The 2019 Coronavirus Disease (COVID-19) has become an unprecedented public crisis. We retrospectively investigated the clinical data of 197 COVID-19 patients and identified 88 patients as disease aggravation cases. Compared with patients without disease aggravation, the aggravation cases had more comorbidities, including hypertension (25.9%) and diabetes (20.8%), and presented with dyspnoea (23.4%), neutrophilia (31.5%), and lymphocytopenia (46.7%). These patients were more prone to develop organ damage in liver, kidney, and heart (P < 0.05). A multivariable regression analysis showed that advanced age, comorbidities, dyspnea, lymphopenia, and elevated levels of Fbg, CTnI, IL-6, and serum ferritin were significant predictors of disease aggravation. Further, we performed a Kaplan–Meier analysis to evaluate the prognosis of COVID-19 patients, which suggested that 64.9% of the patients had not experienced ICU transfers and survival from the hospital. Sequence analyses of coronaviruses from lower respiratory tract samples have shown a structure typical of other coronaviruses, such as SARS and MERS coronaviruses. Afterwards, the WHO named the unidentified coronavirus SARS-CoV-23. SARS-CoV-2 belongs to the clade of the sarbecovirus subgenus of the Orthocoronavirinae subfamily. Evidence has been found that SARS-CoV-2 is extremely contagious to humans and can be transmitted through respiratory droplets, contact, and even via faecal-oral transmission4.In December 2019, an outbreak of unidentified pneumonia aroused great attention throughout the world and is considered to be not only a pandemic, but also a disaster. On January 27, 2020, the World Health Organization (WHO) issued worldwide surveillance and vigilance recommendations of highly contagious respiratory diseases5. Therefore, an investigation of the risk factors that are associated with disease aggravation is greatly warranted. In this study, we investigated the clinical characteristics and relevant factors that are associated with the outcomes of patients with COVID-19 infections, which may provide considerable value for the early identification of individuals who are at risk of disease aggravation and who are most likely to benefit from intensive care treatment.As has been reported in the literature, patients with COVID-19 mainly presented with fever, cough, fatigue, myalgia, and dyspnoea. Several studies have shown that the majority of patients are considered to have a favourable prognosis; however, elderly men and those with underlying diseases, including hypertension, diabetes, and chronic obstructive pulmonary disease (COPD), have a higher risk of developing acute respiratory distress syndrome (ARDS), which may be the leading cause of deathIn this study, a total of 197 patients with COVID-19 were confirmed from the designated hospital, and the study follow diagram were shown in Fig. The symptom onset of the patients is shown in Table d-dimer (27.2%), and fibrinogen (Fbg: 21.3%).The laboratory findings of the confirmed patients are summarized in Table +, CD4+, and CD8+ T cells in the aggravation group were significantly lower than those of the non-aggravation group (P < 0.05), but there was no difference in the counts of CD16+CD56+ T (P = 0.13) and CD19+ B cells (P = 0.06). Blood biochemical examination results suggested that patients with disease aggravation demonstrated higher levels of AST, ALT, DBIL, BUN, creatinine, CK-MB, CTnI, ProBNP, and LDH. Table d-dimer and Fbg were markedly elevated in the aggravation patients.There were many differences with regard to laboratory parameters between the non-aggravation and aggravation patients, as shown in Table Mycoplasma pneumonia , influenza B virus , parainfluenza virus , and cytomegalovirus . There was no significant difference in the incidences of these pathogens between the disease aggravation group and the control group.The chest imaging features upon admission are summarized in Table 6. However, some research has suggested that this method usually shows lower positive rates due to poor RNA stability, different specimen positions, and quality7. Therefore, the IgM-IgG combined assay has been recommended to increase the sensitivity of COVID-19 diagnoses, especially in patients with suspected COVID-19 infections8. In this study, dynamic changes in IgM-IgG antibody levels were detected. Specific IgG and IgM antibodies can be detected 4–7 days after the onset of the illness. The IgM antibody titres sharply and significantly increased in the initial 2 weeks, peaked at 1–2 weeks after symptom onset, and significantly declined after 21 days. The IgG antibody titres increased over time, peaking at 4–5 weeks after the onset of the illness, after which higher levels were maintained for the entire observation period , Atractylodis Macrocephalae Rhizoma (Baizhu), Glycyrrhizae Radix Et Rhizoma (Gancao), Saposhnikoviae Radix (Fangfeng)13. Of the 197 patients, more than two-thirds of the patients required oxygen therapy. A total of 105 (53.3%) patients were treated with high-flow oxygen. Forty-three (21.8%) patients were given non-invasive mechanical ventilation. Twenty-five (12.7%) patients received invasive mechanical ventilation. The study also indicated that antibiotics, corticosteroids, and oxygen therapy were necessary more often in aggravation patients than in non-aggravation patients (P < 0.05).All of the confirmed patients were isolated and treated in a negative pressure ward. Table By the end of March 15, 164 83.2%) patients had improved and were discharged. The median length of stay of the patients was 15 days (IQR: 9–21). Of the 88 aggravation patients, 49 (55.7%) patients were admitted to the ICU for a median of 8 days, which were significantly higher than that of the non-aggravation patients for a median of 5 days. To evaluate the prognoses of the COVID-19 patients, we performed a Kaplan–Meier analysis, and the composite endpoint (event) was ICU transfer or death within 25 days from the date of admission to the hospital. The cumulative event-free survival curve is plotted in Fig. .2% patied-dimer, IL-6, CRP, procalcitonin, and serum ferritin were all significantly correlated with disease aggravation. Subsequently, these variables were included in the multivariable logistic regression model, which indicated that older age, hypertension, diabetes, dyspnoea, lymphocytes, Fbg, CTnI, IL-6, and serum ferritin were all independently associated with disease aggravation , dyspnoea, and chest tightness were associated with a higher risk of disease aggravation. Lymphopenia, neutrophilia, and high levels of AST, BUN, CK, LDH, CTnI, ProBNP, Fbg, on Table .17. These results showed that advanced age is an independent risk factor for disease progression. Growing evidence demonstrates that males were more susceptible to acquire viral infections and disease progression. A study from China firstly reported the presence of gender differences which suggested that males account for 60% of the COVID-19 patients18. And then a study examining 799 patients found that 73% of patients who died (113 patients) were male. Another study suggested that male was a significant risk factor for disease progression19. According to the last sex-related study conducted in Italian on 239,709 patients, lethality is more prevalent in males compared to females (17.7% vs 10.8%), and males account for 59% of deaths from COVID-1920. Further, a system review with 59,254 patients from 11 different countries demonstrated that males had much higher mortality risk than females21. In the present study, we found that males were identified more likely to be contaminated with the virus (60.9% vs 39.1%), and had inferior outcomes than the female patients. Therefore, our study further confirmed that males were more susceptible to acquire viral infections and disease progression.The present study included a total of 197 patients who were hospitalized with COVID-19 from January 25, 2020, to March 15, 2020. All of the patients were evaluated for therapeutic efficacy after at least 1 week of hospitalization. The results identified disease aggravation in 88 patients and non-aggravation of the disease in 109 patients. We summarized the clinical characteristics and identified several risk factors that were associated with disease aggravation in the patients. As of March 15, 2020, 164 (83.2%) patients improved and were discharged, and 33 (16.8%) patients died. Although all of the age groups have been affected by COVID-19, elderly patients (over 60-years-old) have a greater risk for infection and a relatively high proportion of severe and disease aggravation cases23. Recently, a retrospective cohort study also demonstrated that these comorbidities have been responsible for 60.9% of deaths caused by COVID-1924. A large number of studies have indicated that the angiotensin-converting enzyme 2 (ACE2) receptor is highly expressed in the cardiovascular/cerebrovascular and lung tissues in hypertension patients25. In addition, ACE2 is one of the most important host receptors of H7N9, SARS, and COVID-19, whose activities are closely related to the pathogenesis of inflammatory and acute injuries of lung disease caused by these viruses27. Experimental studies have suggested that the spikes of SARS-CoV-2 could bind to ACE2 receptors, which can mediate virus entry into HeLa cells28. Some scholars have found that most COVID-19 patients showed higher angiotensin II activity than uninfected people, and the abnormal increase in angiotensin II was related to lung failure and death29. It has been established that the expression of ACE2 is significantly upregulated in COPD and in cigarette-smoking patients30. Several studies have demonstrated that smokers and patients with COPD have a high rate of SARS-CoV-2 infection and increased risks of severe complications and mortality31. A meta-analysis by Carlos A. J. included 6487 cases from 19 studies and revealed that smoking status could produce a more serious clinical condition of COVID-19 and can more likely lead to ICU admission and death32. However, several studies have shown that smoking was not associated with an increased risk of critical illness33. In the current study, we found that smoking was not associated with disease aggravation or critical illness. This may be explained by the small number of available studies, small sample sizes, and short lengths of study. ACEIs and ARBs are recommended for the management of hypertension and renal disease associated with diabetes34. Recent studies have reported that the application of ACE inhibitors can induce a marked increase in ACE2 expression, which means that ACEIs/ARBs would increase the risk of COVID-19 infection and disease aggravation of COVID-19 in hypertension and diabetes patients receiving these drugs36. In the present study, COVID-19 patients with ACEI/ARB therapy were enrolled, and we found that there were more patients taking ACEIs/ARBs in the aggravation group. Therefore, taking ACEIs/ARBs may be another potential risk factor for the disease aggravation of COVID-19.Of all of the patients, 72 (36.5%) patients cases had one or more underlying diseases, including diabetes, hypertension, and COPD. The logistic regression analysis confirmed a higher frequency of hypertension and diabetes in patients with disease aggravation. It has been reported that patients with hypertension or diabetes accounted for 20–30% of the total infected patients and comprised over half of the patients in the ICU22, the most common symptoms were fever, cough, fatigue, chest tightness, and myalgia or arthralgia. The proportions of patients with myalgia or arthralgia, chest distress, and dyspnoea were significantly higher in the disease aggravation group. Remarkably, few patients exhibited some of the less common symptoms, including abdominal pain, vomiting, and conjunctival hyperaemia, which may result in missed diagnoses and transmissions to other people. Previous studies have found that the virus could be detected in stool samples in patients with symptoms of abdominal pain and vomiting, as well as in asymptomatic patients38. Therefore, further