Iran has recently included integrase (INT) inhibitors (INTIs) in the first‐line treatment regimen in human immunodeficiency virus (HIV)-infected patients. However, there is no bioinformatics data to elaborate the impact of resistance-associated mutations (RAMs) and naturally occurring polymorphisms (NOPs) on INTIs treatment outcome in Iranian patients.In this cross-sectional survey, 850 HIV-1-infected patients enrolled; of them, 78 samples had successful sequencing results for INT gene. Several analyses were performed including docking screening, genotypic resistance, secondary/tertiary structures, post-translational modification (PTM), immune epitopes, etc.The average docking energy  of different samples with elvitegravir (EVG) and raltegravir (RAL) was more than other INTIs. Phylogenetic tree analysis and Stanford HIV Subtyping program revealed HIV-1 CRF35-AD was the predominant subtype (94.9%) in our cases; in any event, online subtyping tools confirmed A1 as the most frequent subtype. For the first time, CRF-01B and BF were identified as new subtypes in Iran. Decreased CD4 count was associated with several factors: poor or unstable adherence, naïve treatment, and drug user status.As the first bioinformatic report on HIV-integrase from Iran, this study indicates that EVG and RAL are the optimal INTIs in first-line antiretroviral therapy (ART) in Iranian patients. Some conserved motifs and specific amino acids in INT-protein binding sites have characterized that mutation(s) in them may disrupt INT-drugs interaction and cause a significant loss in susceptibility to INTIs. Good adherence, treatment of naïve patients, and monitoring injection drug users are fundamental factors to control HIV infection in Iran effectively. •Integrase inhibitors (INTIs) are the first‐line treatment in HIV-infected patients.•The average docking energy in elvitegravir and raltegravir was more than other INTIs.•Phylogenic tree analysis revealed HIV-1 CRF35-AD was the predominant subtype.•For the first time, CRF-01B and BF were identified as new subtypes in Iran. Plasma samples were collected for viral load assay from 850 HIV-infected patients originating from the south of Iran; they were either antiretroviral therapy-naïve or RTIs and/or PIs treatment-experienced patients with a viral load above 1000 IU/ml enrolling at the Behavioral Diseases Consultation Center (BDCC) affiliated with Shiraz University of Medical Sciences. Medication adherence means a ratio of the number of pills doses taken in patients to the number of doses prescribed over a given period . Accordi2.2Using “FACSPresto Near-Patient CD4 Counter (BD Biosciences)”, CD4 T-cell count was performed. Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) levels (IU/L) in the serum samples were measured using the commercial enzymatic kits of Biorex-Fars Company  and DIRUI Automatic Biochemistry Machine.2.3Serum RNA was extracted by the QIAamp Viral RNA Mini Extraction Kit , and Artus HI Virus-1 RT-PCR kit (Qiagen) was utilized to define viral load in all samples. HIV-INT region was amplified by RT-nested PCR using the primers listed in 2.4Based on the RAMs and the high frequent NOPs detected in the INT sequences, 78 patients were clustered into 11 groups (1-11) and 7 models including five INTIs mutated models  and two mutants: mutant 1 and mutant 2. The high frequent NOPs in the INT protein is shown in In this study, INTs of all 11 groups and 7 models are called mutated INT genes/proteins.AB703607). For example, to build CAB mutated model, all RAMs presented in our integrase genes that were attributed to the failure of CAB were inserted in INT reference gene. In addition, mutant 1 and mutant 2 were built by the insertion of all RAMs and high prevalent NOPs in the INT reference sequence. Nucleotide and amino acid sequences of mutated INTs genes/proteins are shown in Each group encompasses distinct mutations ; also, f2.5“Expasy's ProtParam”  Table 5Table 5a 2.6PTMs of proteins are involved in the attachment of functional groups or small proteins such as disulfide bridging, phosphorylation, glycosylation, etc. to specific residue in the protein that alters the charge and the structure of the protein. NetPhos ,12, DISP2.7Secondary structural features can change the binding pocket properties or affect the stability of the whole protein  that inf2.8To determine the tertiary structure, amino acid sequences of the mutated INT proteins were submitted in “I-TASSER” ,33. Amon2.9https://go.drugbank.com/)  .Table 8M3.4Escherichia coli and yeast. Grand average hydropathy (GRAVY) signified that all INT proteins had been hydropathy with a negative score of −0.359 , C-glycosylation sites (19), and some O-glycosylation sites  was amon3.6The results of secondary structure prediction of mutant model 1 (A) and reference gene, AB703606 (B) was analyzed , and the3.7The best-refined models were regarded for validation analysis; then, the qualified models were employed for docking analysis .3.8To evaluate the effect of RAMs  and that of high frequent NOPs ] on INTIs treatment outcome, we prepared the list of amino acids involved in protein-drug interaction and the related docking scores of INTIs and mutated proteins interaction, as shown in S230 N and Q95K mutations caused a substantial loss in docking energy of groups 7 and 10 for all five INTIs. In addition, reduction in the docking value related to some of the INTIs is displayed in generated models 1, 2, groups 1 and 2. In brief, major, minor, and some of the accessory (Q95K and S230 N) mutations were attributed to E value declined; nonetheless, high frequent NOPs did not influence the binding affinity. Thus, specific mutations in the mentioned groups and models might have a pivotal role in drug resistance.Although RAMs and NOPs were distributed in different INT domains, all mutated INT proteins were docked through CCD with all INTIs. More importantly, none of the RAMs and NOPs was involved in interaction with INTIs.Of note, some motifs of amino acids  and the residues at the positions of 74, 116, 117, 118, 127, 137, 139, 141, and 199 very actively participated in many docking interactions.3.9Among suggested B cell epitopes, only those that were not placed in α-helix and β-sheet structure of protein were analyzed for antigen properties. Finally, the probable antigens were chosen as favorable epitopes .Table 123.10Based on the phylogenic Neighbor-Joining tree of HIV-INT gene sequences of HIV infected patients in Iran which was generated with the corresponding INT gene of 89 subtype reference strains  and StanConforming to the phylogenetic tree result, some new subtypes including CRF-01B and BF were introduced in Iranian patients for the first time. Of note, our data displayed RAMs found in our patients including V72I, I201V ,54, M50I4INTIs regimens are highly efficient antiretroviral agents with long‐lasting potency and reduced toxicity, which are globally accepted in treating naive and experienced individuals. The presence of mutations in INT genes that can change the structural stability and flexibility of these proteins can impact the treatment outcome ,5.In this study, we completed several bioinformatics analyses on integrase genes and proteins of the most prevalent subtype in Iran, CRF-35-AD. INT genes failed to amplify in 73 out of 151 plasma samples via an effective RT-nested-PCR. It can be inferred that the emergence of new HIV strains may influence the efficiency of nested-PCR that highlights the importance of whole-genome sequencing of HIV-1 circulating in Iran periodically.In this study, we found that good adherence even to previous ART regimes was a significant factor to decline the new mutations. Similar to our data, some studies indicated that reduced adherence was linked to some situations and signs such as digestive symptoms ,56, deveTo control HIV infection and achieve better medical outcome, we need to upgrade the surveillance at three levels: identification and treatment of naïve treatment patients, medication adherence improvement, and regular monitoring of IDUs.From the present analysis, most of NOPs lied in the catalytic core domain that is target for INTIs ; yet, suHere, RAMs including major (R263K) , minor  might confer substantial reductions in susceptibility to a wide range of INTIs .R263K appeared with G134 N ,69 and Kin accordance with some studies that observed R263Ksustained a moderate loss in potency against DTG, EVG, RAL, and BIC about 2-fold and had a detrimental effect on CAB susceptibility [The E value of interaction between INTIs with integrase genes in group 5 carrying R263Kdeclined for four INTIs: EVG, DTG, RAL, and BIC. This result is tibility ,71.In addition, one minor RAM, L74I, was found in group 8 that reduced the energy value of BIC, CAB, and EVG. This result is somehow in line with a study that illustrated L74I would have a slight effect on INTIs susceptibility . The preIn our samples, accessory mutations including G163R, Q95K, S230 N, M50I, V72I, V249I, and L74 M appeared, which can cause a substantial loss in susceptibility to INTIs alone or in combination. INTI drugs usually select G163R ; this muQ95K and S230 N mutations were another accessory mutation that had a significant reduction in susceptibility to all INTIs in the CRF35-AD subtype, while no major, minor, or other accessory mutations were identified in this group. Evidence demonstrated S230 N conferred drug resistance and reduced DTG susceptibility by 3-fold.Some studies revealed that Q95K did not affect INTIs susceptibility or viral replication, but in the presence of some other mutations enhanced resistance to RAL and EVG ,78.Other investigations need to be done to clarify the functional role of G163R, and Q95K in response to INTIs.In addition, the effect of L74 M was evaluated alone or in combination with other mutations. From our data, L74 M retained the docking energy and similar to M50I, may be responsible for the increase in free energy of binding values.In this study, M50I and R263K were not present in one patient simultaneously. Hence, M50I and R263K were inserted in INT reference, resulting in generation of 5 models, including mutant 1, 2, BIC, DTG, and CAB mutated models followed by the evaluation of the effect of these mutations on the E value. The reduction in energy value was only found in the interaction of BIC drug with BIC-mutated models and mutant 1; it can be concluded that combination of M50I and R263K possibly hurts the sustainability to BIC. In contrast to our data, some studies declared M50I along with R263K was responsible for remarkable loss in DTG ,80, BIC Ceccherini-Silberstein et al. reported that G134S in conjunction with some other mutations resulted in INT catalytic core destabilization and reduction in INTIs efficiency [The high frequent NOPs  were identified in our patients not previously reported in two earlier studies in Iran, except for G134S . In our ficiency ,77. ClinOne of the NOPs in our patients was L101 M, which has not been described previously as RAMs, but L110 M in groups 5, 7, and 9 decreased the docking energy in some INTIs that may be correlated with drug resistance in vivo. Developing the mutant virus carrying L101 M can clarify the influence of this mutation on viral fitness, integration steps, etc.According to our data, the presence of RAMs and NOPs may cause slight effect on binding energy and drug efficacy; thus, INTIs are likely to be capable of treating Iranian patients infected with the CRF35-AD subtype. This may be due to the location of the amino acid substitutions that were not in the conserved parts of the INT core domain  .To understand the effect of the NOPs and RAMs on binding affinity, performing molecular dynamics can provide a better explanation; nevertheless, the docking data in this report can also be helpful to declare the efficiency of different INTIs on Iranian patients.Based on the predicted amino acids involved in molecular docking, some motifs in the integrase proteins  and some amino acids  were involved in INTIs interaction. Therefore, these conserved regions may provide an absolute opportunity for drug development to target