The cells in the upper chamber were gently wiped with a cotton ball and the migrated cells were photographed under a high-magnification field of view using an inverted microscope (×200).All statistical calculations were carried out using R 3.4.1. One-way ANOVA was used to ascertain statistical significance in the experimental data, which are presented as the mean standard deviation. Statistical significance was set at a bilateral p value of 0.05.The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed at the corresponding author.All authors have agreed on the contents of the manuscript.Supplementary Figure 1Supplementary Table 1


Body mass index (BMI) , physical activity , serum calcium levels , hypertension , and neuropathy  were significantly associated with sarcopenia following multivariate regression analysis. (4) Conclusions: T2DM individuals are prone to sarcopenia, especially those with a low BMI, low physical activity, hypertension, neuropathy, and low serum calcium levels. Hence, by modifying these risk factors among the elderly T2DM, sarcopenia can be prevented.(1) Background: Sarcopenia has gained much interest in recent years due to an increase in morbidity. Sarcopenia is associated with type 2 diabetes mellitus (T2DM) and vice versa. There is a paucity of information regarding the prevalence and predictors of sarcopenia among T2DM individuals. The aim of the present study was to determine the prevalence and predictors of sarcopenia among T2DM individuals. (2) Methods: This study included 159 diabetics (cases) and 79 non-diabetics (controls) aged >50 years. The subjects were assessed for demographic and anthropometric parameters. Sarcopenia (according to the Asian Working Group for Sarcopenia 2019 criteria) was assessed using Jammer’s hydraulic dynamometer for handgrip strength, dual-energy X-ray absorptiometry for muscle mass, and 6m gait speed. The biochemical investigations included glycated hemoglobin; fasting and prandial glucose; fasting insulin; lipid, renal, liver, and thyroid profiles; serum calcium; phosphorous; vitamin D; and parathyroid hormone (PTH). Appropriate statistical methods were used to determine the significance of each parameter, and a multivariate regression analysis was applied to determine the predictors. (3) Results: The prevalence of sarcopenia was significantly higher among the cases than the controls (22.5% vs. 8.86%,  Sarcopenia is defined as a loss of muscle mass, muscle strength, and performance . Of thesThe prevalence of sarcopenia varies widely across the world due to the different age groups studied, different ethnicities, associated comorbidities, and different diagnostic criteria employed. The prevalence among community-dwelling elderly individuals ranges between 9.9 to 40% ,7, whereSarcopenia is associated with increased morbidity and mortality due to physical disability, falls, fractures, poor quality of life, depression, and hospitalization . The cauApart from age, multiple factors are involved in the causation of sarcopenia among individuals with diabetes. These include HbA1c levels, degree of insulin resistance; microvascular complications, particularly neuropathy; and macrovascular complications . Other fThe diagnostic criteria for sarcopenia have been evolving over the past few years. Various working groups  on sarcopenia have distinct cutoffs for the parameters. The three components defining sarcopenia include muscle mass, muscle strength, and functioning, while a few other criteria use only two components. A comparison of the various diagnostic criteria is summarized in Various studies from different parts of the world have used different criteria to define sarcopenia, and it had many cutoffs based on race and gender. Only a few Indian studies have been conducted on sarcopenia, taking subjects of variable ages with or without T2DM. Most Indian studies have used the European criteria to define sarcopenia.These European cutoffs are higher than the Asian criteria cutoffs, thus leading to an overestimation of the prevalence of sarcopenia among Indians. In addition, the methods used to assess the components of sarcopenia were different amongst various studies. There is a paucity of studies from the Indian subcontinent on elderly T2DM investigating sarcopenia using the Asian criteria and standard methods for the assessment of the components of sarcopenia. Hence, the present cross-sectional observational study was conducted among elderly T2 DM patients using standard methods to assess sarcopenia and covering all the three aspects of sarcopenia. We assessed the prevalence using the AWGS 2019 criteria and the predictors of sarcopenia among elderly T2DM.2/min (stages 4 and 5), (iii) chronic liver disease, (iv) heart failure, (v) malabsorption, (vi) malignancies, (vii) autoimmune disorders, (viii) inflammatory diseases, (ix) stroke, (x) severe hip or knee osteoarthritis (xi) cognitive impairment (xii) amputations (xiii) spinal cord injuries, (xiv) acute and chronic ongoing infections, (xv) myopathies, (xvi) BMI ≥ 40 or ≤18 kg/m2, (xvii) those on hormonal or nutritional supplements with high protein content, (xviii) illicit drug users, (xix) athletes, and (xx) non-consenting individuals, were excluded from the study. Both the case and control groups were subjected to clinical evaluation and detailed history taking. The sample size was calculated based on the prevalence from the previous Indian study and using the formula n = 4pq/d2. Permission from the ethics committee of our institution was obtained prior to the beginning of the recruitment, and informed consent was obtained from all the participants.The present cross-sectional study included individuals with T2DM aged ≥50 years as cases and age, sex, body mass index (BMI) matched healthy non-diabetic individuals as controls. The source of the samples was the outpatient Department of Endocrinology and Metabolism of our institution. Individuals with (i) secondary and type 1 diabetes, (ii) chronic kidney disease with an estimated glomerular filtration rate (eGFR) ≤ 30 mLmEach patient was asked in a detailed interview about their age, sex, duration of diabetes, past medical illness, physical activity [using the International Physical Activity Questionnaire—Short Form (IPAQ-SF) score ], smokin2 (in kg/m2). The cutoff values of <7.0 kg/m2 in men and <5.4 kg/m2 in women were taken as low muscle mass. Handgrip strength (HGS) was measured using JAMAR’s hydraulic handheld dynamometer. The dominant hand was tested. The subjects were asked to relax for 5 min and tested in a sitting position with their shoulders adducted and neutrally rotated, elbow flexed at 90°, forearm in a neutral position, and wrist slightly dorsiflexed and deviated in the ulnar direction. The subjects were asked to squeeze as hard as possible in one go and then relax. The scores of three successive trials were recorded, and an average was taken. Cutoff values of <28 kg for men and <18 kg for women were taken to describe decreased muscle strength. For gait speed, the participants were asked to stand still behind the start line and asked to walk at their normal pace until they were beyond the finish line. The walkway distance was 6 m. Timing started when the participant stepped on the start line and stopped as soon as they crossed the stop line. Their speed was calculated as distance/time in seconds. A cutoff value of <1 m/s was taken to define a decreased gait speed.Sarcopenia was evaluated using the Asian Working Group for Sarcopenia