research is still required to determine whether faecal oral transmission exists.In our cohort, no significant difference in the median days from symptom onset to hospital admission was found between the disease aggravation and non-aggravation patients. Consistent with the symptoms that have been previously reported40. In the present study, 92 (46.7%) patients showed significant neutrophilia and lymphopenia with pronounced reductions in peripheral blood CD3+, CD4+, and CD8+ cells. Lymphocyte subsets, especially CD3+, CD4+, and CD8+ cells, were more severely impaired in disease aggravation patients. This is consistent with a previous study by Qin et al., which demonstrated that the percentages of memory, as well as the percentages of regulatory and effector T cells, were significantly decreased in severe cases, compared to non-severe cases41. Furthermore, we found that CD19+ cell counts also exhibited a decreasing trend, compared with matched controls, but this difference did not reach statistical significance. These results were consistent with the study conducted in Spain, which demonstrated that there were no differences in CD19+ B cell percentages between the survivor and non-survivor groups42. The plausible explanation for this discrepancy may be due to the direct infection of T lymphocytes by SARS-CoV-2, cytokine-mediated lymphocyte trafficking into infected tissues, or lymphocyte exhaustion in the peripheral blood43. All of these hypotheses remain to be proven in further investigations.SARS-CoV-2-induced immune responses and infection cytokine storms are believed to play major roles in disease aggravation. The destruction of lung cells can recruit macrophages and monocytes and release cytokines to resolve the infection and even simultaneously mediate widespread excessive inflammation40. In this study, compared with patients without disease aggravation, the disease aggravation patients showed significantly increased expression of inflammation-related factors, including IL-6, CRP, serum ferritin, and procalcitonin levels. Multivariate analysis revealed that elevated levels of IL-6 and serum ferritin were risk factors for disease aggravation in the patients. It has been previously established that patients with severe SARS and MERS have a higher incidence of multiple organ dysfunction syndromes, including liver damage, acute heart/kidney injury, coagulation dysfunction, and even death45. Many studies have demonstrated the clinical characteristics and laboratory findings associated with different degrees of multiple organ dysfunction in patients with COVID-1946. In our study, liver damage and acute heart/kidney injury were considered to occur in more than one-third of patients. The levels of AST, ALT, direct bilirubin, LDH, creatinine, BUN, CTnI, and ProBNP seemed to be significantly higher in patients with disease aggravation. The multivariate analysis revealed that elevated levels of LDH, CTnI, and ProBNP were risk factors for disease aggravation. Currently, there are three potential mechanisms for this observation. First, the binding of SARS-CoV-2 to ACE2-positive cells mediates direct damage. Second, systemic inflammatory response syndrome includes a cytokine storm, dysregulated immunocytes, and uncontrolled inflammation. Third, exogenous drugs induce organ metabolizing burden or worsen organ function48. Recently, coagulation dysfunction has attracted increased attention among scholars. It has been reported that the incidence of coagulopathy in all patients showed abnormalities of varying degrees. A previous study revealed that Fbg and d-dimer elevations were related to the severity of the disease49. Ji et al. demonstrated that coagulation activation and hyperfibrinolysis coexisted in patients with severe COVID-19 infections50. Patients with elevated plasmin and Fbg levels may have an increased risk of ARDS and mortality51. In this study, we found that coagulation function parameters  were higher in patients with disease aggravation. A further analysis revealed that a high level of Fbg was significantly associated with the outcome. Therefore, measurements of these coagulation function parameters may be important biomarkers of disease aggravation and outcomes.Previous studies have shown that serum inflammation-related indices are closely related to the degree of inflammation and disease severity53. In our study, most patients received combination therapy with oxygen support, antibiotics, antiviral agents, and glucocorticoids. Several antiviral agents previously used to treat SARS and Middle East Respiratory Syndrome (MERS) and HIV have been considered as the most potential candidates for COVID-19 patients. With the spread of COVID-19, a large number of clinical studies all over the world have been underway to investigate the potential agents for COVID-19. Some preclinical research has suggested that remdesivir has broad-spectrum antiviral activity against MERS-CoV and SARS-CoV-1/2 infections54. Several studies indicated that COVID-19 patients would benefit from the combination of lopinavir and ritonavir with fewer adverse clinical outcomes56. However, further studies on a larger set of clinical specimens will be required to assess the efficacy and safety of antiviral drugs58. It has been reported that coinfection with bacteria has a great influence on the disease progression and prognosis of COVID-19 patients, which may lead to increased needs for ICU care, and increased mortality rate especially in severe patients60. Brockmeier et al. reported that coinfection with bacteria could significantly upregulate the levels of inflammatory cytokines, especially IL-6 and MCP-1 in COVID-19 patients61. Martins- Filho et al. demonstrated that the bacterial and fungal coinfection had an increased mortality by 2.5 folds in COVID-19 patients62. Therefore, antibiotic treatment for COVID-19 patients based on the experience of overlapping bacterial infections and results of antimicrobial susceptibility test seemed to be the basic principle. In our study, 101 (51.3%) patients received antibiotics treatments, and the aggravation patients were more likely to receive antibiotics than the non-aggravation patients. Due to the cytokine storm, corticosteroids are widely used in the treatment of patients with severe illness to attenuate inflammation that is associated with lung injury. In the present study, the disease aggravation patients were significantly more likely to receive glucocorticoid therapy. Nevertheless, some researchers have stated that the use of glucocorticoids did not reduce mortality but could easily lead to disease aggravation and increase the risk of secondary infections63. Therefore, the rational use of an appropriate dose of glucocorticoids could suppress the excessive activation of the immune system and the secretion of inflammatory cytokines. Almost all of the patients received respiratory support, including nasal cannula oxygen and continuous positive air pressure. The aggravation group was significantly more likely to receive higher levels of respiratory support. Undoubtedly, patients with disease aggravation have a higher mortality rate than patients without disease aggravation. Additionally, ARDS remained to be the most common cause of death, followed by multiple organ failure. Therefore, the prevention and treatment of ARDS represent an important strategic objective for the reduction of mortality and morbidity.Currently available treatment approaches for COVID-19 include symptomatic and supportive therapies, such as oxygen therapy, antivirals, prevention and treatment of infections, and combination treatment with glucocorticoidsThis study also has severe limitations. First, the inherent shortcomings are attributed to a retrospective observational study, and the small sample size and short-term follow-up make it difficult to obtain a firm conclusion. Second, the present study had possible patient selection bias. Therefore, a larger cohort study of patients from China and other countries may help to further investigate the clinical characteristics and risk factors for the outcomes. Finally, the actual duration of viral clearance was overrated, which is due to the frequency of respiratory specimen collection. In addition, the viral load of SARS-CoV-2 was not accurately quantified, and false-negative results for upper respiratory samples have also been reported. Therefore, studies on the dynamic characteristics of the viral load are still warranted.Investigating the potential factors of advanced age, comorbidities and elevated level of Fbg, CTnI, IL-6, serum ferritin enables early identification and management of patients with poor prognosis. In addition, detection of the dynamic antibody may offer vital clinical information during the course of SARS-CoV-2 and provide prognostic value for infected patients. More related studies are needed in the future.This study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of Taihe and Jinzhou central hospital. All of the patients were consecutively enrolled from January 18, 2020, to March 15, 2020, in Taihe and Jinzhou Central Hospital, and the patients had been confirmed to have COVID-19 infections according to World Health Organization interim guidance. All of the participants provided written informed consent before the data were retrospectively collected.We reviewed the clinical medical records of all of the patients to collect the demographic data and clinical and laboratory findings of the patients. All of the data, including age, sex, occupation, onset of symptoms, underlying comorbidities, laboratory results, treatment, and outcome data, were retrospectively extracted with standardized data collection forms. The clinical outcomes were followed up to March 15, 2020.64. ARDS and shock were defined according to the interim guidance of the WHO for the novel coronavirus65. Acute kidney injury was identified and classified based on the highest serum creatinine level or urine output criteria according to the kidney disease improving global outcomes classification66. Cardiac injury was diagnosed if serum levels of cardiac biomarkers  were above the 99th percentile upper reference limit or if new abnormalities were shown in the electrocardiography and echocardiography tests67. Finally, a total of 197 subjects were enrolled in the study and were divided into two groups: 88 cases with disease aggravation and 109 cases without disease aggravation. The flow chart of the study is presented in Fig. To identify the COVID-19 cases, nasal or pharyngeal swab samples were obtained from the patients at admission and were tested via real-time reverse transcriptase polymerase chain reaction (RT-PCR). The exact date of onset of symptoms was defined as the day when the symptom was first noticed. A weekly assessment of disease aggravation was performed that provided details about the patients’ clinical statuses. All of the study participants were divided into either the disease aggravation group or the non-aggravation group, based on the following specific criteria: (1). aggravation group composed of patients who had a higher body temperature than before, aggravated symptoms, and a varied aggravation of imaging examination manifestation; and (2). non-aggravation group composed of patients who had decreased body temperature, symptom and imaging improvements, or no significant changes in body temperature, as well as respiratory symptoms and imaging manifestations. Smoking history was defined as the smoking of at least one cigarette per day and a smoking duration lasting for more than 1 yearThe categorical variables were expressed as frequencies and numbers (%) and were analysed using the chi-squared test or the Fisher’s exact test. The continuous variables are presented as median or interquartile range (IQR) values, and two-sided unpaired t-tests or Mann–Whitney tests were used to compare the groups. All of the statistical analyses and graphs were generated and plotted by using GraphPad Prism (version 7.0) and SPSS (version 21.0). A confidence level of P < 0.05 was considered to be statistically significant for all of the analyses.