the integrase protein to increase sensitivity to INTIs leading to a favorable clinical treatment.Based on the affinity of the ligand-receptor complex, EVG and RAL were linked to higher free binding energy in our samples that can be considered for the optimal INTIs treatment in Iranian population.One of the reasons for the high score in our patients with EVG and RAL could be attributed to the presence of H bonds that exhibited a strong type of interaction.Viral infection use PTMs to enhance protein antigenicity and virulence properties; plus, increase protein solubilization, interferon response inhibition, and viral replication that have a significant role in viral pathogenesis.Therefore, host machinery cells remove the PMTs from viral proteins to activate immune response pathways, control the virus replication, and inhibit the viral protein synthesis to eliminate the virus.INT undergoes multiple PMTs that play versatile roles in the functions of INT and HIV-1 viral replication . PhosphoGiven the different disulfide bonds that can be formed between cysteines, special linkages are shaped according to the energy and structural constraints . Once foTherefore, disulfide bonds degradation may degrade viral proteins and provide a new area for antiviral drugs development.In this report, none of RAMs and NOPs in our sequences provided the new SUMOylation target site. Hence, other factors may have affected the position of SUMOylation. INT is susceptible to be SUMOylated at three SUMOylation sites  on three Lys residues . INT SUMOylation impairment correlated with a significant drop in integration events and inhibited replication ,90,91.Unlike the second SUMOylation site, the first and third SUMOylation sites were conserved in all our samples that can be a desired target for designing drugs to destabilize HIV-INT.Stabilization of INT is required for efficient genomic interaction which can be done via blockade of ubiquitination associated with proteasomal degradation .Here, different lysines were suggested as ubiquitin targets using bioinformatics tools. Among them, K186 and K240 were essential residues since they play a significant role in the structure and functions of INT protein . To usurVarious agents have been categorized as carbohydrate-binding agents (CBAs) that impede virus infection. To suppress the vast majority of viruses, the development of antivirals components to target special deglycosylation sites in viral proteins may provide an absolute opportunity for clinical therapies.This report defined N, O, and C glycosylation sites and, to the best of our knowledge, the influence of glycosylation on integrase proteins and HIV pathogenesis is not described clearly; thus, experimental studies are needed to elaborate on this matter .The data of ProtParam determined the notable similarity in INT mutated proteins in all patients with reference.However, the pI in different integrase proteins was slightly different, which can be described by the diversity in the number of basic amino acids, as mutated INT proteins are basic proteins. Accurate prediction of the pI of viruses is beneficial for physical/chemical treatment processes and modeling virus behavior in environment . MoreoveAttributing to the short half-lives of proteins assessed by Protparam, all INT proteins in this study were a kind of fast degradation proteins in humans, yeast, and E-coli. The rate of protein degradation is dependent on a few factors such as molecular weight, size, and surface charge ., and vaccine development. There is no general expression system to use optimally for all mentioned purposes;thus, various expression host systems should be applied for each purpose.In recent years, HIV-INT recombinant protein were used for different approaches including serological diagnostic methods, therapeutic applicationsThe effect of the RAMs and NOPs on the secondary structure was analyzed and our finding revealed that these mutations did not change various properties of the secondary structure. Accordingly, significant changes in the binding pocket of the mutated INT proteins did not happen and drug potency was retained. This finding is in the same line with docking results in this study.In our study, only epitopes located in β-turn and random coil structures were considered for further analysis because such secondary structures are mostly placed in the surfaces of the protein and are more likely to be favorable for binding to antibodies . HoweverHere, the subtyping result of our samples is different according to the kind of applied methods. . From our data, INT gene can be considered as an appropriate region for HIV subtyping in Iranian patients if only the phylogenetic tree or Stanford HIV subtyping program was applied for subtyping.Subtyping results of our patients showed that CRF-35AD subtype was the most frequent subtype in Iran, which is in agreement with other studies in Iran ,99,100. In this report, the phylogenic analysis revealed some new subtypes; CRF-01B and BF; therefore, whole-genome sequencing is essential for such samples and those that were not amplified via RT-nested-PCR to confirm the emergence of the new strains.Till now, the list and role of RAMs are not identified for the CRF35-AD subtype. By comparing the mutations that developed in our CFR-35 AD subtypes with other subtypes , it can No compelling evidence was found that HIV-1 subtype, but effectiveness, toxicities, and tolerability of ART regimens need to be considered in choosing first-line or second-line therapy, in low-income and middle-income countries . HoweverAdvanced In-silico researches in drug discovery and vaccine designing have a considerable role in HIV studies that have accelerated the rapid advancement in the production and manufacture of medicine to balance the therapeutic options by clinicians.In other words, choosing a treatment strategy is attributed to the various factors including fewer side effects, availability, acceptable tolerability, short treatment duration, efficacy, and pan-genotype activity. This study could act as a stepping stone to designing novel experiments:Applying mentioned bioinformatic approaches may be very useful to examine the efficacy of antiretroviral drugs periodically in different epidemiological settings to anticipate the most efficient HIV inhibitors. Plus, such results can be even more beneficial if all predictions will be evaluated in-vitro. Furthermore, such tools can optimize new drugs to suppress HIV infection that may lead to the restoration of strong immune responses that aim to eliminate the virus.Moreover, the molecular docking methods enable the researchers to reveal the interaction between host factors and the HIV proteins to suggest the evolutionary relationship between them that is helpful to recognize the virus behavior.INT post-modifications are essential for HIV pathogenesis; therefore, applying bioinformatics tools to unveil PTM sites, pathways, and the underlying mechanisms can propose pharmacological inhibitors. Accordingly, to disclose PMT issue bioinformatic studies are suggested for new therapeutic approaches.5Higher E value of EVG and RAL of mutated INT proteins showed these drugs may help achieve optimal treatment response in Iranian patients. Our bioinformatics analysis showed that RAMs and NOPs led to zero to modest loss in INTIs potency, suggesting that INTIs can be considered in the first-line and salvage therapy in treatment of patients infected with CRF35-AD subtype. Among different NOPs, Q95K, S230 N, and L110 M lowered the strength of INTIs docking energy in interaction with INTs that may consider such mutations as the major or minor ones in the CRF35-AD subtype. Various post-translation modifications and B-cell epitope prediction suggested particular target sites and epitope regions for future antiretroviral drugs and vaccines design, respectively. PTM sites and pathways are possible pharmacological targets for new therapeutic approaches. RT-nested-PCR test failed to amplify INT genes in 50% of the samples that might be due to the emergence of new HIV subtypes in Iran; whole-genome sequencing is strongly recommended to clarify this point. More focus on improving the quality of HIV care, good medication adherence to any types of anti-HIV drugs, and treatment of naïve patients along with better management of HIV-positive IDUs are essential factors to achieve continuous HIV care and better medical outcomes in Iranian patients.10.13039/501100004320Shiraz University of Medical Sciences with the Certificate Reference Number of REC 270710028RA. All participants signed written informed consent before participating in the study, in accordance with the Declaration of Helsinki.The ethical permission for this research study was granted by the ethics committee of the Not applicable.10.13039/501100004320Shiraz University of Medical Sciences with the Certificate Reference Number of REC 270710028RA.This work was supported by the AH: design of the study, conceptualization & supervision,FGH: performing the experiments, FGH, AH, and ZH: gave scientific suggestions and controlled experiments, MJSH, SB and ZH: data collection, NKH, AH, FGH, and ZH: data analyzing AH: resources & funding acquisition; PK, AH and NKH: statistical analysis of data, AH, FGH: writing the original draft; AH: review & editing.All authors have read and approved the final manuscript.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


N = 13,668). The latent class modeling method was used to identify trajectory groups of selenium intake. A total of 4039 respondents developed hypertension. The incidence of hypertension was 30.1, 30.5, 30.6, and 31.2 per 1000 person-years among participants with cumulative average selenium intake of 21.0 ± 5.1, 33.2 ± 2.8, 43.8 ± 3.6, and 68.3 ± 25.2 µg/day, respectively. Region and selenium intake interaction in relation to hypertension was significant. In the multivariable model, cumulative intake of selenium was only inversely associated with the incident hypertension in northern participants (low selenium zone), and not in southern participants. Compared to selenium intake trajectory Group 1 (stable low intake), all three trajectory groups had a low hazard ratio for hypertension among the northern participants. However, Group 4 (high intake and decreased) showed an increasing trend of hypertension risk in the south. In conclusion, the association between selenium intake and the incidence of hypertension varied according to regions in China. In the low soil selenium zone, high selenium intake might be beneficial for hypertension prevention.The effect of selenium on hypertension is inconclusive. We aimed to study the relationship between selenium intake and incident hypertension. Adults (age ≥20 years) in the China Health and Nutrition Survey were followed up from 1991 to 2011 ( Hypertension is one of the most critical risk factors for cardiovascular disease and even mortality. In 2014, the prevalence of hypertension in adults (aged ≥18 years) was approximately 22% worldwide .Selenium (Se) is an essential trace element for human health. In selenoprotein, it plays a variety of physiological functions on the body, such as antioxidant activity, prevention of viral mutations, protection of intestinal mucosa, anti-inflammatory activity, glucose metabolism effects, and insulin sensitivity effects . The orgSelenium affects the prevention and treatment of hypertension because of its antioxidant capacity ,10. ManyMost present studies on the relationship between selenium status and hypertension used serum selenium concentration as the indicator of selenium status . Few popUsing data from the China Health and Nutrition Survey (CHNS), we wanted to evaluate the association between selenium intake and hypertension. Furthermore, we aimed to assess the selenium intake trajectory in the general population and its association with hypertension.n = 4982), those who had incredible estimates of their daily energy intake  (n = 113), pregnant or breastfeeding females (n = 169), individuals with implausible BMI , and those who participated in only one wave (n = 8470). A total of 15,479 samples were used as a baseline, and their selenium intake was estimated. Focusing on newly discovered hypertension at follow-up, we excluded patients who had hypertension at baseline (n = 1811). 