Criteria 2019 [Various biochemical parameters were assessed. Fasting plasma glucose (FPG), postprandial plasma glucose (PPG), fasting lipid profile, serum calcium, serum phosphorous, serum albumin, serum alkaline phosphatase and serum creatinine, thyroid stimulating hormone (TSH) were performed by an automated analyzer. Glycated haemoglobin (HbA1c) was measured using high performance liquid chromatography as per the norms of National Glycohemoglobin Standardization Program. Sample for the serum intact parathyroid hormone (iPTH) was collected in fasting and transported in a cold chain, centrifuged and stored at −80 °C till processed and measured using electrochemiluminescence immunoassay (ECLIA). Serum 25-hydroxy vitamin D (25-OH-Vit D) and fasting insulin were measured using enzyme linked immunosorbent assay (ELISA). Insulin resistance was calculated using homeostatic model assessment for insulin resistance (HOMA-IR). p < 0.05 was considered statistically significant for all statistical tests. To study the association between various factors and sarcopenia, univariate logistic regression analysis was used first, then a multivariate logistic regression analysis was performed. All the data analyses were carried out using the statistical product services solution IBM SPSS version 29.0. Tests of the normality assumption of continuous data were performed using an appropriate statistical test. For normally distributed continuous variables, descriptive statistics such as mean, standard deviation, and range values were calculated. Comparisons between two group means were tested using a Student’s independent t-test. For non-normal data, the median values were calculated. The median values were compared using the nonparametric Mann–Whitney U-test. Regarding categorical variables, the data were presented as frequency and percent values. Frequency data across categories were compared using a Chi-square/Fisher’s exact test as appropriate. A two-sided probability of The present study included 159 individuals with T2DM as cases and 79 non-diabetic individuals as controls. The controls were age, sex, and BMImatched to the cases. The mean age of our study population was 57.4 ± 6.04 for the cases and 56 ± 5.82 for the control group. The sex distribution, male to female ratio, was 1.06 for the cases and 1.024 for the controls and mean BMI of the cases and controls were 27.4 and 26.9, respectively. The rest of the demographic parameters of the cases and controls are shown in The prevalence of sarcopenia among the individuals with T2DM was 22%, which was significantly higher compared to 8.8% among the individuals without T2DM. According to the 2014 AWGS criteria, the prevalence of sarcopenia was 17.6%, which was less compared to that of 2019 criteria, which was 22%. However, this was not statistically significant. The comparison of cutoffs of the parameters of sarcopenia is shown in All the characteristics of the diabetic individuals, including the medications used and microvascular complications, are summarized in The mean appendicular mass index (AMI), handgrip strength (HGS), and gait speed (GS) were less in females than the males, which was expected and is shown in A univariate regression analysis revealed a significant association between sarcopenia and age, alcohol intake, physical activity, BMI, waist circumference, hypertension, insulin usage, sulphonylureas, neuropathy, nephropathy, retinopathy, triglyceride levels, VLDL (very low density lipoprotein) levels, eGFR, UACR, serum calcium, and parathyroid hormone (iPTH), as shown in 2 (p—0.005). Of the microvascular complications, only neuropathy showed a significant positive association, with an odds ratio of 5.57 (CI of 1.258–24.66). Amongst all the biochemical parameters, only serum calcium was significantly negatively associated with sarcopenia, with an odds ratio of 0.155 (CI of 0.035–0.687), implying that the lower the serum calcium levels, the higher the risk of sarcopenia.After applying the multivariate regression analysis shown in , we foun2 . HyperteOther parameters like age, alcohol intake, insulin and sulphonylurea use, waist circumference, serum triglycerides, eGFR, urine ACR, serum iPTH, nephropathy, and retinopathy showed a significant association based on the univariate analysis; however, they were insignificant after the multivariate regression analysis. Lower physical activity, lower BMI, the presence of hypertension, presence of neuropathy, and low serum calcium levels were significant predictors of sarcopenia among the diabetics.To the best of our knowledge, this was the first study using AWGS 2019 criteria among elderly T2DM individuals in India. The prevalence of sarcopenia according to this criterion was 22% among elderly T2DM individuals compared to 8.8% among the age-, sex-, and BMI-matched healthy controls. This indicates that diabetes itself is an independent risk factor for sarcopenia. Earlier studies that followed the AWGS 2014 criteria reported a prevalence of 28.5% and 27.4%, which were slightly higher than in our study ,24. ThisWe also compared the 2014 and 2019 AWGS criteria cutoffs and found the prevalence of sarcopenia to be 17.6% and 22% using the 2014 and 2019 criteria, respectively. The absolute prevalence of low AMI was equal due to the same cutoffs in both criteria, whereas the absolute prevalence of low HGS and low GS was higher with the 2019 criteria, of which the prevalence of low GS was statistically significant. This was due to a change in the cutoff of HGS from 26 to 28 kgs for males and GS from 0.8m/s to 1m/s for both the sexes. The mean AMI in diabetic males and females was 7.75 and 6.26, respectively, which was similar  to an earlier report by Zengin et.al  among olDemographic characteristics like age, gender, and duration of diabetes did not show a significant association with sarcopenia. These findings were similar to reports by other researchers . NeitherThe duration of diabetes did not show any significant association with sarcopenia. This was contrary to earlier findings reported by Sazlina et al., who showed a significant association, with an odds ratio of 1.85 with a duration >10 years . This diIn the present study, physical activity had a significant association with sarcopenia, showing that moderate physical activity decreases the risk of sarcopenia (odds ratio of 0.045), which was persistently seen after multivariate logistic regression, also with an odds ratio of 0.45. This means that with regular physical activity, the risk of sarcopenia was decreased by 45%. This finding was similar to earlier published studies ,23. As pIn the present study, BMI and waist circumference had a significant negative association with sarcopenia. Having a BMI within the overweight or obese ranges resulted in an odds ratio of 0.154 or 0.041, respectively. Waist circumference showed an odds ratio of 0.147. This is in agreement with the study by Sazlina et al., who reported odds ratios of 0.16 and 0.03 for the overweight and obese ranges, respectively . FollowiThe presence of hypertension was significantly associated with sarcopenia, with an odds ratio of 8.7; that is, individuals with T2DM and hypertension had an 8–9 times higher risk of sarcopenia. This could be due to the synergistic effect of both diabetes and hypertension on muscle health. This finding was different from previous published studies ,26. SeveAmong the various antidiabetic agents, we found a positive association between insulin use and sarcopenia