Air pollution is one of the world’s leading environmental causes of death. The epidemiological relationship between outdoor air pollution and the onset of health diseases associated with death is now well established. Relevant toxicological proofs are now dissecting the molecular processes that cause inflammation, reactive species generation, and DNA damage. In addition, new data are pointing out the role of airborne particulates in the modulation of genes and microRNAs potentially involved in the onset of human diseases. In the present review we collect the relevant findings on airborne particulates of one of the biggest hot spots of air pollution in Europe , in the largest urban area of this region, Milan. The different aerodynamic fractions are discussed separately with a specific focus on fine and ultrafine particles that are now the main focus of several studies. Results are compared with more recent international findings. Possible future perspectives of research are proposed to create a new discussion among scientists working on the toxicological effects of airborne particles. Similar results were also found from the monitoring stations located in Milan Metropolitan Area, where particulate matter (PM) still represents one of the most critical environmental issues for its impact on human health [https://www.eea.europa.eu/data-and-maps/data/aqereporting-8#tab-figures-produced and http://discomap.eea.europa.eu/map/fme/AirQualityExport.htm and https://www.eea.europa.eu/data-and-maps/indicators/exceedance-of-air-quality-limit-3/assessment-5) confirm that annual averages of fine particulate matter, i.e. particles with aerodynamic diameter lower than 2.5 µm (PM2.5), measured at an urban traffic (Via Senato) and at an urban background  station, are not significantly different. These data also show that the annual limit of 25 µg/m3 set by the European Union (EU) Directive 2008/50/EC is not always respected. Therefore, due to its peculiar geomorphological conditions, and as the biggest city in the north of Italy, the city of Milan represents a significant case study at the European level for comprehension of the potential impacts of air pollution in an exposed population [in vitro and in vivo toxicological effects, as well as clinical and epidemiological studies [Milan is the biggest city in the Po Valley area with 1.3 million inhabitants, the second largest in Italy after Rome, and one of the most populous metropolitan cities in Europe with more than 7.5 million inhabitants (considering the Milan Metropolitan area). Air pollution in the Po Valley region is a matter of concern. The latest air quality report from the European Environment Agency (EEA) [n health . Since t studies ,7,8. 2.5.Nowadays the presence of micro and nano-plastic debris in the environment is attracting increasing scientific interest. A recent study  reportedin vitro studies on the toxicological impact of airborne PM, also considering the international literature, and proposes the focal research points that, in the coming years, researchers, who deal with the mechanisms and modes of action related to airborne pollution and health risks, should take in consideration. This review summarizes the most relevant results obtained through 10 was the first fractional dimension subject to EU environmental regulation, with limits for atmospheric concentrations since 1999 . As a consequence, it was also the fraction that first received attention from the community of environmental toxicologists. However, in the last years, the interest on PM10 toxicological potential has received less and less attention if compared to fine PM ), and ultrafine PM ). PMin vitro and in vivo, has been largely reported [10 and coarse PM ) relevant to explain adverse human health effects. Although out of the scope of this review, it is worth mentioning that coarse airborne particles deposited in the tracheobronchial respiratory tract may end into the gastrointestinal system [10 particles should mainly deposit in the upper respiratory tract affecting the bronchial and thoracic regions [10. The reported effects, which include inflammation, oxidative stress, DNA damage, and cell death, have been related to the activation of different biological pathways [10 was performed comparing the potency of samples obtained during winter and summer seasons. The experience collected over a decade showed two main different impacts of these particulates: summer PM10 was able to induce an increase in inflammatory mediators in the exposed in vitro systems [10 was a more potent inducer of DNA damage and reactive oxygen species (ROS) formation [10 and coarse PM has received significant interest during the last decade of 20th century and the beginning of 21st century. The capability of PM10 to induce inflammation has been identified by several authors [The reasons for this reduced research interest stand in the increased number of studies focusing on the effects of selected emission sources  and by the increasing evidence from epidemiological studies showing a strong correlation between fine PM exposure and adverse health effects ,13. Nonereported ,15. Indel system ,17. Cilil system  that tral system ,20,21. Il system ). Consid regions . The effpathways ,25,26. Gpathways ,27, an e systems , also ac systems . On the ormation . These dormation ,25, and ormation . These rormation ,33,34,35 authors ,37,38,39 authors ,31,32, m authors ,34, and  authors ,41,42. H authors  showed s10 to induce damages to the DNA, oxidative species, and activation of the xenobiotic responsive element (XRE) is also a well-learned lesson  is reviewed in detail by Donaldson and colleagues [10 oxidative properties and DNA damage. More recently a study by Morales-Bárcenas and co-workers [10 on protease activity and cell invasion. The authors also concluded that this alteration, combined with inflammatory insults also promoted by airborne PM, is likely to increase the possibility of chronic lung diseases, including cancer. Accordingly, Quezada-Maldonado and colleagues reported [10-associated deregulation of micro-RNAs (miRNAs) relevant for cellular pathways usually modified in cancer cells. In this context, future research should focus on the consequence of long-term exposure to PM from different seasons and with clearly different toxicological potency, for example, summer (inflammogenic) and winter PM10 (genotoxic)  . 