13,668 samples were finally analyzed. Those excluded in the analysis were older (44.5 vs. 39.2 years), had high BMI (23.3 vs. 22.0) and were more likely to have high income (44.8% vs. 37.5%) and live in the urban area (54.0% vs. 34.4%).The CHNS is a prospective cohort study reviewed by the University of North Carolina (USA) and the National Institute of Nutrition and Food Safety (China). This study was designed to monitor the Chinese population’s health and nutrition status using a multistage random cluster sampling method to survey nine provinces of China. Geographically, we defined Heilongjiang, Liaoning, Shandong, and Henan as the northern regions and Jiangsu, Hubei, Hunan, Guizhou, and Guangxi as the southern regions. The respondents in the cohort included all the family members of the selected family. By 2011, the CHNS had conducted nine surveys . The 1989 survey did not collect data for each age group. Thus, we only analyzed the data collected from 1991 to 2011. Due to the high loss to follow-up caused by the accelerated urbanization process, residents of new communities were invited to participate in the survey as supplementary samples since 1997. The response rate for respondents with informed consent from 1989 to 2006 was more than 60 percent. In eight surveys from 1991 to 2011, 29,220 participants aged ≥20 years old were included in the study. We excluded subjects who had no dietary intake information , including those with hypertension at baseline. After excluding those with hypertension at baseline (n = 1169), the remaining 10,025 participants were included in the analysis of the association between the trajectory of selenium intake and incident hypertension  to measure blood pressure in participants’ right arms. Three measurements were made for each person, and the average value was considered to be the final value. For patients with diagnosed hypertension, we measured their blood pressure before taking medication. Participants were labeled as having hypertension when their average systolic blood pressure >140 mmHg and diastolic blood pressure >90 mmHg or being diagnosed with hypertension.During the three-day diet survey, trained investigators weighed and recorded food and condiments from household stock, market, garden, and leftover food waste. According to China Food Composition Tables, the intake of nutrients, including selenium and protein, was collected based on the average of three-day food consumption information. Regional differences of nutrient content in food  grown in different regions were reflected in the China Food Composition Table by assigning different food codes . We calc2 (Chinese standards) [In each survey, sociodemographic characteristics and health-related lifestyle information must be obtained through questionnaires. The socioeconomic variables were as follows: education ; per capita annual family income ; and urbanization levels  . The metandards) ,18. MeanSelenium intake was categorized into quartiles. The Chi-square test was adopted to analyze the differences of classification variables. The one-way analysis of variance was used to compare the differences of continuous variables. Cox proportional hazard model with time-varying cumulative selenium intake and covariates was used to calculate the hazard ratio (HR) for incident hypertension. Three models were established, as follows. Model 1 was adjusted for age, sex, and energy intake. Model 2 was further adjusted for fat and sodium intake, smoking, alcohol consumption, income, urbanization, education, physical activity, and BMI. Model 3 was further adjusted for two dietary patterns: the traditional southern and the modern patterns, based on a previously published factor analysis . The traThe latent class modeling method was applied to trajectory analysis. We constructed a group-based trajectory modeling to identify distinctive subgroups of individual trajectories of selenium intake within a population and profiling characteristics of individuals within the subgroups using the Stata command, traj. In the analysis, we included all participants who had data on their dietary intake collected at least three waves during 1991 and 2011. For each trajectory, we estimated several possible combinations of trajectory shapes  to identify the model. The number of trajectory groups was finally determined based on two criteria: (1) the smallest value of Bayesian information criterion and (2) each group should have at least 3% of participants. Finally, the Cox proportional hazards model was adopted to analyze the relationship between selenium intake and incident hypertension risk. Model was adjusted for age, sex, intake of energy, fat and sodium, smoking, alcohol drinking, income, urban, education, physical activity, and BMI. The results were visually presented. p < 0.05 (two-sided).STATA 16.1 was used. Statistical significance would be identified when At baseline, participants in the highest quartile of cumulative selenium intake had higher income and BMI than those in the lowest quartile . Energy During a median follow-up of nine years, 4039 of 13,688 participants  developed hypertension. Across the cumulative selenium intake quartiles, hypertension incidence was 30.1, 30.5, 30.6, and 31.2 per 1000 person-years . In the No interactions were observed between selenium intake and sex, income, and urbanization level to hypertension risk . A signin = 7149, 67.0%) had lower-than-average selenium intake profiles during all the waves; Group 2  had a stable medium intake of Selenium; Group 3  had a medium and increasing selenium intake; and Group 4  had a high intake that decreased over time. The mean baseline selenium intakes in Groups 1, 2, 3, and 4 were 35.7, 51.2, 51.2, and 87.6 µg/day, respectively  was used as the reference in multivariable Cox regression.In this large prospective cohort study, the selenium intake of most of the participants was below the recommended nutrient intake (60 μg/day) set by the Chinese Nutrition Society , and nonTo the best of our knowledge, this is the first longitudinal study that examines the association between selenium intake and hypertension. A cohort study conducted in Western Europe showed that low blood selenium was a risk factor for hypertension in males . WesternIn contrast, using a sub-cohort of 2530 adults who attended the CHNS between 2009 and 2015, low serum selenium level was related to the increased risk of developing hypertension . CompariThe interaction between region and selenium intake to hypertension is interesting. Such interaction may explain the inconsistent findings on the association between selenium intake and hypertension in studies conducted outside the country. Dietary intake is the primary way for humans to consume selenium. The level of selenium is positively correlated with the protein content of the food. Selenate in plants needs to be converted into an organic form before it can be consumed by humans . SeleniuSeveral mechanisms may explain the inverse association between selenium intake and hypertension in participants from the northern region. First, animal study results have suggested that Selenium inhibits adipocyte hypertrophy and adipogenesis . In a laThe positive association between selenium intake and hypertension in participants from the south region is puzzling. While a similar association between serum selenium and hypertension was found in other studies in the USA ,40, the Our study has several strengths. First, this is a longitudinal study with multiple waves of dietary assessments using three-day food recall, combined with household food inventory records. We used cumulative mean selenium intake in our data analysis to reflect the long-term selenium intake . Second,This study has several limitations that need to be acknowledged. First, it was unclear whether long-term intake can be adequately reflected in a three-day diet survey. The survey was not spread over four seasons. Thus, we were unable to adjust for seasonal variations in selenium intake. Second, although we adjusted for income, BMI, and dietary patterns in the multivariable analysis, residual confounding was still possible. Third, the subgroup analyses  were explorative. Furthermore, there was no data on serum selenium.In conclusion, selenium intake is relatively stable over the past two decades in the Chinese adults included in the study. Selenium intake was inversely associated with the risk of hypertension in participants from the north, but was positively associated with hypertension in participants from the south. High selenium intake appeared to be a protective factor for blood pressure in the low-selenium region. Thus, dietary supplement use should take the region into consideration.


To evaluate the success and complication rates of ultrasound-guided renal biopsy at a tertiary care hospital.This was a retrospective analysis of 97 ultrasound-guided renal biopsies, all performed by the same radiologist, between 1 March, 2017 and 31 October, 2019.Of the 97 biopsies evaluated, 87 had a definitive pathological diagnosis. In five cases (5.4%), the biopsy results were inconclusive and a second procedure was required. In seven procedures (7.6%), there were complications, all of which were properly resolved.Ultrasound-guided renal biopsy has proven to be a safe, effective method for the diagnosis of nephropathies, with high success rates. Its use has grown, especially in the last 50 years, aiming mainly to stratify risks and avoid unnecessary ablative surgeries and therapies, as well as to clarify the diagnosis and prognosis of glomerular diseases,3. In experienced hands, the procedure is associated with low complication rates (< 5%), which has made it a routine instrument for clinical evaluation,4.The use of renal biopsy as a diagnostic tool has enabled a greater understanding of the pathogenesis of several kidney diseases, from glomerulopathies to focal lesions, such as nodules, tumors, and complex cysts,5.The most frequently used imaging methods for guided procedures are ultrasound and computed tomography (CT). The choice of methods depends on many factors, including equipment availability, patient age, and the risks of exposure to ionizing radiation, as well as the physician and patient preferences. The advantages of ultrasound are that it allows real-time multiplanar imaging, can be used at the bedside, is versatile, does not use ionizing radiation, and is more affordable than are traditional methods, such as surgical biopsy. One disadvantage of ultrasound is that it is operator-dependent,4. Currently, the use of 16-gauge needles is recommended for the procedure. The needle gauge has a direct influence on the number of clusters obtained, as well as on complication rates. The outer diameter of a 16-gauge needle is 700 µm, whereas that of an 18-gauge needle is 350 µm. When considering the glomerular diameter in a normal adult (250 µm), the 18-gauge needle may not be of a caliber sufficient to obtain adequate samples. However, the use of a larger gauge needle  is associated with a higher risk of complications without obtaining a higher number of glomeruli. Therefore, in order to strike a balance between lower complication rates and higher success rates in the collection of adequate samples, the 16-gauge needle is the most attractive choice.Image-guided percutaneous renal biopsy may be used in order to diagnose suspected malignant lesions, defining whether they are of primary or secondary origin, as well as being used as a tool for the differential diagnosis of parenchymal nephropathies, clinically significant hemorrhage is rare. Macroscopic hematuria and hematomas have been observed, respectively, in 2-4% and approximately 20% of patients. Most of these incidents evolve to spontaneous resolution and are treated conservatively. The most feared complication is massive hemorrhaging, which occurs in 1-2% of procedures, especially in elderly patients, in patients with renal dysfunction (glomerular filtration rate < 30 mL/min/1.73 m2), and in patients with uncontrolled hypertension. Other, less common, complications include urinary clots (in 0.3% of procedures), tumor spread through needle-track seeding (in < 0.01%), and intra-abdominal organ perforation, which is extremely rare,7.The most common complication of image-guided percutaneous renal biopsy is hemorrhage. Although small hematomas can be detected in up to 91% of the proceduresThe present study aims to evaluate the success rates and complication rates of ultrasound-guided renal biopsies in patients with parenchymal nephropathies performed at a tertiary hospital. We also describe the pathological findings.This was a retrospective study in which we analyzed 97 renal biopsies performed between March 1, 2017 and October 31, 2019 at the University Hospital of the Federal University of Vale do São Francisco, located in the city of Petrolina, in the Brazilian state of Pernambuco.All of the biopsies analyzed in this study were performed in patients being followed at the glomerulopathy outpatient clinic of the hospital or in patients who developed acute kidney injury due to undefined causes during hospitalization. Patients previously submitted to renal biopsy were excluded, as were those in whom biopsy was performed to investigate a suspected malignant lesion.Data were collected from renal biopsy reports and from the hospital medical records department. The study was approved by the Human Research Ethics Committee of the Federal University of Vale do São Francisco (Reference no 83840317.4.0000.5196). Because the study involved only a review of electronic medical records, the requirement for written informed consent was waived. The procedures and materials cited in this project are compliant with the Declaration of Helsinki.The main indications for renal biopsy in this study were as follows: urinary anomalies, defined as hematuria or non-nephrotic proteinuria; nephrotic syndrome; nephritic syndrome; acute kidney injury/rapidly progressive glomerulonephritis; and chronic kidney disease. After the exclusion criteria had been applied, the sample comprised 92 renal biopsies. We studied variables related to the techniques employed in and the complication rates related to those biopsies. In addition, we analyzed the pathology findings for the biopsies, as determined by a renal pathologist..All procedures were performed by the same radiologist, who had more than five years of experience, using a 16-gauge needle , and were guided by ultrasound with a Logiq P5 system . For the biopsy of native kidneys, 16- or 18-gauge needles are typically usedIn accordance with the institutional protocol at our hospital, the ultrasound-guided renal biopsy procedure involved the following steps: a) placing the patient in the prone or lateral position; b) asepsis and antisepsis of the procedure site; c) local anesthesia with lidocaine 2%; d) ultrasound study to identify the region to be biopsied (posterior lower pole of the kidney); e) choice of the needle path and marking of the needle entry site on the skin surface; f) introduction of the needle under real-time ultrasound guidance ; g) coll. The procedure was performed with a sterile technique . In addition, patients were considered candidates for renal biopsy only if they were not hypertensive (with a blood pressure < 140 × 90 mmHg) and were not using anticoagulant or antiplatelet medications.In accordance with the protocol adopted by our department, all renal biopsies were performed only after laboratory confirmation of conditions favorable to the procedure  were excluded because they were repeat biopsies. Of the 92 remaining biopsies, 87 (94.6%) produced a definitive histopathological diagnosis, thus being considered appropriate, and 5 (5.4%) were inconclusive, thus requiring a new procedure. The mean number of fragments collected per procedure was 2.42, and the mean number of glomeruli per sample was 14.89 .The mean age of the 92 patients whose biopsies were evaluated in the present study was 36.2 ± 16.0 years . Less than half (38.0%) of the patients were male, with a mean age of 37.1 ± 16.9 years . Despite being predominant in the sample, the females had a mean age of 35.6 years ± 16.3 years , comparable to that of the males.In our sample, the main indication for renal biopsy was nephrotic syndrome (in 50.0%), followed by acute kidney injury/rapidly progressive glomerulonephritis (in 19.6%), nephritic syndrome (in 13.0%), chronic kidney disease (in 9.8%), and urinary anomalies (in 9.8%). Among the 46 cases of nephrotic syndrome, the main pathological findings were segmental/focal glomerulosclerosis and lupus nephritis, which were diagnosed in 14 and 13 cases, respectively. Among the specific diagnoses found, the most prevalent was lupus nephritis, which was diagnosed in 32 biopsies (36.8%). The remaining diagnoses are shown, in descending order, in Immediate complications related to the procedure occurred in seven patients, manifesting as macroscopic hematuria in three (3.3%), perirenal bleeding in three (3.3%), and subcapsular bleeding in one (1.1%). Follow-up imaging evaluations (ultrasound or CT) were performed in patients who evolved to worsening of red blood cell counts, hemodynamic instability, flank pain, or macroscopic hematuria. There was no procedure-related death or report of late complications in patients who were followed at the outpatient nephrology clinic.Of the four patients who had hemorrhaging after the procedure, three (3.3% of the sample as a whole) eventually required emergency clinical intervention. Two patients received blood products and, among the procedures performed, surgery for hemostasis and arteriography with embolization  were nec-14. Ultrasound-guided percutaneous renal biopsy is established as a safe routine procedure, with a well-defined risk profile and a low complication rate. Studies conducted at several referral centers have evaluated the application of this type of biopsy, involving real-time ultrasound and an automated biopsy device, and have shown that it is associated with low complication rates.Image-guided biopsies have been evaluated in a number of recent articles in the radiology literature of Brazil-19, which can be explained by the fact that, at the facility where our study was conducted, the interventional radiology department and the outpatient clinic for nephrology and glomerular diseases were established only recently (in March 2017).The size of our sample was small in comparison with the samples evaluated in other studies, Korbet et al., and Muñoz et al., respectively. However, the repetition of the procedure in inconclusive cases increased our success rate to 98.9%, corroborating the findings of other authors, who have reported that diagnostic confirmation is achieved in 80-100% of cases. The mean of 14.89 glomeruli per sample in our study was adequate and is in accordance with much of the literature, in which approximately 10 glomeruli per sample is considered appropriate,21.In the present study, the success rate for obtaining adequate samples by needle biopsy was 94.6%, which is lower than the 100.0%, 99.0%, and 97.5% reported by Manno et al., involving 220 patients, complications occurred in 42 patients (19.1%), major complications, including hypotension and a significant hematoma requiring blood transfusion, occurring in 16 patients (7.3%) and minor complications, including macroscopic hematuria and renal hematoma < 5 cm, occurring in 26 (11.8%).A small portion of the patients in our sample (7.6%) experienced complications inherent to the procedure, which is a promising result, despite the fact that interventional radiology is in the incipient phase at our facility, when compared with the complication rates reported in other studies. However, that result should be analyzed with caution, because follow-up imaging exams (ultrasound or CT) were performed only in cases in which there were clinical repercussions, such as macroscopic hematuria, flank pain, worsening of red blood cell counts, and hemodynamic instability. Therefore, it is possible that complications with less pronounced clinical or biochemical features were not identified. In a study conducted by Azmat et al. evaluated 203 ultrasound-guided percutaneous renal biopsies and reported a complication rate of approximately 8%, which is similar to the proportion observed in our study, although our sample was smaller. The Tang et al. study was also limited by the fact that post-procedure ultrasound was used only in patients who presented changes in clinical parameters . Those authors reported that most of complications were minor, mild hematuria occurring in 4.5% of the cases, whereas only 1.5% presented major complications that required blood transfusion and only 1.0% required percutaneous intervention with embolization.Tang et al. reported a complication rate of 5.6%, an interventional approach being required in only 1.67% of the cases. Their findings are similar to those of Ali et al., who evaluated 527 ultrasound-guided or “hands free” (ultrasound-assisted) renal biopsies, reporting an overall complication rate of 5.64% and a 2.84% rate of major complications. In the Ali et al. study, the use of a 14-gauge needle was identified as a risk factor for complications in the ultrasound-assisted biopsies, whereas there was no statistically significant difference in risk between the use of a 14-gauge needle and that of a 16-gauge needle (as used in our study) in the real-time ultrasound-guided biopsies. In that study, there were 213 patients who underwent ultrasound-guided biopsy with a 16-gauge needle, of whom two (1.29%) required blood transfusion and one (0.60%) required arterial embolization. In our study, only 16-gauge needles were used, the rate of hemorrhagic complications was 4.3%, two patients requiring blood transfusion and two requiring arterial embolization.In another study of percutaneous renal biopsies, Guerrero-Ramos et al., who observed lupus nephritis in 44.7% of cases, focal segmental glomerulosclerosis in 16.4%, and membranous nephropathy in 9.3%. Our findings are also similar to those found in the Pernambuco State Registry of Glomerulopathies, which included biopsies performed in the state capital (Recife) between 1998 and 2016, as well as being similar to those of a study conducted in the south of Brazil.Among the 85 biopsies in which the pathological finding was glomerulopathy in our sample, there was a predominance of secondary glomerulopathies over primary glomerulopathies, which accounted for 56.5% and 43.5%, respectively. The most common pathological diagnosis (seen in 36.8% of the biopsies) was lupus nephritis, followed by focal segmental glomerulosclerosis (in 20.7%), and membranous glomerulonephritis (in 9.2%). Our results are similar to those reported by Muñoz et al.Our study has some limitations. Because our study was based on information collected from electronic medical records, we faced challenges related to missing data and poorly organized archives, as well as difficulty in accessing the results of some previous laboratory tests, which are common limitations of retrospective studies. The small number of samples in our study is another limiting factor. However, the results obtained are relevant, given that the study was conducted at a facility that was established only a few years ago and in a semi-arid region of Brazil . In addition, our data may be included in the Pernambuco Registry of Glomerulopathies and serve to improve understanding of the behavior of glomerulopathies in this region of the country.Ultrasound-guided biopsy has proven to be a safe, effective method for the diagnosis of nephropathies, especially glomerulopathies, with a high rate of success in making a specific diagnosis. In addition, the success rates are even higher when repeat biopsies are performed.In our sample, the main indication for biopsy was nephrotic syndrome and the most prevalent pathological diagnosis was lupus nephritis. The procedure presented a low complication rate and there were no instances of nephrectomy or death as a result of the procedure.