Streptochaeta angustifolia, one of two genera in the grass subfamily Anomochlooideae, a lineage sister to all other grasses. The final assembly size is over 99% of the estimated genome size. We find good collinearity with the rice genome and have captured most of the gene space. Streptochaeta is similar to other grasses in the structure of its fruit (a caryopsis or grain) but has peculiar flowers and inflorescences that are distinct from those in the outgroups and in other grasses. To provide tools for investigations of floral structure, we analyzed two large families of transcription factors, AP2-like and R2R3 MYBs, that are known to control floral and spikelet development in rice and maize among other grasses. Many of these are also regulated by small RNAs. Structure of the gene trees showed that the well documented whole genome duplication at the origin of the grasses (ρ) occurred before the divergence of the Anomochlooideae lineage from the lineage leading to the rest of the grasses (the spikelet clade) and thus that the common ancestor of all grasses probably had two copies of the developmental genes. However, Streptochaeta (and by inference other members of Anomochlooideae) has lost one copy of many genes. The peculiar floral morphology of Streptochaeta may thus have derived from an ancestral plant that was morphologically similar to the spikelet-bearing grasses. We further identify 114 loci producing microRNAs and 89 loci generating phased, secondary siRNAs, classes of small RNAs known to be influential in transcriptional and post-transcriptional regulation of several plant functions.In this work, we sequenced and annotated the genome of  The grasses (Poaceae) are arguably the most important plant family to humankind due to their agricultural and ecological significance. The diversity of grasses may not be immediately evident given their apparent morphological simplicity. However, the total number of described species in the family is 11,500+ , and mor3 photosynthetic pathway , is sister to all other grasses  are annotated. Because of the distinct floral morphology of Streptochaeta, we also investigate the molecular evolution of two major transcription factor families, APETALA2 (AP2)-like and R2R3 MYB, which are known to control floral and spikelet structure in other grasses and are regulated by sRNAs.Of the handful of species in the Anomochlooideae, stifolia , 3 is thStreptochaeta angustifolia is native and restricted to the Atlantic Forest of Brazil, although other species of Streptochaeta can be found as far north as southern Mexico. The reference plant for this project was collected in Brazil by Thomas Soderstrom of the Smithsonian Institution in 1980 of the Smithsonian Institution, though the precise collection location is unknown. The plant has been propagated by division and single seed descent, first at the Smithsonian and more recently at Iowa State University and at the Donald Danforth Plant Science Center in St. Louis, MO. The voucher Clark 1304  represents the plant from which DNA was extracted for the initial molecular phylogenetic studies of this genus. Either this plant or one of its descendants was used for this project, based on the same voucher.Streptochaeta leaf tissue was harvested and used to estimate genome size at the Flow Cytometry Facility at Iowa State University. DNA was then isolated using Qiagen DNeasy plant kits. Three Illumina libraries (paired end and 9- and 11-kb mate pair) were generated from these isolations at the Iowa State University (ISU) DNA Facility. One lane of 150 bp paired-end HiSeq sequencing (insert size of 180 bp) and one lane of 150 bp mate-pair HiSeq sequencing (9- and 11-kb libraries pooled) were generated, also at the ISU DNA Facility . This program uses the alignment of in silico-generated maps (from input contigs) to the consensus optical map (Bionano) to output genome maps. The genome maps are then aligned back to the original in silico maps to output fasta-formatted hybrid scaffolds . The full list of options used for running the alignment and to generate the hybrid scaffolds are provided in the associated GitHub repository . All scripts for assembly and downstream analysis are available at: https://github.com/HuffordLab/streptochaeta.We used MaSuRCA v2.21  to generThe Bionano assembly was screened for haplotigs, and additional gaps were filled using Redundans v0.13a . BrieflyTo annotate the repeats in the genome, we used EDTA v1.8.3  with defab initio predictions , Brachypodium distachyon (Bdistachyon_556_v3.2), Oryza sativa (Osativa_323_v7.0), Spirodela polyrhiza (Spolyrhiza_290_v2), Setaria viridis (Sviridis_500_v2.1), and Zea mays (Zmays_493_APGv4). Sequences of Eragrostis tef were retrieved from CoGe (id50954) (Triticum aestivum were retrieved from Ensembl Plant r46 (Triticum_aestivum.IWGSCv1) (A BLAST database was built using seven grass species including id50954) . SequencIWGSCv1) .2) in an iterative manner. Specifically, 18 Arabidopsis AP2-like proteins  was used to retrieve rice MYBs using Oryza sativa Japonica Group genes (IRGSP-1.0) as the database on Gramene Biomart3. The number of MYB domains was counted by searching for “Myb_DNA-bind” in the output of hmmscan, and 82 proteins with two MYB domains were used as the initial query. Iterative blastp and hmmscan were performed in the same manner as for AP2 except using a domain E-value threshold of 1e-3.AP2 and MYB proteins were identified using BLASTP and hmmscan  genes, following  sequenced sRNAs from leaf, anther and pistil tissues, (ii) compared miRNAs present in anthers to those of three other representative monocots , and (iii) validated gene targets of these miRNAs.To annotate microRNAs (miRNAs) present in the We collected tissues from leaf and pistil as well as 1.5, 3, and 4 mm anthers. Samples were immediately frozen in liquid nitrogen and kept at –80°C prior to RNA isolation. Total RNA was isolated using the PureLink Plant RNA Reagent . sRNA libraries were published previously . RNA seqStreptochaeta genome using ShortStack v3.8.5  with an N50 of 824 kb. Through scaffolding with the optical map and collapsing with Redundans software, the total number of scaffolds dropped to 17,040, improving the N50 to 2.6 Mb and the L50 to 161. A total of 79,165 contigs were provided as input for Redundans for scaffold reduction . With eight iterations of haplotype collapsing, the total size reduced to 1,796 Mbp. Additional rounds of gap-filling using GapCloser reduced the total size of gaps (Ns) from 210.13 to 76.33 Mbp. The improvement in the N50/N90 values with each iteration is provided in Streptochaeta genome and the use of mate-pair sequencing libraries. Merqury’s log-scaled probability of error in the consensus base calls indicated a high QV of 47.2146 (or probability of finding an incorrect base in this assembly was 1.89908e-05). The K-mer completeness measured by Merqury indicated 95.96% of reliable k-mers that occurred in the raw reads were also in the genome assembly, suggesting most of the reads were incorporated in the genome assembly. Dot plots of Streptochaeta contigs aligned to rice revealed substantial colinearity . Of these 2,767  were present as a complete single copy. Only 158 BUSCOs were missing entirely with another 110 present as fragmented genes. The LAI (LTR Assembly Index) score, which assesses the contiguity of the assembled LTR retrotransposons, was 9.02, which is somewhat higher than most short-read-based assemblies , perhapsinearity .BlobTools (v0.9.19)  detectedStreptochaeta genome with < 7% multi-mapped reads. Paired-end reads mapped (uniquely) at a higher rate (88.59%) than the single-end RNAseq (70.38%) reads. Genome-guided transcript assemblers produced varying numbers of transcripts across single-end (SE) and paired-end (PE) data as well as various assemblers. Cufflinks produced the highest number of transcripts , followed by StringTie , and Strawberry . Class2 generated fewer transcripts overall . The best transcript for each locus was picked by Mikado from the transcript assemblies based on its completeness, homology, and accuracy of splice sites. Non-coding (due to lack of ORFs) or redundant transcripts were removed to generate 28,063 gene models . Mikado also identified 19,135 non-coding genes within the transcript assemblies. Further filtering for transposable-element-containing genes and genes with low expression reduced the total number of evidence-based predictions to 27,082 genes .More than 79% of the total RNAseq reads mapped uniquely to the Streptochaeta genome) and the mapped RNA-Seq reads (as a hints file using the bam file), produced a total of 611,013 transcripts on a soft-masked genome. This was then subjected to filtering to remove TE containing genes  as well as genes only found in Streptochaeta . After removing both of these classes of genes, which overlapped to an extent, the