and a negative association between sulphonylurea use and sarcopenia. On the contrary, based on multivariate logistic regression analyses, there was no significant association. None of the other antidiabetic agents, including metformin, voglibose, DPP-4 inhibitors, and SGLT-2 inhibitors, had any significant association with sarcopenia. These findings are similar to earlier findings by Sazlina et al. . HoweverAll three microvascular complications showed a significant positive association with sarcopenia, with odds ratios of 4.13, 3.93, and 3.68 for neuropathy, nephropathy, and retinopathy, respectively. After a multivariate analysis, only neuropathy showed a significant association with sarcopenia, with an odds of 5.57. A recent study among T2DM individuals >50 years showed that neuropathy was associated with decreased muscle strength in the lower extremities . This waGlycemic parameters such as fasting, postprandial blood glucose, and glycated hemoglobin showed no significant association with sarcopenia. These findings are similar to earlier studies that assessed HbA1c ,39. SugiFasting insulin and HOMA-IR (insulin resistance index) were not associated with sarcopenia. Insulin sensitivity as a predictor of sarcopenia is a less well-studied entity. The available literature shows variable results. Lee CG et al. studied non-diabetic individuals and reported that the highest quartile of insulin resistance increased the odds of losing lean mass . On the Among the serum lipids, triglycerides and VLDL showed a significant negative association based on a univariate analysis, which was not evident based on a multivariate regression analysis. Thus, in our study, none of the serum lipid levels showed a significant association with sarcopenia. This is similar to earlier research findings , whereasSerum albumin levels did not have a significant association with sarcopenia. This parameter has not been well-studied in earlier research studies. Sugimoto et al. reported significantly low serum albumin levels in their sarcopenic group compared to their non-sarcopenic group . Renal pAmong the bone mineral metabolic parameters, serum calcium showed a significant negative correlation, and serum intact PTH showed a significant positive correlation with sarcopenia. Serum phosphorous and 25(OH)vitamin D had no significant correlation with sarcopenia, which was similar to an earlier study that showed no association with serum vitamin D levels . HoweverCalcium plays a vital role in muscle contraction, as it facilitates the effective interaction between actin and myosin muscle fibers. In striated muscle, calcium binds to troponin c on actin filaments, causing a shift in the position of troponin–tropomyosin complexes, resulting in exposure of the myosin binding sites. This allows myosin bound by ADP and inorganic phosphate (Pi) to form cross bridges with actin. The subsequent release of ADP and Pi generates the power stroke that drives muscle contraction . ChangesIn the present study, neither the duration of diabetes, the glycemic control parameters, nor the use of diabetes medications influenced the risk of sarcopenia. However, these parameters should be studied in a longitudinal design to assess their causal role in sarcopenia. The present study provides a few recommendations by which sarcopenia can be prevented to some extent by simple, feasible lifestyle modifications. A particular emphasis should be given by both clinicians and national policy makers on the importance of physical activity, calcium intake, adequate control of hypertension, and maintaining a healthy BMI to prevent sarcopenia.The strengths of the present study were that it included healthy age-, sex-, and BMI-matched controls, the use of the dual X-ray absorptiometry for appendicular mass index, Jammar’s hand-held dynamometer for handgrip strength, an assessment of the effects of antidiabetic medication, and assessment of all possible biochemical parameters that are responsible for muscle health. The present study, despite being done very meticulously using standard criteria and methods to assess sarcopenia, is not completely devoid of limitations. First, this was a cross-sectional study, which does not allow for the evaluation of causal relationships. Second, the prevalence in the study could be underestimated because we excluded all the patients with other chronic systemic illnesses who are usually more prone to sarcopenia. Third, protein intake was not included because the precise amount could not be elucidated; however, the nutritional status was also reflected by the serum albumin levels, which were assessed in our study. Fourth, the results of our study may not be applicable to the general population because the study population was taken form a single tertiary care center. Hence, large population-based and long-term follow-up studies are warranted to precisely determine the risk factors. In the future, interventional trials aimed at treating sarcopenia should be conducted. The modifiable risk factors for T2DM may help for achieving better outcomes. It should not be forgotten that individuals with T2DM are more prone to sarcopenia. These include individuals with hypertension, a low BMI, less physical activity, neuropathy, and low serum calcium levels, who are at high risk and should be screened for sarcopenia. Every individual with T2DM should be educated about these modifiable risk factors and should be encouraged to maintain a healthy BMI; regular physical activity, especially muscle strengthening exercises; control of hypertension; and adequate calcium intake to prevent sarcopenia. Further longitudinal studies are warranted to assess the causal relationships between various factors and sarcopenia.


Only one fetus was positive in the liver and kidney. Leptospirosis would have been misdiagnosed if only kidney/liver samples had been tested. Therefore, we recommend the investigation of multiple organs, beyond the liver and kidneys, especially in fetuses with anicteric lesions.Abortion is one of the main signs of bovine leptospirosis. The necropsy of the fetuses followed by molecular analysis of the tissues is recommended for the diagnosis. In general, the target sites for diagnosis are the kidneys and liver. However, due to the high diversity of leptospiral strains and anatomic lesions caused by those, this may not be valid for the major agents of bovine leptospirosis. In this context, this study aimed to analyze unconventional sites for the presence of leptospiral DNA in bovine anicteric aborted fetuses. Five anicteric fetuses of a dairy herd with seroreactive cows were submitted for necropsy. After conventional PCR of the lipL32-PCR was performed in the fetuses’ kidneys, livers, lungs, hearts, spleens, subcapsular kidney content, abomasal fluid, and in the cavity’s hemorrhagic contents. To complete the investigation, the sera of 30 cows of the herd were collected to perform the serologic screening by Microscopic Agglutination Test. In addition, six subfertile non-pregnant cows from the same herd were selected due to their low reproductive performance, and genital samples  and urine were collected for lipL32-PCR. PCR-positive samples were submitted to a nested PCR of the secY gene and intended for sequencing. Results: The herd presented seroreactive animals , all against the Sejroe serogroup, with titers between 200 and 1600. In necropsy, four fetuses showed hemorrhagic and anicteric lesions, while one fetus had no macroscopic lesions. Regarding