Long-term exposure to childhood abuse and occurrence of mental illness are positively correlated. Using long-term tracking data in Korea, we identified the characteristics of children and adolescents who experienced abuse and impact thereof on their psychopathology. Using the Korea Welfare Panel data, 354 teenagers in grades 4–6 of elementary school participated, were assessed at baseline, and monitored 3 years later. They were categorized into Never, Occurrence, Continuation, and Discontinuation groups according to changes in the abuse experienced. Psychopathology was evaluated using K-CBCL. Childhood abuse experience significantly affected psychopathology. At the baseline, the Continuation and Discontinuation groups had a higher severity of psychopathology than the Never group. Psychopathology at the baseline was associated with whether the patient experienced abuse that year. In the follow-up observation, the risk of psychopathology in the Occurrence and Continuation groups was higher than that in the Never group. The Discontinuation group had decreased psychopathology, which was not clinically significant in the follow-up observation . Stopping abuse in late childhood reduces adolescents’ psychopathology in the long term, meaning they can recover their normal developmental trajectory according to risk groups and provide effective interventions including discontinuation of abuse. Recent neurobiological research has explored the development of the human brain during childhood and adolescence. This is a critical time in neurological development, and experiences of childhood abuse including physical, sexual, and emotional abuse and neglect, lead to children and adolescents deviating from their normal developmental trajectories. Children who experience abuse have a high risk of developing externalizing symptoms such as aggression and antisocial behavior in adolescence ,2. AbuseThere is a clear positive correlation between the duration of exposure to childhood abuse experiences and the incidence of psychopathology. In particular, the risk of persistent and repeated abuse leading to psychopathology is high ,8,9. WhiThe number of child abuse reports in Korea has steadily increased every year, with a total of 30,045 cases of domestic child abuse in 2019 . Given twww.koweps.re.kr, accessed on 14 March 2020) were combined and analyzed. The Korea Welfare Panel is a long-term survey conducted annually by the Korea Institute of Social Health and Seoul National University Social Welfare Research Institute, which includes representative samples of approximately 7000 households nationwide. The target population of KOWEPS concerns all households living across the country, representing 90% of the census. Trained interviewers visit participants’ homes and collect data through interviews. Additional surveys of children younger than 15 years are conducted every 3 years. In this study, 354 adolescents from grades 4–6 of elementary school were selected at baseline to check the longitudinal relationship of psychopathology according to the changes in abuse experiences. The mean (SD) age of the participants was 10.05 (0.799) years; 167 (47.2%) were male and 187 (52.8%) were female. Among the participants, 12 (3.4%) used alcohol, 3 (0.8%) smoked, and 48 (13.6%) had chronic diseases. A total of 337 (95.2%) were living with both parents, while 17 (4.8%) were living with single parents or grandparents. The household income of 304 (85.9%) participants was below the 60th percentile, and 23 (6.5%) participants received basic livelihood security. The study also employed data tracked 3 years later when they were in grades 1–3 of middle school.In this study, the baseline (taken in 2015) and a 3-year follow-up (done in 2018) of household data and children’s survey data from the KOWEPS . Emotional abuse (EA) consists of three questions pertaining to occurrences over the past year, for example: “I was scolded by parents enough to feel ashamed and insulted,” and “My parents have told me such things as ‘I would feel better without you,’ ‘You are a fool,’ or ‘You are a worse person than a dog.’” The Cronbach’s α for the four questions consisting of one physical abuse and three emotional abuse questions was 0.808 (baseline) and 0.757 (follow-up), respectively.The four groups were delineated according to changes in the abuse experiences over time. The control group  reported no abuse at baseline and at follow-up; the Occurrence group (O) did not report abuse at baseline but reported abuse at follow-up, the Continuation group (C) reported abuse at baseline and at follow-up, and the Discontinuation group (D) reported abuse at baseline but no abuse at follow-up.To assess adolescents’ psychopathology, we used the standardized Child Behavior Checklist in Korean (K-CBCL) ,15,16. I2 test. Moreover, ANOVA was used to compare the mean values of the K-CBCL scores according to whether the adolescents were abused at baseline and follow-up, and the Bonferroni method was employed as a post-test. Logistic regression analysis was used to determine the effect of longitudinal changes in abuse on each of the internalizing and externalizing symptoms at the follow-up point. In the logistic regression analysis, three models were used for covariate correction. The first model was a univariate analysis, and the second was a multivariate analysis including age, sex, academic achievement, alcohol use, smoking, chronic disease, cohabitation, family income, and basic livelihood security. In the third model, the presence of psychopathology in the K-CBCL that was investigated at baseline was used as an additional covariate. It was used to assess the effects of changes that abuse experiences had on psychopathology at the follow-up point. All analyses were conducted using IBM-SPSS 25.0, and schematized using the ggplot2 package of R.To identify the socio-demographic characteristics of the target population, a comparison was performed using a χAt baseline, 102 (28.8%) participants reported experiencing abuse over the past year, and 38 (10.7%) experienced both types of abuse. Furthermore, 58 (16.4%) reported experiencing physical abuse and 82 (23.2%) reported emotional abuse.p = 0.02). However, there were no significant differences between the groups according to age, sex, alcohol use, smoking, chronic disease, cohabitation, family income, and basic livelihood security.p = 0.005, EXT p = 0.001; D: INT p = 0.001, D: EXT p = 0.004). The level of psychopathology at baseline was associated with whether abuse was experienced in the past year. The severity of internalizing or externalizing symptoms could not predict whether abuse would continue in the future.After classifying the groups according to changes in abuse experiences, the K-CBCL score averages of the four groups were compared to determine each group’s psychopathological characteristics . Comparip < 0.01).To distinguish clinical significance, we set the K-CBCL cutoff point to 64 points corresponding to the 2 SD. A logistic regression analysis was conducted to determine the short-term effect of abuse on psychopathology in the same year at baseline . During p < 0.01).A logistic regression analysis was conducted to determine the effect of changes in abuse experiences on follow-up K-CBCL scores and the clinical significance of psychopathology . Model 3p = 0.24), and externalizing symptoms remained significant, but had decreased significantly . The clinical risk of psychopathology of the Occurrence (O) and Continuation group (C) increased significantly in both internalizing and externalizing symptoms , while the Never group (N) showed no significant difference. The subgroup analysis showed that unlike physical abuse, externalizing symptoms no longer exhibited clinical risk compared to the controls, especially when emotional abuse was discontinued .The K-CBCL scores in the Discontinuation group (D) decreased  and compp < 0.01). For the Continuation group (C), the risk of internalizing symptoms and externalizing symptoms increased 6.29 times and 16.41 times respectively .In contrast, the risk of internalizing symptoms and externalizing symptoms increased 9.36 times and 14.93 times respectively in the Occurrence (O) group than in the control group , which is the basis of this study, is cited as a critical time during brain development. Recent neurobiological studies have noted the responsiveness of the amygdala to abusive experiences . In partThe clinical significance of the study was that the discontinuation of abuse in children aged 10–12 years, an age suitable for monitoring, screening, and intervention, confirmed the possibility of reducing psychopathology in early adolescents aged 13–15 years.There are several limitations to this study.First, a relatively small sample of 354 children and adolescents was analyzed. However, it has sufficient clinical significance because it uses long-term follow-up observations from representative samples. This study also reproduced statistically significant results using a larger number of samples than existing studies with similar designs .Second, we established highly sensitive questions that distinguished between the presence and absence of abuse experiences. Further analysis in this study identified a positive correlation between psychopathology and abuse frequency, indicating that the association with the type of abuse was not significant. Although existing studies deal with the link between the severity of abuse and level of psychopathology, this study focused on quantitative relationships based on the accumulation of time exposed to abuse rather than the severity thereof. Existing studies have noted the major impact of the presence or absence of abuse on psychopathology, rather than the type, frequency, and severity of abuse when setting a cutoff for abuse experiences .Third, the types of child abuse did not include neglect, sexual abuse, or witnessing domestic violence. Several existing studies include these types of abuse, such as Adverse Childhood Experiences (ACEs), and have found that neglect is especially as influential as all other types of abuse ,13. In tFourth, there may be confounders that were not considered in this study. In particular, the effects of protective factors have not been sufficiently controlled. Existing studies have reported that individuals’ inner traits, such as positive self-esteem, self-resilience, and control, are protective factors that predict resilience ,31. AlthFifth, as the participants’ races and ethnicities are homogeneous, this study may be limited in generalizing to racial and ethnic groups beyond the Korean population.Despite these limitations, this study has several important implications. Representative samples were monitored for a long period, confirming the possibility that psychopathology in late childhood could suggest an abuse experience. This suggests that the previous abuse experiences of children who present psychopathology at the time of evaluation in late childhood must be intensively evaluated and interventions should be implemented. The study also showed the possibility that the discontinuation of abuse thereof could reduce psychopathology in adolescence in the long term. This suggests that the prevention of child abuse requires screening of risk groups and active, specialized interventions, especially as direct abuse discontinuation can restore adolescents’ developmental trajectory.