total number of ab initio predictions dropped to 40,921 genes .BRAKER, with inputs including predicted proteins from the direct evidence method .Functional annotation was informed by homology to the curated proteins in SwissProt and resulted in the assignment of putative functions for 38,955 transcripts . Of the unassigned transcripts, 41 predictions had pfam domain matches, and 16,918 transcripts had an interproscan hit. Only 3,068 transcripts contained no additional information in the final GFF3 file.Streptochaeta genus and more than 15%  were Poaceae specific. Nineteen percent  of genes’ origins could be traced back to cellular organisms and 15%  to Eukaryotic genes. The distribution of genes based on strata and annotation method is provided in All gene models were classified based on their presumed age. More than 8% of the total genes  were specific to the The repeat annotation performed by the EDTA package comprised 66.82% of the genome, the bulk of which was LTR class elements , followed by DNA repeats , and MITE class repeats .AP2-like genes were divided into euAP2 and ANT clades, as expected from previous work  including SHAT1, ANT1, ANT3, ANT4, BBM1, ANT7, ANT8, and ANT9, and  (implying that the outgroup sequence is lost or was not retrieved by our search) including BBM3, WRI2 and WRI4  soon after the grass duplication. Some subclades contain two Streptochaeta sequences and one copy in other grasses. These Streptochaeta sequences are either sisters to each other with the Streptochaeta clade sister to the other grasses  (RSR1) (WRI1) ( is lost . The twoand WRI4 . These p] (RSR1) , or succ) (WRI1) .IDS1/Q-SNB/SID1, ANT5–ANT6, BBM4–BBM2 and basalANT1-basalANT2, the grass-duplicated gene pairs were retained, and were also in syntenic regions based on a syntelog search of the Brachypodium distachyon, Oryza sativa or Setaria viridis genomes ] ,G2)] )] and , respectively ]  and ANT5S2),G2)] . In subcectively . These tAP2-like tree, the most common tree topology within each subclade is , found in 16 individual subclades, followed by  in 10 subclades. We also found 16 subclades with other tree topologies either without or with one or two Streptochaeta sequences and one copy of the other grass sequences, including  (MYB48),  ,  (MYB78 and MYB92), ,  (MYB56) and  (MYB47 and MYB83)  . Convers105-FDL1 . TogetheMYB53-MYB54 with tree topology of , that supports ρ having occurred after the divergence of Streptochaeta ,,G2))] ] and , respectively ,G2))] in MYB6–MYB7 and SWAM1 and SWAM2, or  in MYB42–MYB43, MYB51–MYB52, MYB65–MYB66, MYB75–MYB76, and MYB105-FDL1.Among the subclade pairs that retain both grass sequences, we found one subclade pair, tochaeta . Convers3),G2))] . In the he other . In subcectively . We obseStreptochaeta, including  (MYB1–MYB2 and MYB62–MYB63),  (MYB22–MYB23) and  (MYB11–MYB12)  . In othes clades . This maAP2-like and R2R3 MYB trees support the inference of ρ before the divergence of Streptochaeta (12 subclades) over ρ after the divergence of Streptochaeta (1 subclade)  , consistubclade) . In addiIn total, 185.3 million (M) sRNA reads were generated  from five sRNA libraries. We annotated 114 miRNA loci, of which 98 were homologous to 32 known miRNA families and 16 met strict annotation criteria for novel miRNAs –10. MostStreptochaeta (GPX6 gene (At4g11600), known to function in the protection of cells from oxidative damage in Arabidopsis  libraries to identify and validate the cleavage of miRNA-target pairs in anther, pistil and leaf of tochaeta , 12. Ovebidopsis . Among tbidopsis , 3. miR1is miRNA , 2008. APHAS loci) including 71 21-PHAS and 18 24-PHAS loci (PHAS to 24-PHAS locus number comparing asparagus (<1), a member of Asparagaceae, to grass species . Second, among Poaceae species, the number of genomic PHAS loci was lower in Streptochaeta than in both maize and rice. Third, several PHAS loci were also expressed in the pistil and leaves. Overall, 23 (32%) 21-PHAS loci and 11 (61%) 24-PHAS loci were expressed in the pistil with a median abundance of 32.9 and 12.3%, respectively, compared to phasiRNAs detected in anther tissue. Similarly, 22 (31%) 21-PHAS loci and 10 (56%) 24-PHAS loci were detected in leaf tissue with a median abundance of 53.3 and 13.2%, respectively, compared to phasiRNAs detected in anthers. The expression of 24-nt phasiRNAs in vegetative tissues is unusual.phasiRNAs from the same sRNA libraries were annotated and the abundance of these loci compared to that in asparagus, maize, and rice. Overall, we detected a total of 89 phasiRNA loci , with the genome size matching predictions based on flow cytometry. The genome-wide LTR Assembly Index (LAI) for measuring the completeness of intact LTR elements, was 9.02, classifying the current genome as “draft” in quality, and is on par with other assemblies using similar sequencing technology .Streptochaeta, also known as orphan genes . Many previous studies have indicated that orphan genes may comprise 3–10% of the total genes in plants and can, in certain species, range up to 30% of the total  an investigation of gene families known to play a role in floral development that have potential relevance to the origin of the grass spikelet. Previous phylogenetic work based on transcriptomes (Streptochaeta shared the same WGD (ρ) as the rest of the grasses but that it might also have its own duplication. Among the large sample (200) of clades in the transcriptome gene trees from Streptochaeta  support the same conclusion.Our highly contiguous assembly in genic regions combined with gene model and functional annotations allowed: (1) evaluation of patterns of orthology between genes in riptomes  or indivriptomes  suggestetochaeta . AdditioAP2-like and MYB subclades through the lens of grass WGD events, we found 12 and one cases supporting ρ before and after the divergence of Streptochaeta, thus confirming previous transcriptomic data  – might have borne its flowers in spikelets, and the truly peculiar “spikelet equivalents” of Anomochlooideae are indeed highly modified.Genome structure and phylogenetic trees of Many transcription factor families are known to regulate spikelet development in the grasses . Of thesFRUITFULL (FUL) duplicated at the base of Poaceae before the divergence of Streptochaeta, but FUL1/VRN1 in Streptochaeta was subsequently lost (LEAFY HULL STERILE1 (LHS1) and Oryza sativa MADS5 are duplicated at the base of Poaceae, but Streptochaeta has only one gene sister to the LHS1 clade (PISTILLATA (PI), Streptochaeta has orthologs in both the PI1 and PI2 clades , extra florets are formed within the spikelets in both male and female flowers  produce repetitive glumes (SUPERNUMERARY BRACT (SNB), which is an ortholog of SID1, also exhibit multiple rudimentary glumes, due to the delay of transition from spikelet meristem to floral meristem. Such mutant phenotypes are somewhat analogous to the Streptochaeta “spikelet equivalents,” which possess 11 or 12 bracts. In situ hybridization studies on FUL and LHS1 showed that the outer bracts 1–5 resemble the expression pattern of glumes in other grass spikelets, while inner bracts 6–8 resemble the expression pattern of lemma and palea ] in IDS1/Q-SID1/SNB subclade pairs, leaving the evolutionary history of Streptochaeta ambiguous  belong to the euANT lineage of the AP2-like genes, and are well known for their function in induction of somatic embryogenesis  mutants show decreased sugar content in floral organs including anthers, resulting in a male sterile phenotype (DEFECTIVE in TAPETAL DEVELOPMENT and FUNCTION1 (TDF1) is required for tapetum programmed cell death ,G] in GAMYBL2, MYB29, and GAMYBL1, or a tree topology of  in GAMYB . Plant genomes encode a variety of sRNA types that can act in a transcriptional or post-transcriptional regulation mode. In this paper, we focused on miRNA and phasiRNA. The list of miRNA annotated in this study is likely incomplete because the Streptochaeta sRNA-seq data were limited to anther, pistil and leaf tissues, and would miss miRNAs expressed specifically in other tissues/cell types or at growth conditions not sampled. Thus, miRNAs missed in our data may well be encoded in the Streptochaeta genome. That being said, our miRNA characterization provides a starting point with which to describe Streptochaeta miRNAs, and our sequencing depth and tissue diversity was likely sufficient to identify many if not the majority of miRNAs encoded in the genome.sRNAs are important transcriptional and post-transcriptional regulators that play a role in plant development, reproduction, stress tolerance, etc. Identification of the complement of these molecules in PHAS loci  are 21-nt or 24-nt sRNAs generated from the recursive cleavage of a double-stranded RNA from a well-defined terminus; these transcripts define their precursor HAS loci . ReproduHAS loci , expressof maize  to thous in