molecular analysis, all the fetuses were positive in lipL32-PCR and the positive sites were the heart, lungs, subcapsular kidney content, thymus, kidneys, liver, and abomasal fluid. Only one fetus presented positive results in the kidney and liver, while three fetuses were positive in the abomasal fluid. Five of six cows were positive for lipL32-PCR, all being positive only in genital samples. Of the fetuses and the cows, seven sequences were obtained and all were identified as Leptospira interrogans serogroup Sejroe serovar Hardjoprajitno. Conclusions: In order to improve the diagnosis of leptospirosis in cows, it is recommended to perform a comprehensive analysis of the samples, beyond the kidneys and liver. Thus, we highly encourage testing multiple organs by PCR to investigate abortions suspected of bovine leptospirosis, particularly in anicteric fetuses.Background: Bovine leptospirosis is an important reproductive disease and abortion is a major sign, leading to economic impacts. Due to its multifactorial etiology, the proper diagnosis of the cause of the abortion is crucial. Necropsy of the fetuses followed by molecular analysis is recommended for diagnosis, and the investigation mainly occurs in the kidneys and liver. This study aimed to analyze unconventional sites for the presence of leptospiral DNA in bovine anicteric aborted fetuses. Methods: Five fetuses of the same herd were received for necropsy and diagnosis. Conventional  Leptospira sp. genus, which infects humans, domestic and wild animals [Leptospirosis is a zoonosis caused by spirochetes of the  animals . In bovi animals ,3. Lepto animals ,5. Due t animals ,7. In th animals ,8. On th animals . Inciden animals ,9,10. Wh animals .Abortions are a major clinical sign in bovines ,13, but The exact mechanism of the pathogenesis of leptospiral abortion is yet unclear, but it is suggested that leptospires pass through the placenta during maternal leptospiremia, causing fetal infection that often results in death ,18,19. ISince different pathogens can lead to abortions in cattle, the proper diagnosis is crucial to implement control measures. The necropsy of aborted fetuses followed by molecular analysis of its tissues and placenta is highly encouraged for the diagnosis of animal leptospirosis ,22,23. DThe fetuses were received in the Laboratory of Veterinary Bacteriology of Federal Fluminense University for diagnosis. The further procedures for investigation that involved cows were approved by the Ethical Committee and Animal Use of Federal Fluminense University under protocol 9527220222.The herd consisted of dairy production, composed of 91 Holstein lactating cows farmed in an intensive production system (compost barn), located in the Zona da Mata region, of Minas Gerais State, Brazil. The herd is certified as brucellosis-free and the cows had never been vaccinated against leptospirosis. The herd presented constant abortions in the last six months and poor reproductive parameters, including many cows with a history of embryonic mortality and consequent estrus repetition. Five fetuses with development ranging from five to nine months were aborted by different cows in six months. The fetuses were kept frozen until necropsy and analysis. The necropsy and macroscopic evaluation were performed, and the organs collected were the lungs, heart, liver, spleen, kidneys, thymus, and uterus. Approximately 2 g of each tissue was deposited in sterile tubes. Abomasal contents, hemorrhagic liquids, and subcapsular kidney content were collected by aspiration with a syringe of 5 mL. All samples were immediately frozen at −20 °C until the DNA extraction. ®, Botucatu, SP, Brazil). Uterine biopsy was conducted with a Yeoman forceps of 55 cm  in a transcervical approach, while the forceps was guided by transrectal palpation. The obtained fragment was of approximately five millimeters and was deposited in sterile tubes and kept frozen (−20 °C) until molecular analysis.After the abortions and necropsies, our team went to the herd to collect new samples for additional tests. Except for the mother of fetus B, the other cows were culled before our visit. Six cows with reproductive failures such as embryonic mortality and a history of previous abortion and stillbirths were selected for further molecular analysis, as well as the fetus B’s mother, and five other subfertile non-pregnant cows. From those, cervicovaginal mucus (CVM) and uterine fragments were collected ,25. BrieBesides the six cows selected for the genital samples collection, other 24 cows of the herd were randomly selected for blood collection, in order to complete the sampling of 30 animals for serology. Blood samples were obtained with venopunction of the coccygeal vein and its serum was immediately separated and stored frozen (−20 °C) until analysis.Leptospira using an antigen panel composed of 11 reference strains: Leptospira interrogans serovars Copenhageni (strain M20), Hardjoprajitno (strain OMS), Pomona (strain Pomona), Icterohaemorrhagiae (strain Verdun), Canicola (strain Hond Utrecht), Bratislava (strain Jez Bratislava), Australis , Autumnalis (strain Akiyami), Grippotyphosa (strain Moskva V), Leptospira borgpetersenii serovar Hardjobovis (strain Sponselee), all originated from Institut Pasteur, Paris, France; and Leptospira santarosai serovar Guaricura (strain FV52), a local Sejroe strain of bovine origin, from Federal Fluminense University, Rio de Janeiro, Brazil. Those strains were selected for the antigen panel because they are known to be predominant in cattle in southeast Brazil [For the serology screening of the herd, the serum samples from 30 cows were analyzed by Microscopic Agglutination Test (MAT). The serum samples were tested for antibodies against t Brazil  and reprt Brazil . A twofot Brazil .lipL32 gene, exclusive of pathogenic leptospires [2, and 1.25 μL dNTP, 1.25 μL Buffer, in a final volume of 12.5 μL. The reaction was conducted with one cycle of initial denaturation at 94 °C for two minutes, followed by 40 cycles of denaturation at 94 °C for 30 s, annealing the primers to 54 °C for 30 s, and one-minute extension at 72 °C and final extension cycle at 72 °C for five minutes [L. interrogans serovar Copenhageni (Fiocruz L1-130) were used as reaction negative and positive control, respectively. After amplification, lipL32-PCR products were submitted to electrophoresis in a 2% agarose gel and visualized under UV light after gel red staining. The DNA extraction from the samples was performed with DNeasy Blood and Tissue Kit  following the manufacturer’s recommendations. Molecular analysis proceeded by conventional PCR, targeting the tospires . The PCR minutes . UltrapulipL32-PCR were submitted to a nested PCR of the secY gene [2, and 1.5 μL dNTP, 2.5 μL Buffer, in a final volume of 25 μL. The reaction was conducted with one cycle of initial denaturation at 94 °C for five minutes, followed by 40 cycles of denaturation at 94 °C for 30 s, annealing the primers to 54 °C for 30 s, and one-minute extension at 72 °C and final extension cycle at 72 °C for five minutes. The second reaction was performed with the primers secY_inner_F (5′-CCTCAGACGATTATTCAATGGTTATC-3′) and secY_inner_R (5′-AGAAGAGAAGTTCCACCGAATG3′). Those primers were used in a concentration of 1 μL, and the other concentrations of the reagents were 0.3 μL Taq polymerase, 3 μL MgCl2, and 2 μL dNTP, 5 μL Buffer, in a final volume of 50 μL. The reaction was conducted with one cycle of initial denaturation at 94 °C