Faster gait speed was associated with decreased risk of falling  for every 10 cm/s increase in gait speed adjusted for age, gender, study arm, site, and MCI status. (4) Conclusions: The relationship between gait speed and risk of falling did not vary by MCI status .(1) Background: Falls are common in older adults and result in injuries, loss of independence, and death. Slow gait is associated with falls in older adults, but few studies have assessed the association between gait speed and falls among those with mild cognitive impairment (MCI). (2) Methods: The association between gait speed and falls was assessed in 2705 older adults with and without MCI participating in the Ginkgo Evaluation of Memory Study. Gait speed was measured via a 15-foot walk test and fall history through self-report. We used data collected at the 12-month (2001–2003) and 18-month visits (2002–2004). (3) Results: Participant average age was 78.5 years (sd = 3.2); 45% were female, and 14% had MCI at baseline. The average gait speed was 0.93 m/s (sd = 0.20). Sixteen percent ( Falls in older adults are common, affecting 20 to 33% of those over the age of 65 ,2. DeathNumerous studies have demonstrated an association between slower gait speed and increased fall risk in the general population of older adults ,10,11. HGinkgo biloba decreased the risk of Alzheimer’s disease [Ginkgo biloba on MCI. GEMS received Institutional Review Board (IRB) approval from all involved sites and this study was additionally approved by the University of Montana IRB.Our study population included 3069 adults aged 75 years and older participating in the Gingko Evaluation of Memory Study (GEMS) in four communities in the United States: Sacramento County, CA, Washington County, MD, Forsyth County, NC, and Pittsburgh, PA ,19. GEMS disease ,20. Stud disease ,20. ExclGait speed in GEMS was assessed annually as part of the Functional Assessment. The gait speed measurements used for this analysis were from the 12-month study visit. The gait speed measurement at the 12-month study visit aligns with the beginning of the 6-month period for falls reported during the fall history at the 18-month visit . Gait spFall history was ascertained from the Medical History questionnaire completed every 6 months over the course of the study. Participants were asked, “In the past six months since we last saw you, have you had a fall?”, with “yes, no, or don’t know” as possible responses. Any participant who responded “yes” to the question was considered to have had a fall in the past 6 months. Additional information about the definition of a fall was not provided to participants, however at the screening visit, participants were given instructions to not include falls that occurred during skiing, skating, or other activities that may affect balance, but these instructions were not provided at subsequent visits. For this analysis, fall occurrence as a dichotomous variable from the 12-month study visit was assessed as a potential confounder, and fall occurrence as a dichotomous variable from the 18-month study visit was the outcome of interest .MCI was ascertained at the screening visit and was determined based on criteria from the International Working Group on MCI ,23. StudIn addition to MCI, treatment arm assignment, and study site; age and gender were selected a priori for inclusion in the modified Poisson regression models for their demonstrated association with falls and gait speed ,21. We aParticipants with data for falls at the 18-month visit, gait speed at the 12-month visit, and MCI, determined at the screening visit, were included in the analysis. Participants were excluded if they were missing data for fall history at the 12-month visit or responded “don’t know” when asked if they had fallen at the 12-month or 18-month visit. Characteristics of excluded and included participants were assessed for statistically significant differences using t-tests and Chi-square tests. We summarized selected characteristics overall and by gait speed quartiles. We used modified Poisson regression with robust standard errors to evaluate associations between gait speed and risk of falling . We chosA total of 2705 study participants were included in the analysis . Of the p < 0.01), were more likely to be female (p < 0.01), were more likely to be from Forsyth County (p < 0.01), and were less educated (p < 0.01). There were no statistically significant differences in race or treatment arm assignment.Within the complete data set of 2705 participants, 45% of participants were female, the mean participant age at baseline was 78.5 (SD = 3.2) years, and 96% of participants were white . For heaWe observed evidence of a relationship between our exposure of interest (gait speed) and age, gender, MCI, study site, a fall reported at the 12-month study visit, education level, 3MSE score, use of an assistive device, history of a stroke, and alcohol use .We observed evidence that our primary outcome of interest  was associated with age, study site, fall reported at 12-month study visit, MCI, 3MSE score, use of an assistive device, history of stroke, and history of a heart attack .p-value = 0.66) , report of a fall at the 12-month study visit was selected as a confounder in the modified Poisson regression models because it changed the relative risk of falls associated with preferred gait speed by 10% . In the  = 0.66) .We observed a significant association between slower gait speed and increased risk of falling in older adults including those with MCI. This association persisted in models adjusted for age, gender, treatment assignment, study site, and MCI, but not after adjusting for falls at the 12-month visit. Although those with MCI are at a higher risk of falling ,25, we fThe magnitude of the association between gait speed and falls in our study is similar to those of another study evaluating gait speed as a continuous variable and fall risk . Other sOur study provides evidence that gait speed is a valuable predictor of fall risk even in those with cognitive impairment despite the fact that other factors might influence risk of falls among those with MCI. While these findings are not consistent with our hypothesis, that gait speed may be more strongly associated with falls in cognitively healthy older adults than those with MCI, these findings are consistent with previous work . In a stOur study had a number of strengths. Specifically, it benefitted from inclusion of a relatively large and geographically diverse population of older adults for who we have a robust determination of MCI, including multiple diagnostic tests and expert evaluation. In addition, the study included participants who are considered “old (75–84)” and approaching the status of “oldest-old (85 and older)”. These age-groups are at increasingly high risk of falls , and scrWe acknowledge some limitations. While a strength of the study was the inclusion of older adults in the oldest-old age group, the results may not be generalizable to the young-old (65–74 years). In addition, participants were also predominantly white, and the results might not be generalizable to people of other races. Given GEMS exclusion criteria, some chronic conditions were not represented in the data. Missing data were another limitation, with 12% of GEMS participants excluded from the analysis for missing values for falls, gait speed, and MCI. Those excluded from the analysis were more likely to be female, older, and have less education. Fall risk was ascertained by self-report, and was therefore likely underreported in this study; 18% of participants reported a fall; whereas the prevalence of falls found in other studies of adults over 65 is 20% to 33% ,28. HoweOur findings add to the evidence that gait speed and fall risk are associated for older adults with and without MCI. Importantly, from our study there was no evidence that the relationship between gait speed and fall risk varied by MCI status, providing support for the use of gait speed as a screening tool for falls for people with and without MCI.


Modern imaging techniques, such as TTE with cardiac strain, are contributing to the evaluation of patients with HFrEF, increasing the chances of CRT success, and allowing physicians to anticipate and plan for case management.Cardiac resynchronization therapy (CRT) represents an increasingly recommended solution to alleviate symptomatology and improve the quality of life in individuals with dilated cardiomyopathy (DCM) and heart failure (HF) with reduced ejection fraction (HFrEF) who remain symptomatic despite optimal medical therapy (OMT). However, this therapy does have the desired results all cases, in that sometimes low sensing and high voltage stimulation are needed to obtain some degree of resynchronization, even in the case of perfectly placed cardiac pacing leads. Our study aims to identify whether there is a relationship between several transthoracic echocardiographic (TTE) parameters characterizing left ventricular (LV) performance, especially strain results, and sensing and pacing parameters. Between 2020–2021, CRT was performed to treat persistent symptoms in 48 patients with a mean age of 64 (53.25–70) years, who were diagnosed with DCM and HFrEF, and who were still symptomatic despite OMT. We documented statistically significant correlations between global longitudinal strain, posterolateral strain, and ejection fraction and LV sensing (r = 0.65, 0.469, and 0.534, respectively,  Dilated cardiomyopathy (DCM) carries a significant burden for the patients and for the health care system and physicians as well. As the disease progresses, treatment options involving medication become limited as heart failure (HF) continues to worsen, severely limiting the patient’s ability to perform even the smallest of daily tasks . FortunaThis study aims to investigate whether several TTE parameters characterizing cardiac performance, and especially the strain imaging techniques such as LV global longitudinal strain (LV-GLS) and LV postero-lateral strain (LV-PLS) could be considered as predictors for suboptimal CRT functional parameters, namely low sensing, and high pacing, in patients with DCM and HFrEF.A single cohort of 48 patients suffering from DCM and HFrEF, who were implanted during January 2020 and June 2021 with three-lead pacemaker CRT (CRT-P) and three-lead intracardiac defibrillator (CRT-D) devices at the Institute of Cardiovascular Diseases from Timisoara, Romania, represent our study population. All patients were symptomatic despite optimal medical therapy (OMT), all had LBBB and were implanted with these devices according to current European Society of Cardiology (ESC) guidelines . PatientAll patients signed the standardized informed consent form required by the Health Authority of Romania at hospital admission, and their data were anonymized before data collection. The Ethics Committee of the Institute of Cardiovascular Diseases, Timisoara approved this study, Nr. 4052/19.06.2020.A complete bidimensional TTE evaluation was performed by a single blinded cardiologist, with advanced training in echocardiography, in all patients approximately 1 day before CRT intervention, using the General Electric VIVID E95 ultrasound system . Several parameters reflecting LV  and right ventricular  morphological and functional status, as well as atrial morphological status , were selected to be analyzed for this study.The left atrial volume (LAV) was measured by planimetry in a biplane manner, in 4 chambers and 2 chambers views, respectively, according to the European Association of Cardiovascular Imaging (EACVI) recommendations .The LV volumes (end-diastolic volume—EDV and end-systolic volume—ESV) were measured by planimetry (tracing the interface between the myocardium and the cavity), also in a biplane manner, in 4 chambers and 2 chambers views, respectively. The LV ejection fraction (LVEF) was measured by using the biplane modified Simpson’s method ,19.The longitudinal strain was evaluated by using the speckle tracking technique, in 4, 2, and 3 chamber views. The region of interest (ROI) was automatically rendered, with subsequent manual adjustments to obtain the best delineation of the myocardium to be analyzed. Care was taken not to include the pericardium in the ROI, and thus obtain falsely decreased absolute strain values. The LV was divided into 6 segments to be analyzed-basal, mid-ventricular, and apical, in each view. Three cardiac cycles were recorded for offline analysis in each view, at a frame rate between 40 and 90 frames/s, depending on the heart rate (higher frame rate for higher heart rates). The global longitudinal strain was calculated on the resultant bull’s eye model ,20,21.The longitudinal contraction of the RV was evaluated by measuring the tricuspid annular plane systolic excursion (TAPSE). This parameter was measured according to the EACVI chamber quantification recommendations, by M-mode, between end-diastole and peak-systole ,19.The systolic pulmonary artery pressure (sPAP) was evaluated by measuring the tricuspid regurgitation flow