rice , barley  in rice , and bre in rice . Additio in rice  and rice in rice  showed t tapetum , 2012 suStreptochaeta. We annotated tens of PHAS loci in Streptochaeta showing that anthers express phasiRNAs even in the absence of the middle layer. Likewise, in maize, PHAS to 24-PHAS loci from asparagus (<1), an Asparagaceae, to grass species (>1), although the implications of this shift are as yet unclear.Given that most of our data (>100 M reads) were collected from anthers, we have good resolution for annotation of phasiRNAs in this tissue. We characterized their absence/presence in the three-layer anthers of PHAS loci are likely trans-acting-siRNA-generating (TAS) loci, important in vegetative tissues, but typically there are only a few TAS loci per genome  and the PACMAD clade , but, because these comparisons involve two sister clades, it is impossible to determine whether the BOP or the PACMAD clade character state is ancestral. Streptochaeta functions as an outgroup in such comparisons and can help establish the direction of change. Here, we highlight just a few of the traits whose analysis may be helped in future studies by reference to Streptochaeta and its genome sequence.The four species of Anomochlooideae contribute to understanding the evolution of the grasses and the many traits that make them unique. We have highlighted the unusual floral and inflorescence morphology of The grasses, including not only Anomochlooideae, but also Pharoideae and Puelioideae, the three subfamilies that are successive sister groups of the rest of the family, appear to have originated in environments with low light and high humidity . The shiStreptochaeta, like other forest grasses, has broad, spreading leaf blades and a pseudopetiole that results in higher leaf angle and increased light interception (Streptochaeta may provide insight into how leaf angle is controlled in diverse grasses. Leaf width in maize is controlled particularly by the WOX3-like homeodomain proteins NARROWSHEATH1 (NS1) and NS2, which function in cells at the margins of leaves (NS1 and NS2 genes in the Streptochaeta genome could test whether the models developed for maize were present in the earliest of grasses.rception . Leaf anrception . A closef leaves . DuplicaJoinvillea develops colorless cells in the mesophyll (Streptochaeta will be a useful system for understanding the development of fusoid cells in early diverging and other grasses.The grass outgroup esophyll . These aesophyll . While t2). In Streptochaeta the silica bodies are a distinctive shape, being elongated transverse to the long axis of the blade  in details of its anthers and pollen development, and the current genome provides tools for comparative analyses. The sRNAs described above are produced in the epidermis, endothecium and tapetum of most grasses and we presume they are also produced in those tissues in Streptochaeta. In all grasses except Anomochlooideae and Pharoideae, the microsporangium has four concentric layers of cells – the epidermis, the endothecium, the middle layer, and the tapetum – which surround the archesporial cells (Streptochaeta and Pharus, however, the middle layer is absent (Streptochaeta and Pharus. In known mutants of maize and rice, loss of the middle layer leads to male sterility (Streptochaeta are unclear.al cells . Cells ial cells . In Stres absent , 2012 anterility  so the fStreptochaeta is unclear, with contradictory reports in the literature (Development of microsporangium layers may also be related to the position of microspores inside the locule. In most grasses, the microspores and mature pollen grains form a single layer adjacent to the tapetum, with the pore of the pollen grain facing the tapetum, unlike many non-grasses in which the microsporocytes fill the locule and have a haphazard arrangement. The condition in terature , 2012.Streptochaeta and its grass sisters have several GAMYB genes, which are known to be involved in exine formation in rice (The exine, or outer layer, of grass pollen is distinct from that of its close relatives due to the presence of channels that pass through the exine. While controls of this particular aspect of the pollen wall are unknown in the grasses, we find that  in rice  and to h in rice .Streptochaeta and other early diverging grasses will be useful for resolving this open question, but will require pseudomolecule-quality assemblies. Two other species of Streptochaeta have been reported to have n =11 chromosomes (Anomochloa marantoidea, n = 18 (Joinvillea plicata and Ecdeiocolea monostachya have n = 18 (n = 19 (Estimates of the chromosome number and karyotype in the common ancestor of grasses have reached different conclusions e.g., , in partomosomes , well be, n = 18 . The oute n = 18  and n =  (n = 19 , respectStreptochaeta, with additional insights possible in, for example, the study of embryo development, caryopsis modifications, endosperm/starch evolution and branching/tillering. We have demonstrated that genomes of targeted, non-model species, particularly those that are sister to large, better-studied groups, can provide out-sized insight about the nature of evolutionary transitions and should be an increased focus now that genome assembly is a broadly accessible component of the biologist’s toolkit.Finally, these are but a few of the opportunities for understanding trait evolution in the grasses based on investigation of PRJNA343128. The final genome assembly, annotation and Bionano optical maps are also available in CyVerse data commons at: https://datacommons.cyverse.org/browse/iplant/home/aseetharam/Streptochaeta_v1_publication_release_2021-05-13. The scripts and commands used for generating assembly, annotations, small RNA analyses and phylogenetic analyses are documented in the GitHub repository: https://github.com/HuffordLab/streptochaeta.The sRNA-seq data were reported in a previous study . Also, oMH, AS, EK, and LC designed the project. LC and EK provided the plant material. MH and AS generated the sequence data and assembled the genome. SB and BM analyzed the data on small RNAs. YY analyzed the AP2 and MYB sequence data. All authors drafted and edited the manuscript, and produced figures and tables.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Bearing vibration signals typically have nonlinear components due to their interaction and coupling effects, friction, damping, and nonlinear stiffness. Bearing faults affect the signal complexity at various scales. Hence, measuring signal complexity at different scales is helpful to diagnosis of bearing faults. Numerous studies have investigated multiscale algorithms; nevertheless, multiscale algorithms using the first moment lose important complexity data. Accordingly, generalized multiscale algorithms have been recently introduced. The present research examined the use of refined composite generalized multiscale dispersion entropy (RCGMDispEn) based on the second moment (variance) and third moment (skewness) along with refined composite multiscale dispersion entropy (RCMDispEn) in bearing fault diagnosis. Moreover, multiclass FCM-ANFIS, which is a combination of adaptive network-based fuzzy inference systems (ANFIS), was developed to improve the efficiency of rotating machinery fault classification. According to the results, it is recommended that generalized multiscale algorithms based on variance and skewness be examined for diagnosis, along with multiscale algorithms, and be used to achieve an improvement in the results. The simultaneous usage of the multiscale algorithm and generalized multiscale algorithms improved the results in all three real datasets used in this study. Bearings are among the most important and useful rotating machinery components . A lack The vibration signals of bearings usually exhibit a nonlinear behavior due to the effects of coupling and nonlinear interactions, friction, damping, and stiffness , and fauEntropy is a measure of the disorder and predictability of the signal. It is one of the most powerful concepts used to evaluate signal characteristics . SeveralDisorder and complexity have different physical meanings ,11. AccoWang et al. utilized MDE for feature extraction in bearing diagnosis . CongzhiVarious techniques have been used along with RCMDispEn for bearing diagnosis. These techniques include the fast ensemble empirical mode decomposition , adaptivCosta et al. introduced generalized multiscale entropy (GMSE) in 2015 . GeneralBecause of the advantages of DispEn-based algorithms over the SampEn-, FuzEn-, and PerEn-based algorithms , the preA combination of several classifiers was used to overcome the limitations of each classifier and achieve higher efficiency ,32,33. IThe rest of the paper is organized as follows. N can be calculated in six steps [The DispEn for the time series ix steps :Step 1. The signal is normalized between 0 and 