for five minutes, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing the primers to 55 °C for 30 s, and one-minute extension at 72 °C and final extension cycle at 72 °C for five minutes. The obtained amplicons were purified using the Wizard® SV Gel Kit and PCR Clean-Up System , following the manufacturer’s instructions, and were submitted to sequencing. Sequencing reactions were performed using the Big Dye Terminator v. 3.1 Cycle Sequencing Kit  on a 3100 automatic DNA sequencer according to the manufacturer’s protocol. The sequences analysis was edited and analyzed by Pairwise/Blast/NCBI software, SeqMan v. 7.0, ClustalW v. 1.35 [Leptospira interrogans, Leptospira borgpetersenii, Leptospira santarosai, Leptospira noguchii, and Leptospira biflexa serovar Patoc.The positive samples in ecY gene , initial v. 1.35 , as it wLeptospira antibodies titers of 200, three presented titers of 400, one of 800, and three cows had titers of 1600. Only one cow  was seroreactive to a non-Sejroe, presenting a titer of 200 against Icterohaemorrhagiae. A total of 11/30 (36.6%) cows were seroreactive and ten  were against the Sejroe serogroup, with titers ranging from 200 up to 1600. Of the 10 positive cows for Sejroe, four presented anti-Of the 30 cows analyzed in the herd screening serology, six were selected for molecular diagnosis of their CVM. Of those, four cows were seroreactive, three positives to Sejroe serogroup , and one positive to Icterohaemorrhagiae serogroup (titer of 200).Fetus A had five months of development and presented subcutaneous edema and serosanguineous content on the thorax and abdomen. Lungs presented hemorrhagic suffusions, the liver was enlarged with dark areas and the abomasum had bloody content. The right kidney was friable, with the presence of subcapsular liquid. Fetus B had six months of development and presented hemorrhagic suffusions in the lungs and serosanguineous content in the thoracic cavity. Fetus C had approximately eight months of development and presented only mild hemorrhagic lesions in the lungs. Fetus D had nine months and showed the presence of serosanguineous content in the thoracic cavity, hemorrhagic suffusions in the left lung lobes, and the heart’s left auricle. The liver presented slightly coppery areas. Although the kidneys had normal characteristics, hemorrhagic subcapsular content was present. Fetus E had nine months and did not present any macroscopic alterations in the organs. None of the fetuses presented jaundice or icteric lesions. lipL32-PCR showed the presence of leptospiral DNA in different organs . The t organs . Fetus AlipL32-PCR (L. interrogans with an identity > 99% with strains from serogroup Sejroe (Genotype Hardjoprajitno), being similar to the sequences obtained from the fetuses. Five out of the six cows selected for molecular analysis were positive in pL32-PCR . From thlipL32-PCR, three were also positive in serology for the Sejroe serogroup, while the other two PCR-positive cows were negative in MAT , as they were included in a highly supported clade (bootstrap = 100%) with L. interrogans sequences, separated from other common pathogenic Leptospira species , and, when comparing sequences from the present study with the UF24 sequence, the genetic similarly was 100% .Nucleotide sequences were deposited in GenBank with the accession numbers OR338746-OR338752. Phylogenetic analysis based on the ML-TN92 tree, including  species . Additio species . It is a species . The oveLeptospira antibodies, reinforcing that MAT lacks in diagnosing chronic infections, which are very common in bovine leptospirosis. Due to the limitations of MAT in the detection of chronic carriers, instead of performing serology of only the dam of the aborted fetus, we encourage the sampling of multiple cows, with and without reproductive problems, from the same herd, to understand the background of leptospiral infection. Herd serology provides epidemiological information, and when it is employed as the first step in the diagnosis of abortions, confirming or excluding the first suspect, it helps to recognize the source of infection and to enforce the control measures. Abortions are one of the main clinical signs of bovine leptospirosis . In thisBesides the herd serology, the individual molecular diagnosis of dams is also recommended to understand the cause of abortions. Unfortunately, we could not access all the dams of the studied fetuses, but other cows with reproductive failures of the same herd subgroup were selected. Almost all 5/6) of the analyzed cows were positive, reinforcing the leptospiral infection in the same subgroup of cows. It is also interesting that those animals were only positive in genital samples, and not in urine samples, characterizing the syndrome named Bovine Genital Leptospirosis /6 of the, beyond Regarding the fetus analysis, the first step toward diagnosis was the necropsy and analysis of the macroscopic alterations in the organs. Although the main referred lesions of leptospirosis are jaundice and pronounced coppery liver and kidneys , none ofLeptospira spp. infection, the key organs recommended for molecular diagnosis are the liver and kidneys [lipL32-PCR in the liver and kidney, while the other four fetuses were negative in those organs. If only those samples were analyzed, as commonly recommended, leptospirosis would have been misdiagnosed. At the same time, unconventional sites such as lungs, heart, thymus, subcapsular kidney content, and abomasal content were positive for the presence of leptospiral DNA. In fact, abomasal content and cardiopulmonary organs were the sites with more positive results. The evidence of DNA of Leptospira spp. in the abomasal liquid of a bovine aborted fetus was reported by Otaka and colleagues [Brucella sp, Campylobacter foetus spp., Coxiella burnetii, Listeria monocytogenes, and mycotic agents [lipL32-PCR and, therefore, complemented the diagnosis. The presence of leptospires in those different sites highlights that several organs should be collected simultaneously for diagnosis, beyond the kidney and liver. When available, molecular analysis of the placenta has been encouraged as well [Due to the classic pathogeny of the  kidneys ,23. Nonelleagues , also inlleagues . The fetlleagues ,21, reflc agents . Other u as well ,14,38. UL. interrogans, with 100% homology with the UF24 sequence, which is characterized as serogroup Sejroe genotype Hardjoprajitno, confirming the serology results. This sequence was first identified in the uterus of a slaughtered non-pregnant cow, in which the reproductive history is unknown, from Rio de Janeiro, Brazil [The genetic analysis demonstrated high genetic proximity with sequences from the Sejroe serogroup identified in bovines. Not surprisingly, this high genetic homogenous cluster was formed by sequences from the same geographical region—southeastern Brazil. More specifically, the involved agent in the present study was , Brazil . Similar, Brazil ,41 and g, Brazil . Those oAbortions are common in the reproductive syndrome caused by leptospirosis. In the case of Sejroe strains, such as Hardjo, fetuses are often anicteric and with no evident visible lesions. In order to increase the diagnostic sensitivity of those fetuses, it is mandatory to perform a comprehensive analysis of samples, beyond the kidneys and liver, especially abomasal liquid, among other unconventional sites. If only the conventional sites are tested, bovine leptospirosis will most probably be misdiagnosed.