maximal pressure. The estimated right atrial pressure (RAP) was added to this value (5 mmHg if the diameter of the inferior vena cava (IVC) was <2.1 cm with >50% inspiratory collapse, 10 mmHg if the diameter of the IVC was >2.1 cm with >50% inspiratory collapse or the diameter of the IVC was <2.1 cm with <50% inspiratory collapse and 15 mmHg if the diameter of the IVC was >2.1 cm with <50% inspiratory collapse).In most patients, the implantation technique consisted of inserting a defibrillator/RV pacing lead into the RV cavity via the cephalic vein which was isolated in the deltopectoral groove. If the vein permitted, the atrial lead was inserted using the same route, if not, the atrial lead was inserted via the subclavian vein after positioning the coronary-sinus (CS) lead. Using the cephalic vein for the insertion of the RV lead allowed us to avoid difficulties in positioning because of lead interactions via the route of the subclavian vein, thus facilitating our control in cannulating the CS and maneuvering the CS lead into the optimal position. Arguably the most difficult step of the procedure is positioning the LV lead. This is mainly due to two anatomical variabilities (1) CS ostium and (2) cardiac veins themselves. Once the CS has been cannulated using the purpose built catheters, contrast is injected to visualize the particular anatomy of the cardiac veins, which presents significant interindividual variety . A guideWe consider ideal functioning parameters for pacing around 1 ± 0.5 V for the RV and right atrium leads and around 2 ± 0.5 V for the CS lead with ideal sensing parameters exceeding 10 mV in the case of the CS and RV lead and over 3 mV for the right atrium lead.p < 0.05, exclusion criteria p > 0.10) so as to obtain the most appropriate theoretical model that fit the collected data. The quality of the model was described using the accuracy of prediction and R squared. Data analysis was performed using the Statistical Package for the Social Sciences v.26 . A p-value < 0.05 was considered statistically significant.We employed the Shapiro–Wilk test to evaluate the distribution of numeric variables. Numeric variables were presented as the median and interquartile range (IQR), and categorical variables were presented as frequency and percentages. We employed Wilcoxon test to compare the QRS duration before and after CRT implantation. To evaluate the association between LV sensing/pacing and different echocardiographic parameters we used the Spearman correlation test. To assess the independent factors that influenced the LV sensing/pacing we built several multivariate linear regression models. In the final regression equations, the predictors were accepted according to a repeated backward-stepwise algorithm  years. Thirty-seven of them were men (77%) and 11 women (23%). Referring to the etiology, 37 had idiopathic DCM (64.6%) and 17 ischemic forms (35.4%). According to their symptomatology, most patients were in class NYHA III (47.9%), followed by class II (41.6%) and class IV (10.4%). The characteristics of the study population, as well as their therapy, are presented in All patients were treated according to the ESC guidelines  with OMTp < 0.001, Wilcoxon test). The median values of sensing and pacing parameters of CRT’ devices are presented in We observed a statistically significant shorter QRS duration after the CRT implantation (p = 0.049).By analyzing the existence of statistically significant correlations between several echocardiographic results and LV sensing and pacing, we identified that the most significant ones were evidenced between the strain parameters, followed by the LVEF and LAV both for LV sensing, but especially for LV pacing, To determine which independent factors, namely the LV sensing and LV pacing thresholds, could be predicted in our study group we employed a multivariate linear regression model. We used the forward stepwise method, and the best models were selected based on Akaike information criteria (AIC). We included in our models parameters such as age, sex, echocardiographic parameters, and patients’ comorbidities.2 = 0.721), the details being presented in The regression equation for the LV sensing proved to be adequate for the model, explaining 72.1% of the LV sensing variance (R2 = 0.446). Lower values of LV-GLS, RV-GLS, and TAPSE increase the LV pacing threshold, as well as the presence of diabetes mellitus.Referring to the LV pacing, the model presented in According to the newest ESC guidelines, CRT represents a Class I, Level A recommendation for the treatment of HFrEF, with LVEF under 35%, regardless of the NYHA class, in patients who are still symptomatic, despite OMT, to reduce morbidity and mortality, and especially, to alleviate their complaints and to improve their quality of life ,10,23. TAs they were evaluated before the procedure, a comprehensive TTE exam, followed by strain imaging was performed to characterize LV performance, to observe the severity of kinesis abnormalities, and the existence of mechanical asynchronism ,26. The As debated in the medical literature ,24,29,31Our results are explained by the fact that GLS  reflects the fibrosis burden in the myocardium. It is demonstrated that scar tissue does not have any electrical activity therefore no pacing and/or sensing is possible ,33. It iIn the same manner, even if there is no consistent scar on the LV lead targeted wall, a high amount of diffuse/interstitial or replacement fibrosis explain the altered of the pacing and sensing thresholds. The low response to CRT in these patients is induced, as demonstrated previously by several authors ,33,34,35It is true that longitudinal strain evaluates the longitudinal fibers contraction, fibers that are best represented (but not exclusively) in the subendocardial layer , and theThe main limitation of our study is represented by the small number of patients treated with CRT. The low case numbers were due to the COVID-19 pandemic with admission of non-COVID patients and reduced access to the interventional laboratory. As a consequence of the small patient population, we could not further analyze the impact of other factors, especially of associated diseases, on our results.Modern imagistic techniques, such as cardiac strain, are contributing to the evaluation of patients with heart failure with HFrEF who are referred for CRT implantation, also suggesting a course of treatment. Cardiac strain imaging results might be able to predict the chances of CRT being successful or not, allowing the interventional cardiologist to anticipate and plan for improved patient outcomes.


Clavibacter michiganensis subsp. michiganensis (Cmm) is the causal agent of bacterial canker of tomato. Differences in virulence between Cmm strains have been reported. The aim of this study was the characterization of nine Cmm strains isolated in Chile to reveal the causes of their differences in virulence. The virulence assays in tomato seedlings revealed different levels of severity associated with the strains, with two highly virulent strains and one causing only mild symptoms. The two most virulent showed increased cellulase activity, and no cellulase activity was observed in the strain causing mild symptoms. In three strains, including the two most virulent strains, PCR amplification of the 10 virulence genes analyzed was observed. In the strain causing mild symptoms, no amplification was observed for five genes, including celA. Sequence and cluster analyses of six virulence genes grouped the strains, as has been previously reported, except for gene pelA1. Gene sequence analysis from the genomes of five Chilean strains revealed the presence of deletions in the virulence genes, celB, xysA, pat-1, and phpA. The results of this study allow us to establish correlations between the differences observed in disease severity and the presence/absence of genes and deletions not previously reported. Clavibacter michiganensis subsp. michiganensis (Cmm) is the most important bacterial disease affecting tomato crops in Chile, due to severe symptoms and its easy dissemination. Disease symptoms include wilting, chlorosis, stem canker, vascular necrosis, and brown spotting surrounded by creamy-white halos on the fruit. However, differences in the expression of symptoms have been observed in the field as well as under controlled laboratory conditions [Bacterial canker of tomato caused by nditions ,2.Cmm [chpC, pat-1, phpA, and phpB), carbohydrate-active enzymes (CAZymes), including cellulases (celA and celB), xylanases (xysA and xysB), and pectinases (pelA1 and pelA2), and a tomatinase, encoded by the tomA gene. Studies on the presence of putative virulence genes carried out in different countries have shown variations among analyzed strains [celA and pat-1) was correlated, in some cases, with a delay in the expression of symptoms or in a reduction or total loss of virulence [Several genes have been associated with the virulence of Cmm ,4 such a strains ,6,7,8,9.irulence ,11,12.On the other hand, it has been demonstrated that genomes of bacterial pathogens are reduced in the process of permanent association with the host . This prCmm was carried out, using a multilocus sequence analysis (MLSA)/multilocus sequence typing (MLST) analysis based on five housekeeping genes [pelA1, which exhibited more variability in different strains, and the phpA and phpB genes, which were absent in one strain [Cmm strains isolated in Central Chile to reveal the causes of their differences in virulence. In this study, we compared the virulence observed in tomato plants and explored the gene repertoire, sequence variability, and presence of pseudogenes of virulence genes in Chilean strains belonging to the three MLST/MLSA groups. The results of this study showed correlations between virulence, pathogenicity genes’ repertoire, and the presence of pseudogenes.Previously, a characterization of 25 Chilean strains of ng genes , which re strain . The aimClavibacter michiganensis subsp. michiganensis (Cmm) strains used in this study are listed in Cmm strains had been previously identified and characterized through microbiological, biochemical, and molecular techniques [Cmm strains were routinely cultured in yeast-peptone-glucose agar  and incubated at 28 °C for 72 h.chniques . Cmm strCmm strains were selected, based on previous pathogenicity tests [Cmm strains were plated on YPGA and incubated for 3 days at 28 °C. Bacterial biomass was resuspended in sterile distilled water and adjusted to 108 CFU mL−1. Bacterial concentration was confirmed, using the dilution plate method. For each Cmm strain, five tomato seedlings were inoculated by puncturing the stem with a sterile needle 1.0 cm above the cotyledons and adding 10 µL of bacterial suspension onto the wound. Plants inoculated with sterile distilled water were used as negative controls. Disease symptoms were monitored every week. The assay was repeated three times. At day 21, plants were classified, based on disease symptoms: 0 = no symptoms; 1 = canker in the site of inoculation; 2 = canker extended by the stem; 3 = yellowing and slight wilting; 4 = wilting in all leaves; 5 = dead plant. The disease index (DI) was calculated, using the following formula:Nine Chilean ty tests , that shty tests . Brieflyp ≤ 0.05) using R version 3.6.3. Uninoculated controls were excluded from statistical analysis.Virulence assay data were analyzed. Normality and homogeneity of variances were first checked through Shapiro–Wilk test and Levene test, respectively. Then, they were analyzed by a one-way analysis of variance (ANOVA). The means were compared by Tukey’s HSD test  in a rotatory shaker at 150 rpm and 30 °C. Turbidity was measured by spectrophotometer and all cultures were diluted in YPGB to adjust the turbidity to 0.3 at 600 nm. Subsequently, 10 μL of each culture were plated onto TCYA medium  for cellulase activity. Plates were incubated for 4 days at 28 °C. Plates of TCYA medium were stained with Congo red (1 g L−1) for 20 min and finally bleached with 1 M NaCl. Enzymatic activity was compared by measuring the size of the halo around bacterial growth. Three replicates for each medium were carried out and the experiment was done three times.Endocellulase activity was evaluated, using the method described by Meletzus et al. , with mochpC and tomA [celA and pat-1 [phpA, phpB, celB, xysA, pelA1, and pelA2 gene amplifications, primer pairs were designed, based on the genome sequence of Cmm strain NCPPB382 as reference [Ten virulence genes were selected for PCR-amplification, using primer pairs for amplification of and tomA , and celnd pat-1  , using the primers described . Sequence comparisons were performed, using MUSCLE. The domain prediction was obtained based on protein sequences, using Interpro [Three available genomes’ sequences of Chilean analyzed . Carbohynd VL527  were chend VL527 ). For ChInterpro , and proInterpro .Cmm strains analyzed in this study exhibited different virulence levels on tomato seedlings cv. San Pedro . The percentage of polymorphic sites among Chilean strains and