1. The series Here, y is mapped to an integer between 1 and c (Equation (2)):Step 2. Each member of the time series c is the class parameter and indicates the number of classes that can be members of the time series ith member of the classified series m and d, respectively, denote the embedding dimension and time delay. The embedding dimension is the dimension of the state space used for reconstruction.Step 3. All the template vectors Step 4. Each series m members, and each of them can be an integer from 1 to c [The number of possible dispersion patterns that can be attributed to each series m 1 to c . m-dimensional series created.Step 5. For every m and number of classes c is calculated according to Equation (6):Step 6. DispEn with the embedding dimension To calculate the normalized DispEn (NDispEn) according to Equation (7), DispEn is divided by the largest possible DispEn.m or c is too large, the computation time is high, although it makes the DispEn values more reliable [m is too small, the dynamic changes may not be detected in the signal, whereas large m may cause DispEn to be unable to observe small variations [m = 2 and c = 8 were used to calculate DispEn. When reliable . In addiriations . Based oriations ,5, the pnth-moment-based generalized MDispEns are displayed as GMDispEnn. They are implemented as follows:Multiscale dispersion entropy (MDispEn) and generalized MDispEn (GMDispEn) compute DispEn in several consecutive scales based on the first and other momenta. The x with the scale nth moment, is constructed [The signal is coarse-grained up to where the time series structed :For MDispEn, based on the first moment:2, based on the second moment (variance): For GMDispEn3, based on the third moment (skewness):For GMDispEnr constant while calculating the multiscale entropy (MSE) such that r = 0.15 The DispEn of the signal With a change in For DispEn, the parameters must be set in such a way that the number of possible dispersion patterns becomes smaller than the signal length nth-moment-based RCGMDispEn (RCGMDispEnn) with a scale factor of kth coarse-grained time series nth moment and the scale In the calculation of the RCMDispEn and the n is defined as follows:Hence, for every scale factor, RCGMDispEnIn RCGMDispEn, 2 and RCGMDispE2, and from 3 for calculating GMDispE3 and RCGMDispE3 [It must be noted that the scale starts from 2 for calculating GMDispEGMDispE3 ,38.ANFIS is a fuzzy model expressed in the form of a neural network . It is cAn ANFIS structure is composed of five layers with nodes in the same layer possessing the same function family, as explained below:First layer: In this layer, the membership degrees of each input with respect to the membership functions are calculated. Various membership functions can be employed here. Because the partial derivatives of the Gaussian function parameters are smooth, the Gaussian function was used in this research:ith rule and jth feature, and jth feature of the sth sample. The parameters Here, ith rule for the sample Second layer: In this layer, the fuzzy implication in each node is computed using the input membership degrees. N denotes the number of input features Here, ith node , which automatically constructs a fuzzy rule base for ANFIS. A combination of the least-squares method and the backpropagation gradient descent method was used to adjust the membership functions and other parameters.FCM is a clustering algorithm that assigns each data point to a cluster with a specific degree of membership. Dunn introduced this algorithm  and BezdN and D represent the number of data points and clusters, respectively. Moreover, ith data point, and jth cluster. jth cluster, and FCM employs the minimization of the objective function :(20)Jm denotes the noise. The vibration signal of ball bearing with an outer race fault was simulated as follows:Based on previous studies ,49, xserk, Two sinusoidal functions were employed for the harmonic part of Equation (23) ,51:(25)xIn this simulation, the characteristic frequency of the fault and the damping factor were assumed to be Fifty independent healthy and faulty bearing signals with a length of 2048 data points and a sampling frequency of 40 kHz were simulated. Moreover, Gaussian noise was added to them with the variance ratio of signal to noise of 0.257 . Figure 2, GMDispEn3, RCMDispEn, RCGMDispEn2, and RCGMDispEn3, were calculated for the simulated signals, with the results displayed in p-values smaller than 0.05 are identified with asterisks. According to 2, and RCGMDispEn3 possess higher fault distinguishing capability than MDispEn, GMDispEn2, and GMDispEn3, respectively, and their results have a smaller standard deviation. Distinguishing abilities of the bearing faults using the generalized methods are also displayed.MDispEn, GMDispEng effect size [2, GMDispEn3, RCGMDispEn2, and RCGMDispEn3 algorithms effectively show the differences between the healthy and the faulty conditions, similar to MDispEn and RCMDispEn. RCMDispEn, RCGMDispEn2, and RCGMDispEn3 have larger size effects and better fault separation capability than MDispEn, GMDispEn2, and GMDispEn3, respectively.Hedges’ ect size  was usedThis section uses datasets from Case Western Reserve University (CWRU), US , with a The signals consisted of 10 different fault conditions: healthy, ball fault, inner race fault, and outer race fault with intensities of 0.021″, 0.007″, and 0.014″. The shaft rotating speeds were 1772, 1750, and 1730 rpm.A detailed description of the data set is shown in 2, GMDispEn3, RCMDispEn, RCGMDispEn2, and RCGMDispEn3 were calculated for all the signals, and their values were used in 20 scales as features for fault detection and classification. A binary vector was used as the target vector for every bearing condition. This binary vector had a length of 10 because 10 conditions were being studied. This research employed 10 FCM-ANFIS, each of which detected one element in the target vector.Specifically, 72, 18, and 90 signals were used for training, validation, and testing, respectively. MDispEn, GMDispEn2, and RCGMDispEn3 performed better at classification than MDispEn, GMDispEn2, and GMDispEn3, respectively. Moreover, the simultaneous use of RCMDispEn, RCGMDispEn2, and RCGMDispEn3 as the classifier inputs produced the most accurate classification. The faulty conditions classification using multiclass FCM-ANFIS was performed 20 times with different inputs. The results of classifying these features are displayed in Part of the PHMAP 2021 data challenge dataset  was usedThree fault conditions were examined: (1) high Looseness of V-belt, (2) faulty bearing, and (3) fault-free condition. Three hundred independent signal samples with a length of 1024 samples were separated for each fault condition. 2, RCGMDispEn2, GMDispEn3, and RCGMDispEn3 were calculated for all the signals, and their values were used in 20 scales as features for fault detection and classification. For each condition, 120, 30, and 150 samples were used for training, validation, and testing, respectively. These data were classified 20 times using multiclass FCM-ANFIS. The results are displayed in 2, and RCGMDispEn3 as inputs. However, the mean accuracy of RCMDispEn and RCGMDispEn2 as simultaneous inputs was greater than that of other inputs. These results confirm the proposal of this paper regarding the use of generalized multiscale entropies with multiscale entropies to improve the results. The best classification results are displayed in MDispEn, RCMDispEn, GMDispEnThe data used in this section were from the ball bearing data collected in the Mechanical Engineering Construction and Drive Technology (KAt) Research data center, Paderborn University, Germany ,57. The classification of the datasets used in the present work is presented in A signal with a length of 1024 was separated from the beginning of every measured vibration signal, with 60 signals separated from each dataset, to obtain a total of 300 signals for each fault condition.2, RCGMDispEn2, GMDispEn3, and RCGMDispEn3 were calculated for all the signals, and their values were used in 20 scales as features for fault detection and classification. For each condition, 120, 30, and 150 samples were used for training, validation, and testing, respectively. These data were classified 20 times using multiclass FCM-ANFIS. The results, displayed in 2, and RCGMDispEn3. Moreover, the smallest classification accuracy corresponds to the features extracted by RCMDispEn, RCGMDispEn2, and RCGMDispEn, separately.MDispEn, RCMDispEn, GMDispEn3 and RCGMDispEn2 are more capable in separating bearing fault conditions compared to GMDispEn3 and GMDispEn2. Moreover, the combined use of RCGMDE, RCGMDE2, and RCGMDE3 produces better results than using one or two of these approaches in bearing fault diagnosis. The authors suggest investigating the potential of simultaneously using generalized multiscale and multiscale algorithms in other fields. The present paper investigated the simultaneous use of refined composite MDE based on different moments  to probe properties of the signals related to higher moments in bearing fault diagnosis. To this end, a bearing simulation example and three real datasets were utilized. Furthermore, the bearing fault classification was performed using multiclass FCM-ANFIS to examine the proposed technique. The results indicated that our developed RCGMDispEn