Lipid nanoparticles (LNPs) are widely used as delivery systems for mRNA vaccines. The stability and bilayer fluidity of LNPs are determined by the properties and contents of the various lipids used in the formulation system, and the delivery efficiency of LNPs largely depends on the lipid composition. For the quality control of such vaccines, here we developed and validated an HPLC-CAD method to identify and determine the contents of four lipids in an LNP-encapsulated COVID-19 mRNA vaccine to support lipid analysis for the development of new drugs and vaccines. SARS-CoV-2 was initially identified as the pathogen that caused the outbreak of pneumonia cases in late December 2019 and spread quickly across the world . The sprHere, we studied an LNP system for COVID-19 mRNA vaccine delivery shown in  using thHigh-performance liquid chromatography (HPLC) is a commonly used modern analytical technique in pharmaceutical analysis. It is widely used in drug analysis due to its high efficiency, speed, sensitivity, and automation. Multiple different detectors can be used in conjunction with HPLC to meet the analysis requirements of different substances, including ultraviolet detectors (UV), refractive index detectors (RID), evaporative light scattering detectors (ELSD), mass spectrometers (MS), and pulse amperometric detectors (PAD).Charged aerosol detection (CAD) is a new type of universal detector that has been developed in the past decade. Its characteristics include compatibility with gradient elution, response values that do not depend on the structural properties and ionization efficiency of the substance, and the ability to detect non-volatile and semi-volatile substances. When the CAD detector is used in conjunction with HPLC, the HPLC eluent is atomized by nitrogen collision in the CAD detector’s spray chamber. Smaller droplets containing the analyte dry at room temperature to form solute particles. The particle surfaces are positively charged by collision with charged nitrogen. The ion-trap device with low negative voltage removes excess charged nitrogen with high migration rates, and the charged analyte particles with low migration rates transfer their charges to a particle collector. Finally, the electric charge of the analyte is measured and converted into an electrical signal by a highly sensitive electrostatic detection device. The intensity of the signal is proportional to the mass of the solute ,17,18.Liquid chromatography with CAD detection for drug analysis has been included in the pharmacopoeias of various countries. For example, both the British Pharmacopoeia 2021 (BP 2021) and the United States Pharmacopeia 2021 (USP 2021) use CAD detectors to investigate substances related to the contrast agent gadobutrol. The determination of deoxycholic acid content in USP43/NF38 also uses CAD detectors. The current version of the Chinese Pharmacopoeia 2020 mentions the CAD detector in the general HPLC method and provides a brief introduction.Lipids do not contain chromophores and do not absorb ultraviolet radiation under non-derivatization conditions. They are usually difficult to volatilize. Therefore, ELSD detectors and CAD detectors are more suitable for lipid content detection . The priWe developed a robust and efficient HPLC-charged aerosol detection method (HPLC-CAD) for lipid analysis of an LNP-encapsulated COVID-19 mRNA vaccine and performed a comprehensive validation of the method. The method is stable, efficient, and sensitive, and can be used to determine the contents of four kinds of lipids in COVID-19 mRNA vaccines. Though some of the lipids mentioned here are proprietary and cannot be disclosed, it is still valuable to highlight this methodology in the growing field of COVID-19 mRNA vaccine technology.Methanol (liquid chromatograph ion spray mass spectroscopy/LCMS-grade), ethyl alcohol (LCMS-grade), and triethylamine acetate (high-performance liquid chromatography/HPLC-grade) were purchased from Thermo Fisher Scientific ; 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K) was purchased from NOF America Corporation ; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (>99%) and cholesterol were purchased from Nippon Fine Chemical ; and 9001 was synthesized by Abogen . Monobasic potassium phosphate, disodium hydrogen phosphate, and sodium chloride were purchased from Sigma-Aldrich . Water (AqDD: <0.55 μS/cm) was dispensed using a Milli-Q filter system from Millipore . The COVID-19 mRNA vaccine samples were maintained in our laboratory.TM DionexTM ChromeleonTM 7 Chromatography Data System Version 7.3 software.Thermo Scientific UltiMate 3000 HPLC system consisting of a vacuum degasser, quaternary pump, autosampler, thermostated column compartment, and charged aerosol detector was used to develop the method. Pure nitrogen from nitrogen generator was used at a preset manufacture pressure of 60.7 psi. Samples were injected using a fixed-loop injection at 10 μL with a 100 μL sample loop. COVID-19 mRNA vaccine samples were separated using a Waters XBridge Peptide BEH C18 Column . The detection temperature was set at 50 °C, the column temperature at 55 °C, and the flow rate at 1.0 mL/min. The injection volume was 10 μL. Data were processed using Thermo ScientificThe mobile phase comprised two eluents in a gradient elution mode. Mobile phase A was 0.01 M triethylamine acetate in double distilled water, while mobile phase B was an organic eluent containing 0.01 M triethylamine acetate in ethyl alcohol. The first step of the gradient began from 80% B to 100% B in the first 5 min, followed by holding at 100% B until 13 min; at 13.1 min, the %B was decreased to the initial condition (80%) and equilibrated for 4.0 min before the next sample injection.First, DMG-PEG2K, DSCP, CHOLE, and 9001 were individually dissolved in pure methanol to prepare stock standard solutions of 1 mg/mL, 2 mg/mL, 4 mg/mL, and 12 mg/mL, respectively. Then, the mother solution of the standard was prepared by transferring 1 mL of each of the four lipid stock standard solutions into a 10 mL volumetric flask, diluting with pure methanol, and fixing the volume to the scale. Finally, 50 μL, 75 μL, 100 μL, 125 μL, 150 μL, and 200 μL of the stock solution were added to the appropriate volume of mobile phase B to reach a final volume of 1 mL for standard solutions of 50%, 75%, 100%, 125%, 150%, and 200% levels, respectively. We also prepared a quality control solution in the same way as the 100% level standard solution.To make the validation program as close as possible to real vaccine samples, we treated mRNA, double-distilled water, DMG-PEG2K, CHOLE, DSCP, and 9001 in line with the production method of the COVID-19 mRNA vaccine to prepare spiked samples. A 100% level spiked sample contained 10 μg/mL DMG-PEG2K, 40 μg/mL cholesterol, 20 μg/mL DSCP, and 120 μg/mL 9001, and 50%, 75%, 125%, 150%, and 200% level spiked samples were prepared by adjusting the contents of the four lipids according to their levels.The specific solutions were prepared by simulated COVID-19 mRNA vaccine without the four lipids, which contained 0.2 mg/mL COVID-19 mRNA, 0.2 mg/mL monobasic potassium phosphate, 3.07 mg/mL disodium hydrogen phosphate, and 8.8 mg/mL sodium chloride.Each COVID-19 mRNA vaccine sample solution was diluted 20-fold with mobile phase B for sample injection analysis.Pure methanol as a blank was injected at least three times until the baseline was stable. Standard solutions were injected in order from low to high level, and then the spiked samples or COVID-19 mRNA vaccine samples were injected. The quality control solution was injected six times