NCPPB382 was low in most virulence genes. The highest value of polymorphism was obtained for pelA1 with 3.3% (16/489), followed by celB  and celA . Less than 1% was obtained for tomA  and chpC . No polymorphism was observed in the pat-1 genes of the eight strains. The nucleotide sequences of PCR products were analyzed for the virulence genes pat-1 gene was not included in this analysis due to the absence of polymorphism. Each gene showed some differences in strain grouping. The celA, celB, and tomA gene analysis clustered Chilean strains in two, three, and one group, respectively, separated from strain NCPPB382. The chpC and pelA1 gene analysis clustered Chilean strains in two and four groups, respectively, one group including strain NCPPB382. Cluster analysis for five virulence genes was carried out , to visucelA and celB was confirmed in strains VL527, MSF322, OP3, and VQ28. In strain VQ143, the celA gene was absent, but celB was detected. The analysis of complete sequences of celB obtained from genome data allowed the detection of a deletion. The celB sequences of strains VL527 and NCPPB382 had 3G in positions 315–317, and in strains MSF322, OP3, VQ2,8 and VQ143 one G was missing (GGG→GG). This deletion caused a frameshift, and a stop codon truncated the protein after the aa 134, in the endoglucanase domain . This deletion changed the frameshift, and a stop codon truncated the protein after the aa 418, almost at the end of the xylanase domain  for pelA2 in strains MSF322, VL527, and VQ28, respectively, and 93.2% (97% sequence coverage) in strain OP3. For pat-1, the percentage of nucleotide identity was 100% (100% sequence coverage) in strains VL527 and OP3, and 99.8% identity (100% sequence coverage) in strain MSF322; no significant match was found for the gene in strain VQ28. The analysis of the gene sequences of pat-1 revealed two deletions. The sequence of pat-1 had 7G in positions 693–699 and 4T in positions 803–806 in all strains analyzed, except for the strain MSF322 that lost a G and a T, respectively . The first deletion caused a frameshift, and a stop codon truncated the protein after the aa 239, in the peptidase domain  for strains MSF322 and VL527, respectively, and no significant match was found for strains OP3 and VQ28. The analysis of gene sequences of phpA showed four deletions in the gene sequence of strain MSF322, at positions 551–556T (TTTTT→TTTT), 695–699T (TTTTT→TTTT), 737–741G (GGGGG→GGGG), and 794–798T (TTTTT→TTTT), where one nucleotide was missing in each case. The first deletion caused a frameshift, and a stop codon truncated the protein after the aa 189, in peptidase domain (phpB the nucleotide identity was 100% for strains MSF322 and VL527 (both 100% sequence coverage). BLAST analysis for the phpB gene in the genomes indicated the absence of this gene in strains OP3 and VQ28. The identities of nucleotide sequences between Chilean Cmm strains and reference strain NCPPB382 and the deletions detected are shown in CAZy.org database was consulted to obtain information about strains NCPPB382, VL527, and MSF322. Cellulase CelA is present in the strains NCPPB382, MSF322, and VL527, although CelB is present only in strains NCPPB382 and VL527. Based on PCR analysis and BLAST, the presence of e domain . The same domain . The same domain . This dee domain . For phpClavibacter michiganensis subsp. michiganensis virulence genes of nine Chilean strains and their potential correlation with expressed symptoms and cellulase activity was carried out. In this study, an analysis of Cmm strains in the expression of plant symptoms, ranging from severe to nearly asymptomatic. Variations in virulence between strains have been reported in other studies [The results of virulence assays showed differences between  studies ,2. Previ studies . Taking  studies .C. michiganensis [celA in strain VQ143 explained the low virulence observed in plants and almost null cellulase activity. The celA gene encodes for an endocellulase, which is a pathogenicity determinant that plays a major role in virulence and is located in the plasmid pCM1 in reference strain NCPPB382. The inactivation of the celA gene resulted in a loss of pathogenicity [pat-1 gene in strain VQ28 was not a factor in loss of virulence, because the plant symptoms observed were similar to those in plants inoculated with other Cmm strains carrying the pat-1 gene. The pat-1 gene encodes a serine protease, which plays a role in pathogenicity and is located on the plasmid pCM2 of reference strain NCPPB382. The deletion of the sequence associated with the pat-1 gene has been shown to significantly reduce virulence but does not resulted in a complete loss of virulence [celA and pat-1 genes are transcribed extensively by Cmm strain NCPPB382 during early stages of tomato infection [phpA and phpB genes are homologs of the pat-1 gene and are also located in plasmid pCM2 of reference strain NCPPB382. It was shown that the phpA and phpB genes do not induce disease symptoms in tomato plants [pelA1 and pelA2 genes encode for pectate lyases and are located in the chp/tomA region of the pathogenicity island (PAI) of reference strain NCPPB382. The pelA1 gene has an important role in pathogenicity, unlike the pelA2 gene, which has no effect on symptom expression [pelA1 gene of strain NCPPB382 during tomato infection has been reported [celA and pat-1 genes. All the strains analyzed, including five non-pathogenic strains, were PCR-positive for the celA gene. Only two non-pathogenic strains were PCR-negative for the pat-1 gene, suggesting that these genes are conserved in C. michiganensis populations. The study of Milijasevic-Marcic et al. [pat-1 gene. The authors suggest that the attenuated virulence is caused by the loss of pat-1 and, possibly, the loss of the plasmid carrying this gene. Bella et al. [pat-1 gene. These strains were able to cause disease, but in a delayed manner. No significant difference in the disease index (DI) was observed between strains with and without the pat-1 gene. In the study of Cmm strains isolated in Sicily, carried out by Ialacci et al. [celA gene PCR-positive, although the pat-1 gene was not detected in four strains. These four strains were still pathogenic, although with reduced virulence. Tancos et al. [phpA, pat-1, and celA genes, reporting that phpA is the most common gene to be lost, whereas celA is the most stable gene. No correlation with virulence and loss of phpA was observed. Interestingly, one strain lacking celA and phpA was pathogenic for tomato, while two strains that lacked celA showed a lower disease incidence. All pathogenic Cmm strains analyzed by Thapa et al. [celA gene); plasmid cured derivatives failed to induce bacterial canker symptoms on tomato, indicating the essential role of pCM1 in pathogenicity. In the same study, the absence of the pCM2-like plasmid (carrying the pat-1 gene) in some pathogenic strains indicated that this plasmid is not required for pathogenicity. The variable results obtained by different authors, especially in the importance of the pat-1 gene, could be resolved through the study of the complete genome sequences of the strains analyzed, which allows us to search for genes with greater efficiency and detect variations or deletions in the gene sequences and correlate this information with disease symptoms.Virulence is known to be dependent on the presence of virulence genes on the chromosome and plasmids of ganensis . In ordegenicity ,12. The irulence . The celnfection . The phpo plants . The pelreported . Other rreported  analyzedc et al.  revealeda et al.  analyzeds et al.  determinpelA1 gene, wherein the strain VQ519 separated from the group of strains belonging to ST32. Some studies of variability of virulence genes have reported low polymorphism values. In the study of Milijasevic-Marcic et al. [chpC gene and only two variations in the tomA gene. Tancos et al. [celA, tomA, chpE, pat-1, and phpA. No polymorphic sites in the chpE, pat-1, and phpA genes were observed. Low variation was observed in the celA and tomA genes, with polymorphism levels of 1.33% and 0.786%, respectively. Interestingly, high variability has been reported for the pat-1 gene [Cmm. This study revealed that most virulence genes are highly conserved, having more sequence variability in the pat-1, celA, and pelA1 genes. In the same study [Clavibacter michiganensis subsp. chilensis, strain CFBP8217, which is a non-pathogenic strain associated with tomato seed [xysB. The comparison between strain CFBP8217 and strain VQ143 showed that VQ143 also lacked most of the virulence genes studied, but the genes pat-1, celB, and xysA were present.Cluster analysis using virulence gene sequences showed the same strain grouping of strains obtained in previous analysis ,16,28, et-1 gene . Mendez ato seed . This stphpA and phpB genes in strains OP3, VQ28, and VQ143. However, the pelA2 gene was present in strain OP3, although the sequence similarity was lower compared with other genes. This lower similarity might explain why it was not amplified by PCR. The pelA1 and pelA2 genes were also confirmed to be absent in the strain VQ143. Interestingly, deletions were found in the sequences of genes celB, xysA, pat-1, and phpA, which truncated the corresponding proteins. Deletion in the nucleotide 316G of the celB gene was found in strains MSF322, OP3, VQ28, and VQ143, but not in strain VL527. Deletion in the nucleotide 1243G of the xysA gene was found in strains VL527, OP3, VQ28, and VQ143, but not in MSF322. Four deletions were found in the phpA gene of strain MSF322. In strain VL527, the phpA gene showed 100% identity with reference strain NCPPB382 and, as mentioned above, it was absent in the strains OP3, VQ28, and VQ143. The deletion in the position 316G in the celB gene was reported already for the type strain LMG7333T and confirmed for other three Cmm strains by Hwang et al. [Cmm strains, other than Chilean strains, available in the NCBI database. The celB gene encodes for an endocellulase that is located in the chromosome outside of the chp/tomA PAI [celB gene was up-regulated during infection of tomato with Cmm strain NCPPB382, and the level of transcription was diminished by the presence of the celA gene [celB might not be important in disease development and cellulase activity, because it lacked signal peptide (SP) and, therefore, it cannot be secreted. However, the analysis of domain prediction based on protein sequences of the Chilean strains showed the presence of a signal peptide domain . However, this deletion also resulted in a stop codon (TGA) in the position 706–708 of the pat-1 gene sequence truncating the protein. The phpA gene deletions in the Chilean strain MSF322 and the absence of the phpA and phpB genes in strains OP3 and VQ28 were in accordance with the absence of these genes in most Cmm strains with available genome in NCBI, as has been reported by Mendez et al. [The r et al.  determinz et al. .celA/celB, pat-1/phpA/phpB/chpC, and xysA/xysB. Gartemann et al. [Cmm NCPPB382 was low compared to other closely related pathogens, such as C. sepedonicus and Leifsonia xyli subsp. xyli, and speculated that Cmm is a “recent” pathogen that is in the process of adapting to the host plant. It is known that the genomes of bacterial pathogens experience a genome reduction during the process of permanent association with the host and, in this process, there is an increase in the number of pseudogenes [The results showed that virulence genes are suffering deletions that lead to an increase of pseudogenes, even causing the loss of virulence genes in some cases. This phenomenon was observed with genes that had homologs in the genome, such as n et al.  analyzedudogenes ,13,14,31Clavibacter michiganensis subsp. michiganensis was analyzed. Virulence assays showed differences between Cmm strains in the expression of plant symptoms, ranging from severe to nearly asymptomatic. A correlation was observed with endocellulase activity, where the most virulent strain demonstrated the highest cellulase activity and no cellulase activity was observed in the less virulent strain. The analysis of the repertoire and sequence variation of virulence genes among the strains revealed additional diversity among the strains that belonged to the same group determined previously by MLST analysis. The strain that was nearly asymptomatic and without cellulase activity lacked the celA gene, confirming the essential role of the celA gene in pathogenicity. On the other hand, the absence or non-functionality of the pat-1 gene did not show to be a determining factor in the virulence of the strains. Several deletions were found in different virulence genes, most of them reported for the first time, and the complete loss of virulence genes in some cases. This phenomenon was observed with genes that had homologs in the genome. Based on our observations, we postulate that Clavibacter michiganensis, as a species, continues experiencing a genome reduction in its process of permanent association with the tomato plant host.In this study, the virulence of nine Chilean strains of