Galea spixii). Forty-eight rodents  were randomly divided into the treated and control groups. Letrozole administration started at 15 days of age and continued weekly until 30, 45, 90, and 120 days of age. The body, testis, and ovary weights were analyzed, as well as the morphological progression of spermatogenesis and folliculogenesis. Macroscopically, body weight gain and gonads weight were increased in the letrozole group. Microscopically, the ovaries of treated females showed stratified epithelium and a cellular disorder of the tunica albuginea. In the testes of treated males, the development of seminiferous tubules was delayed and sperm was absent. The collective findings indicate that the prolonged use of letrozole alters secondary sexual characteristics, and causes weight gain, reproductive changes, and male infertility.Letrozole is used as a therapeutic agent in reproductive disorders caused by high estrogen levels. Letrozole inhibits cytochrome P450 aromatase and reduces estrogen levels. However, the effects of long-term use on reproductive traits are unknown. The aim of this study was to evaluate the prolonged use of letrozole in the gonads of rodents (Spix's yellow-toothed cavy;  Letrozole is a selective P450 aromatase inhibitor that decreases the amount of estrogen produced without changes in other steroidogenic pathways . AromataP450 aromatase is involved in the development of nuclei of sexual dimorphisms and aromatization zones. These zones seem to be related to the control of reproduction and sexual behavior, as described in mammals, amphibians, birds, fish, and reptiles . FurtherLetrozole is widely used as a therapeutic alternative to treatments for reproductive disorders, including neoplasms . ComparaLetrozole is therapeutically effective in reversing reproductive illnesses related to infertility caused by hormonal imbalance, such as hypogonadism and polycystic ovary syndrome . HoweverThe blocking of aromatization in the prenatal androgen stage is important in establishing the pattern of sexual preference, sexual behavior, and male arousal . Even soThe aim of this study was to evaluate the effects of prolonged use of letrozole on the postnatal development of biometric reproductive parameters of rodents . This rodent species has been used as an experimental model for other rodents and humans to identify steroidogenic events related to reproduction and preservation .The animals were collected from the Centro de Multiplicação de Animais Silvestres” (IBAMA 1478912/2011) of the Universidade Federal Rural do Semiárido, located in Mossoró, Brazil. The research was authorized by the Ethics Committee of the School of Veterinary Medicine and Animal Science of the University of São Paulo, São Paulo, Brazil (protocol number: CEUA FMVZ/USP nº 7781180516/2013) and by the Biodiversity Information and Authorization System (SISBIO nº 41910-4).G. spixii of each sex  were equally divided into treated and control groups. Age was established according to the phase of sexual development previously defined in previous studies as immature (15 days-of-age), pre-pubertal (30 days), pubertal (45 days), post-pubertal (90 days), and mature sexual (120 days) , 2017b.The animals were bred in enclosures measuring 5 × 5 m2 and were paired in boxes according to sex. Each box (2.5 m2) had a sand floor, screen, and was covered with ceramic tiles. After delivery, the birth dates and gender identification of the puppies were recorded. The females and their puppies were separated and placed in individual enclosures (2.5 m2) to start the experiment. The diet available ad libitum included commercial rabbit feed, corn, local fruits, and water.The experiment was conducted from June to December of 2016. According to the Laboratório de Metereologia e Climatologia at Universidade Federal Rural do Semiárido the average temperature during this time was 35.47°C, the relative humidity was 55.61%, rainfall amount was 5.70 mm, and average wind speed was 3 m/s. The duration of the days was approximately 12 h, with an average variation of 25 min.The first dose of letrozole  was at 15 days. Dosing continued weekly for 30, 45, 90, and 120 days. Each letrozole tablet (2.5 mg) was macerated and homogenized in 0.5 ml distilled water to produce a dose of 0.01 mg/kg body weight. The dose was delivered orally to each animal via a syringe.All animals were anesthetized with 0.3 ml ketamine  and 0.3 ml xilasin . They were then euthanized with 0.4 ml of intrathoracic potassium chloride. The animals were weighed and the right testis and right ovary were collected, weighed, fixed in 4% paraformaldehyde  for 24 h, and sectioned.Weight was used to indirectly assess the peripheral and/or tissue effects of letrozole's long- term action. Each rodent was weighed immediately prior to euthanasia using an WT21- LCD balance . The right and right ovaries were weighed during collection using a M214A analytical balance .Sections of the testes and ovaries were fixed in 4% paraformaldehyde  for 24 h, dehydrated in dilutions (70%–100%) of ethanol (Sigma-Aldrich), and embedded in paraffin. Sections of 5 μm were cut and mounted. Sections were stained using hematoxylin and eosin (Sigma-Aldrich) to investigate testicular and ovarian morphology. Following deparaffinization in xylol  at room temperature (RT) using two solutions for 10 min each, rehydration in a descending series of ethanol concentrations was performed , followed by distilled water . The sections were then stained with hematoxylin for 30 s at RT, washed in running water for 10 min, stained with eosin at RT for 15 s, and washed in running water for 10 min. The slides were then dehydrated in increasing dilutions of ethanol , cleaned in xylene (Sigma-Aldrich) at RT, 2 × 10 min, and mounted on Permount® . The analyses were performed by light microscopy.Body, testicular, and ovarian weights of 30-day-old animals were compared with the weight in 45, 90 and 120 day-old animals within and between the letrozole-treated and control groups using Shapiro Wilk and Bartlett test for normality and homoscedasticity , ANOVA fBody weight gain was greater in the group that received letrozole in both sexes and at all ages . Body weOvarian weight gain was greater in the group that received letrozole at 30, 45, 90, and 120 days . HoweverTesticular weight gain was greater in the letrozole group, except at 45 days-of-age . There wIn females not treated with letrozole, the surface of ovaries comprised simple cubic epithelium. The ovary was divided into two regions: a cortical zone with ovarian follicles at different stages of development, and a medullary zone with stroma and vessels. Letrozole altered the morphological development of the ovary . The conThe testicular parenchyma of 30-day-old seminiferous tubules underwent a luminal process, with cells at different stages of the seminiferous epithelial cycle. At 45 days-of-age, the seminiferous tubules and interstitial tissue were well defined. The seminiferous tubules had a lumen, with a germinative epithelium being formed at different stages of the seminiferous epithelial cycle. In 90-day-old animals, seminiferous tubules contained lumina, seminiferous epithelial cells at different stages, and spermatozoa. At 120 days-of-age, the seminiferous tubules displayed luminal areas with cells at different stages of division, allowing their classification into stages of cellular associations as described previously . HoweverProlonged use of letrozole increased body weight gain and gonads, induced infertility in males, and caused dysplasia in females. Letrozole causes an imbalance in the sex hormone steroid ratio, because it induces the reduction of estrogen levels by blocking the action of cytochrome P450 aromatase . This deOur findings suggest that the likely increase in androgen levels at both the tissue and peripheral levels may result in an anabolic effect caused by the action of letrozole , which pSimilarly, the use of the estrogenic inhibitor fulvestrant resulted in heavier male rats, similar to the testes . In contThe present study is clinically important, since letrozole is widely used as an alternative therapy for hormonal decompensation in both sexes . Long-teLetrozole affects the sexual development of Spix’s yellow-toothed cavy by promoting male infertility and inducing dysplasia in females upon prolonged use.