after the blank, and one time at the end of the sequence or after each ten injections of samples.X-axis, the peak area was taken as the Y-axis, and the standard curve of the quadratic function was generated (Y = aX2 + bX + C). The contents of each lipid in the spiked samples and the COVID-19 vaccine samples were calculated through regression.The peak areas of the lipids in standard solution were normalized and integrated. The concentration of standard solution was taken as the R2 of the standard curve for each lipid had to not be less than 0.98. Each validation laboratory injected the spiked samples of each concentration three times; the recovery rate of each injection was then calculated, and the mean value was calculated to verify the accuracy, precision, and linearity. The average recovery rates of the six concentrations of spiked samples given through three parallel injections were calculated by two validation laboratories. Recovery rates of each concentration level within 80–120% were considered acceptable. The precision included intra-laboratory reproducibility, inter-laboratory reproducibility, and overall precision, and RSDs (relative standard deviations) not more than 10% were considered acceptable. The linearity of the HPLC-CAD method was calculated to fit the measured and theoretical concentrations of six spiked samples in two validation laboratories, and the acceptable range was that the correlation coefficient R2 was greater than or equal to 0.98. The specificity was verified by a specific solution. The acceptable criteria were that the specific sample had no interference at the retention time of any of the lipids, and the accuracy of the spiked samples at each concentration had to meet the standard requirements above.Method validation was performed by two technicians in two independent laboratories. They prepared three repeats of the standard solutions, quality control solution, and spiked samples of all levels  for each of the 6 days. The system suitability requirements were as follows: the blank sample had no interference peak at the retention time of the four lipids and the R2 of all standard curves was greater than 0.99 throughout the validation process, indicating a good curve fit . The results indicated that the average recovery of DMG-PEG2K was between 100.98% and 110.55%; the average recovery of cholesterol was between 98.10% and 108.05%; the average recovery of DSCP was between 97.34% and 102.99%; and the average recovery of 9001 was between 97.66% and 102.99%. All the recoveries of spike samples were between 90% and 110%  and Y = 0.9945X + 2.4063 (R2 = 0.9982). This indicates that the method has good linearity.The measured values of the total content of DMG-PEG2K, CHOLE, DSCP, and 9001 in two validation laboratories were set as the Comparison of the chromatograms of pure methanol, standard solution of level 50%, and specific solution indicated that the specific solution had no interference during the retention time of DMG-PEG2K, CHOLE, DSCP, and 9001 . The accBased on the above validation results, the linear range of this HPLC-CAD method for the determination of the content of DMG-PEG2K, CHOLE, DSCP, and 9001 in the COVID-19 mRNA vaccines was 5–20 μg/mL, 20–80 μg/mL, 10–40 μg/mL, and 60–240 μg/mL, respectively. The accuracy and precision met the requirements in To further verify the applicability of the HPLC-CAD method, we used this method to determine the contents of four lipids in five batches of COVID-19 mRNA vaccine in two validation laboratories. The samples were prepared as in Novel coronavirus SARS-CoV-2 has become a global health problem and has infected a significant portion of the world’s population. Over the course of the COVID-19 pandemic, people all over the world have faced significant challenges, anxiety, and stress regarding healthcare because until December 2020, there were no vaccines available for this pandemic and there is still no specific treatment. In mid-December 2020, the US Food and Drug Administration (FDA) granted emergency use authorization for Pfizer/BioNTech and Moderna COVID-19 vaccines. Many studies have analyzed and compared these two vaccines. Both vaccines can offer protection against SARS-CoV-2 infection by developing humoral immunity and possibly cellular immunity. Moreover, both vaccines are effective and offer hope for an end to the COVID-19 pandemic. The dose of the Pfizer vaccine is slightly lower (30 μg), while that of the Moderna vaccine is 100 μg. The Pfizer vaccine is approved for people aged 16 years and older, while the Moderna vaccine is approved for those 18 years and older. According to reports, the efficacy of the Pfizer vaccine is 95%, which is slightly higher than the 94.5% efficacy of the Moderna vaccine. Both vaccines can cause some adverse reactions, including pain, swelling, vomiting, nausea, fever, fatigue, headache, muscle pain, itching, chills, and joint pain at the injection site, and in rare cases, they may also cause allergic reactions. According to reports, the incidence of these adverse reactions is lower with the Pfizer/BioNTech vaccine compared to the Moderna vaccine ,24. HoweBoth authorized COVID-19 mRNA vaccines use modified mRNA to encode the spike protein of SARS-CoV-2, which triggers an immune response and produces neutralizing antibodies, and both use an LNP delivery system. This is a new type of vaccine that has potential advantages over traditional replicating or non-replicating viral vector vaccines because mRNA vaccines are highly effective, can be produced rapidly, and are relatively inexpensive. Compared with viral vaccines, they have good safety profiles because they are not made from actual pathogens and do not integrate with the host DNA.Of course, both COVID-19 mRNA vaccines also have their drawbacks. Due to the instability of mRNA and the lipid nanoparticle, the quality control during the release process is difficult, and extreme cold storage is required to maintain stability during distribution . The stoIn the early 21st century, LNP technology for scalable production began to develop based on cationic lipid technology. It has been shown that LNP can deliver mRNA vaccines to mice, and the clinical studies have gradually attracted attention. LNPs usually consist of multiple lipid components, which are used as delivery systems to package and enhance the stability of mRNA. This can effectively avoid the degradation of mRNA outside the cell, promote its uptake by the cells, and release it into the cytoplasm. Lipid components include but are not limited to ionizable/cationic lipids, helper lipids such as neutral lipids and/or cholesterol, and lipids modified by polyethylene glycol (PEGylation). The appearance of LNPs is a milestone in the development of RNA therapy, which successfully solves the problem of protecting and delivering RNA. Currently, the two COVID-19 mRNA vaccines on the market, BNT162b2 (Comirnaty) and mRNA-1273 (Spikevax), use ionizable lipids ALC-0315 and SM-102 (Lipid H), respectively, and PEGylated lipids ALC-0159 and PEG2000-DMG , respectAs more and more COVID-19 mRNA vaccines are approved for use, the global demand for mRNA vaccines for the coronavirus presents a severe challenge for vaccine quality control. Regulatory agencies, such as the World Health Organization (WHO), the US Food and Drug Administration (FDA), the European Medicines Agency (EMA), and the Center for Drug Evaluation (CDE), have issued corresponding guidelines to standardize and guide the declaration and market application of such products. Among them, the recently released WHO document, “Evaluation of the