COL III, with median values of 9.5 (IQR 3.63)% (OA group) and 17.10 (IQR 11)% (control cases), respectively, showed significant reduction in OA patients (p = 0.002). HA showed a median value of 10.01 (IQR 8.11)% in OA patients, denoting significant decrease (p < 0.0001) with respect to the control group median 39.31 (IQR 5.62)%. The observed differences suggest a relationship between fascial pathology and hip OA. The observed increase in COL I in OA patients, along with the reduction of COL III and HA, could lead to fascial stiffening, which could alter fascial mechanics and be linked to the development and symptoms of OA.The present study compares the structure and composition of fascia lata in healthy subjects and in patients with hip osteoarthritis (OA), to evaluate any differences in the amount of Collagen type I, Collagen type III, and Hyaluronan. Fascia lata samples from voluntary healthy subjects and patients with OA were harvested during surgery. Collagen type I (COL I), III (COL III) antibody, and biotinylated hyaluronan binding protein (HABP) immunohistochemistry stainings were used to evaluate fascial morphology and COL I, COL III, and Hyaluronan (HA) content in both groups. Ten samples from healthy subjects and 11 samples from OA patients were collected. COL I was significantly more abundant in the OA group ( Osteoarthritis (OA) is a degenerative pathology of the joint , charactIt can be secondary to pre-existing conditions leading to structural alterations of the joint, such as congenital diseases , traumasUp to now, no treatment have been proven to stop or delay satisfactorily the progression of the disease . NonoperDespite several mechanisms having been proposed to explain its onset and progression, the aetiopathogenesis of osteoarthritis is still doubtful. Recent literature focuses on intra-articular environment, evaluating chondrocyte or synovial alterations, while little interest has been reserved to extra-articular structures. In particular, the role of fascia in the pathology is still unclear, as a characterization of fascial morphology and composition in hip OA is currently lacking, although fascial alterations have already been related to several musculoskeletal pathologies, as non-specific low back pain, sacro-iliac joint pain, and chronic shoulder pain ,29,30,31Fascial composition and organization, responsible for fascial properties , can be Moreover, Fede et al. demonstrated that fasciae are rich in hyaluronan (HA), which guarantees the gliding ability of the fasciae in respect to the adjacent tissues. The amount of HA varies according to the anatomical site and fascial function, showing higher concentration in aponeurotic fasciae, particularly in the retinacula, which do not adhere to underlying muscle, thus, being able to glide over them . ChangesThis study hypothesizes an association between fascia lata alterations and hip OA; in particular, changes in the production of collagen and HA could result in altered fascial structure and behaviour in OA patients.Hence, this research is aimed at comparing the content of type I collagen, type III collagen, and HA between healthy subjects and patients with hip OA, in order to understand if fascia lata composition can be altered in OA cases.The present research was approved by the Istitutional Ethical Committee (n. 3722/AO/16) and performed in accordance with the ethical standards of the 1964 Declaration of Helsinki as revised in 2000 and those of Good Clinical Practice. All subjects participating in the study received a thorough explanation of the risks and benefits of inclusion and gave their oral and written informed consent to publish the data.Patients undergoing hip arthroplasty, either following hip OA or femoral neck fracture, or intertrochanteric hip fixation at the Orthopaedic Clinic of the University of Padua were included in the study. Exclusion criteria were malignant neoplasms, previous hip surgery, acute inflammatory diseases, or infectious diseases. For each patient, the presence and severity of hip osteoarthritis were evaluated on pre-operative X-rays according to the Kellgren–Lawrence classification . Patient2 sample was harvested from the anterolateral portion of fascia lata, proximal to the greater trochanter. All specimens were immersed in phosphate buffered saline and transported to the laboratory within less than one hour since harvest.For each patient, during surgery, a 1 cmAfter formalin fixation, samples were dehydrated in graded ethanol, embedded in paraffin, and cut into 5 mm-thick sections. On dewaxed and hydrated sections, Hematoxylin and Eosin (H&E) and Picrosirius stainings were performed for histological analysis.2O2 in order to inhibit endogenous peroxidase activity. For collagen I (COL I) and collagen III (COL III), after three washings in phosphate buffered saline (PBS), samples were incubated with bovine serum albumin (BSA) 0.2%. They were then treated overnight with the primary antibody  in BSA. After repeated washings in PBS, the sections were incubated with the secondary antibody . After three washings in PBS, the reaction was developed with 3,3′-diaminobenzidine . Samples were finally counterstained with Hematoxylin, dehydrated, and mounted with a coverslip using Eukitt (Agar Scientific). For each staining, a negative control was obtained by omitting the primary antibody.After deparaffination and hydration, sections were treated with a solution of H2O2 to block any endogenous peroxidase activity. They were then washed with 0.2% Triton-X, in PBS and incubated in 0.2% bovine serum albumin, BSA. Sections were then treated overnight with biotinylated HABP, 2 mg/mL (Millipore), in the same pre-incubation buffer, 1:900 dilution. After repeated washings in PBS, samples were incubated with the secondary antibody . After three washings in PBS, the reaction was developed with 3,3′-diaminobenzidine . Procedure was completed with Hematoxylin staining, dehydration in graded ethanol series and coverslip mounting with Eukitt. Negative controls were checked with similarly treated sections, omitting the primary antibody.To assess the amount of hyaluronan, an immunohistochemical staining for Hyaluronan Binding Protein (HABP) was performed. After deparaffination and hydration, samples were treated with avidin and biotin solutions  to block any endogenous avidin biotin activity. Sections were then incubated with a solution of 0.5% H2,k: 0.9 (0.85–0.95).Specimen pictures, taken with a digital camera  were analyzed using ImageJ software . At a map ≤ 0.05.Data were analysed using IBM SPSS version 25.0 software . The resulting effect size was calculated by G Power 3.1 according to Cohen’s d  and inteTwenty-one patients were recruited for the research, seven males and 14 females, with a mean age of 86 ± 11.68 years. Group A was composed by 10 patients, four males and six females, with a mean age of 76.9 ± 12 years. Group B included 11 patients, three males and eight females, showing a mean age of 85.82 ± 10.06 years. Sample characteristics are represented in p = 0.0015)  in the parallel fibrous fascicles of fascia lata. The amount of COL I was increased in group B, with a median percentage antibody positivity of 75.2 (IQR 13.11), while group A showed a median percentage of 67 (IQR: 8.71). There was statistical significance between the two groups ( 0.0015) .p = 0.02), with a median value of 9.5 (IQR 3.63)% (Collagen III (COL III) showed a median percentage positivity of 17.10 (IQR 11)% in group A; among arthritic patients it was significantly reduced (R 3.63)% .p < 0.0001)  showed a median percentage positivity of 39.31 (IQR 5.62)%. In group B, the median percentage was 10.01 (IQR 8.11)%. These data show a significant reduction of HA content in OA patients ( 0.0001) . Despite the diffusion of OA and the several proposed aetiologic mechanisms, most literature focuses on alterations of intra-articular environment, while the role of extraarticular structures in the development of the disease has been mostly overlooked. To our knowledge, no previous research has assessed any possible alterations of deep fascia in OA joints.Data from immunohistochemical analysis showed that collagen type I is significantly increased in the fascia lata of patients with OA. This alteration could result in a more rigid fascia lata in patients with OA, as this molecule is the main component of the parallel bundles of dense connective tissue responsible of fascial stiffness and force transmission. Indeed, the increases in COL I content following mechanical or hormonal stimuli  have alrCollagen III was mainly located at the periphery of collagen fascicles and in the loose tissue between adjacent fascicles. It showed significant reduction in OA subjects. Considering that this collagen type is present above all in the loose connective tissue and permits a better adaptability of the fascial tissue, its reduction can cause a stiffer fascia. Fede et al.  have alrHA was concentrated in loose tissue between adjacent fibrous fascicles. It showed a significative reduction in cases with OA, as represented by the reduced positivity to HABP. Since this molecule, mainly located in the loose connective tissue between adjacent fascicles, is fundamental for the correct sliding of fascial planes ,42, its The present study highlights for the first time a structural alteration of fascia lata in patients with hip OA. The observed increase of COL I, along with the reduction of COL III and HA, probably suggests a change in fascial mechanics, with stiffness and reduction of its physiological sliding properties, and the development of OA. A rigid fascia could indeed distort hip posture and gait, possibly resulting in joint overload, thus, contributing to disease pathogenesis and progression. Moreover, reduced fascial compliance could further limit joint range of motion, thus, worsening symptoms of OA as pain and movement reduction. Alternatively, the observed alterations could be a consequence of OA. In fact, altered joint mechanics subsequent to the disease could stimulate increased synthesis of COL I and decreased production of COL III and HA; the resulting altered composition could determine a fascial disfunction which could further worsen symptoms.On the basis of the present study, it is not possible to discriminate whether the observed fascial changes can be a contributing factor in the pathogenesis of OA or if they should be regarded as a consequence of the disease. Further research, particularly focused on initial stages of the pathology, is needed to determine the causal relationship between fascial composition and hip OA.Nevertheless, the correlation between fascial stiffness and hip OA can give several hints for the future treatment of OA. Firstly, it could give a rationale for nonoperative treatments aimed at treating fascial disfunction and restoring normal gliding properties of extraarticular tissues either in initial stages, where they could slow disease progression and reduce symptoms, and for patients not suitable for surgery, in which they could decrease pain and joint stiffness, thus, helping symptoms’ control. Despite that several studies ,18,20 haBesides, unrecognized fascial disfunctions, both preceding surgery or related to post-intervention fibrosis, could have a role in the persistence of pain and stiffness after hip arthroplasty, since they could maintain an altered limb mechanics, at least partly contributing to failure of surgical treatment. A proper detection of these conditions could, thus, allow proper treatment of these complications.The present research has several limitations.First, age is different between the analysed group, with the OA group showing an average older age. As age can also determine a modification in the fascial ECM ,49, it cFurthermore, we analysed only three components of the fascial ECM; study of other fascial ECM elements will be crucial to understand the complex multifactorial aspects of their alterations in hip osteoarthritis.Finally, another limitation of this study is the lack of specific hip functional scores that took in account the fascial alteration and of a clear diagnostic to confirm and quantify the involvement of fasciae. Certainly, it is necessary to develop specific scales that consider fascial disorders to better characterize the role of this structure in hip osteoarthritis.Hip OA is related to a structural alteration of fascia lata, which shows an increase in COL I content, along with a reduction in COL III and HA. These results suggest that hip OA is associated with a dysfunctional, stiffened fascia lata, with impaired sliding. An altered fascial behaviour could play a role in the pathogenesis of the disease and contribute to symptoms. Alternatively, fascial alterations could be stimulated by altered hip mechanics, and, thus, be a consequence of hip OA.