Quality, Safety, and Efficacy of Messenger RNA Vaccines for the Prevention of Infectious Diseases: Regulatory Considerations,” is the first global regulation specifically for mRNA products that is significant for guiding national regulatory authorities to evaluate such products and clarify the regulatory requirements. The production of mRNA vaccines involves multiple biological processes and the processing of raw materials, such as large-scale in vitro transcription (IVT), mRNA processing and modification, and mRNA LNP encapsulation. During production, there is a risk of contamination and introduction of impurities, and it is necessary to study and establish multiple novel key quality control parameters and their detection methods and quality standards related to vaccine characteristics, such as identification, content, integrity, encapsulation rate, and LNP content . The detCurrently, there is still a lack of unified testing methods and reference materials for COVID-19 mRNA vaccine quality control, and information on quality control from different research and development companies is difficult to share. How to integrate vaccine regulation, research and development, and production resources and establish unified standards and quality control systems is a key issue facing the development of the mRNA vaccine industry. Companies should design, develop, and produce vaccines based on the concept of risk management, scientifically and reasonably set quality control projects and acceptable standards, and establish simple and accurate testing methods that meet the expected purposes. Regulatory authorities should promote the uniformity of quality control standards and the application of standardized methods to provide a foundation for promoting the development of mRNA vaccines and developing safe and effective vaccines .Over the past few decades, breakthroughs in nucleic acid modification and non-viral delivery vector technology have greatly promoted the development of mRNA vaccines, and the clinical application of mRNA vaccines has also validated their drug efficacy and advantages in scalable production. From a clinical perspective, long-term monitoring of adverse reactions to mRNA vaccines is still needed to respond to potential risks in a timely manner. From a drug development perspective, the current challenges that mRNA vaccines urgently need to overcome include exploring more efficient production processes to ensure their safety, effectiveness, and quality controllability, as well as finding more reasonable storage and transportation methods to improve accessibility.There are a number of COVID-19 mRNA vaccines in the preclinical development and clinical trial stages . To ensu


Enterobacteriaceae, Chloroplast, and Alternaria were the dominant genera in the air-cured CTLs. Aquabacterium, unclassified Burkholderiaceae, Caulobacter, Brevundimonas, and Aspergillus were the dominant genera in the agriculturally fermented CTLs. Acinetobacter, Methylobacterium, Sampaiozyma, and Plectosphaerella first significantly increased, and then significantly decreased during agricultural processing. The changes in microbial communities are mainly related to their different functions during fermentation. This means that when the fermentation effect of the original microbial community in cigar tobacco leaves is not ideal, we can optimize or design the microbial community based on the fermentation function that the microbial community needs to achieve. These results may help adjust and optimize the agricultural fermentation process of CTLs, and help develop the quality of CTLs and increase the income of tobacco farmers.The agricultural fermentation processing of cigar tobacco leaves (CTLs), including air-curing and agricultural fermentation, carried out by tobacco farmers has rarely been studied. In this study, we have investigated the microbial community in the CTLs during air-curing and agricultural fermentation by 16S rRNA and ITS gene high-throughput sequencing. The results showed that the richness of microbial communities gradually increased with the development of agricultural fermentation, which means that not all microorganisms in CTLs come from the fields where tobacco grows, but gradually accumulate into CTLs during the fermentation process.  Total genomic DNA of each sample was extracted using an EZNA® Soil DNA Kit  according to the manufacturer’s instructions. The genomic DNA was amplified and sequenced using the V4–V5 hypervariable region of 16S rRNA genes  (The CTLs (5.0 g) was suspended in 100 mL of sterile PBS and shaken for 2 h at 200 rpm, after which the supernatant was centrifuged at 10,000× T TT-3′)  and the T TT-3′) .Deoxyribonucleic acid libraries were validated by TruSeq Nano DNA LT  and quantified using a PicoGreen dsDNA Assay Kit . Amplicons were pooled in equal amounts, and sequencing was performed using a 2 × 300 paired-end (PE) configuration using an Illumina MiSeq sequencing system according to the manufacturer’s instructions . Operational taxonomic units (OTUs) of qualified sequences were identified using the clustering program VSEARCH version 1.9.6 and the SILVA version 132 database  with 97%2.3.p < 0.05). In order to further investigate the changes in microbial abundance, circular visualization diagrams were used to display the overlapping and differentiating microbial taxa among the three courses of agricultural processing of CTLs at the phylum and genus levels using Circos software online , by one-way analysis of variance (ANOVA) and Duncan’s multiple comparison test (3.3.1.p < 0.05). The Shannon values of the microbes in the air-cured CTLs were higher than those in the freshly harvested and agriculturally fermented CTLs (p < 0.05). These results showed that the diversity of microbial communities gradually decreased with the progress of agricultural fermentation, which means that not all microorganisms in CTLs come from the fields where tobacco grows, but gradually accumulate into CTLs during the fermentation process.In total, 1,741,054 and 2,095,371 reads of the 16S rRNA and ITS gene sequences were obtained from all CTLs. 3.2.Proteobacteria, Cyanobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Ascomycota and Basidiomycota were dominant at the phylum level , Bacteroidetes and Basidiomycota were mainly present in the air-cured CTLs , and Ascomycota was mainly present in the agriculturally fermented CTLs  (p < 0.05). At genus level, unclassified Enterobacteriaceae, Chloroplast, Pantoea, and Alternaria were the dominant genera in the air-cured CTLs. The Acinetobacter, Sphingomonas, Methylobacterium, Sampaiozyma, and Plectosphaerella increased first and then decreased during agricultural processing. Pseudomonas, Staphylococcus, Aquabacterium, unclassified Burkholderiaceae, Caulobacter, Brevundimonas, and Aspergillus were the dominant genera in the agriculturally fermented CTLs.Differences in the dominant phyla and genera in the different courses of agricultural processing of all samples were shown in the Circos diagrams  can be found in the article/supplementary material.DL and JZ conceived and designed the manuscript. QZ, TZ, ZY, SY, WC, PL, and YH handled samples and conducted experiments. QZ and TZ wrote and revised the manuscript. All authors read and approved the manuscript.This work was funded by China National Tobacco Company [Nos. 110202101062(XJ-11) and 110202201032(XJ-03)] and China Tobacco Sichuan Industrial Co., Ltd. (No. rtx201820).QZ, DL, ZY, SY, WC, PL, and YH were employed by China Tobacco Sichuan Industrial Co., Ltd.The remaining 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.