2.5 exposure is not in operation , the World Health Organization (WHO) recommends a 24 h threshold of 25 µg/m3  for protection of human health. Despite this lack of a daily exposure limit, the EEA  also reports that 6%–8% of the urban population are exposed to concentrations in excess of the EU target value of 25 µg/m3, while 74%–81% are exposed to concentrations above the WHO recommended annual concentration. The significance of these exceedances is related to the latest epidemiological reports that identify fine PM as the fifth leading risk factor for death in the world, accounting for 4.2 million deaths and more than 103 million disability-adjusted life years lost [Although an EU daily limit for PMars lost ,52. 2.5. The research conducted on Milan fine PM unraveled significant biological outcomes, which were only in part previously reported. Significantly, we show the capability of airborne fine PM to interact and impair one of the key cell processes that is cell division [10, a recent study by Santibáñez-Andrade and co-workers [2.5 is able to determine significant modifications in the cell cytoskeleton, such as loss of stress fibers, modified actin structure, and increased membrane ruffling [Cyp1A1, Cyp1B1, AhRR) and antioxidant responsive element (ARE) genes  transcription, which eventually determines the increase of related protein levels [2.5 samples, collected during different months and at different locations, is reported by Lauer and colleagues [Cyp1A1) and oxidative stress . Similarly, another study by Zhou and collaborators [Cyp1A1, Cyp1B1, and AhRR genes after exposure to fine PM, also in relation to increased γH2AX levels, is reported by Borgie and co-workers [It is therefore straightforward to understand the strong effort that has been devoted by many research groups in Europe and all over the world in identifying the key biological processes activated by PMdivision ,54, by a-workers  demonstr-workers , providi-workers ). A typi-workers . We exteruffling . These druffling . These aruffling ,60. Furtruffling ,62, whicn levels ,63. The n levels . Incremelleagues  and show-workers  and a re-workers  shows th-workers ,68. PM-d-workers , which a-workers ,70  directly under environmental conditions. Our results showed, for the first time, the feasibility of using in vitro models for direct exposure to airborne aerosols, and the importance of UFP number and median diameter in triggering oxidative responses in exposed cells. Significant correlations were also found between black carbon and PAHs/organic matter fractional concentration and XRE related genes. These results agree with those reported in another study [10 size fractions to induce DNA damage in a cell-free system, showing that UFP was a potent inducer of damage. Moreover, the direct exposure allowed us to identify the relative importance of primary anthropogenic and secondary PM, and related sources of emissions, on the effects determined in exposed cells and to find interesting correlation with human biomarkers of exposure [STAT3 and related genes ; the oncogene KRAS may be a key node in determining UFP effects by upregulating the expression of EREG; and finally, the hypoxia inducible-factor-1a (HIF1a) may play a central role in UFP-related endothelial dysfunction. Building up on these results, we also show the ability of UFP to induce endothelial modification after exposure of lung epithelial cells to UFP [Epidemiological analyses show a relationship between UFP number concentration and both myocardial infarction  and carder study , which eexposure ,100. Sigexposure ,102. Desexposure ,104 or sexposure ,105,106.exposure  that, afexposure . These rexposure ,111,112 s to UFP  suggestis to UFP ,115, butthe association of ALI exposure of lung cells to fine and ultrafine PM with neural cells, possibly in co-culture systems, is a primary goal in future toxicological studies for understanding the mode of action relating particulates with neurological effects.Given the increasing epidemiological and toxicological reports relating exposure to fine and ultrafine pollution with impairment of cognitive function ,118,119,in vitro models during consecutive days. In this respect, we hope for: a significant increase of studies that will assess, using environmental exposure conditions, the association between PM and toxicological outcomes at doses and under sources directly representative of human exposure.The importance of understanding the biological effects of air pollution under realistic exposure conditions, which represent the real particle concentration exposure experienced by a population, has been largely evoked in recent papers . Nonethethe promotion and funding of a research effort (at European level at least) voted at the understanding of the molecules and, consequently, the sources of PM that are responsible of the observed toxicological effects, by means of a common platform for toxicological and chemical analysis, with the intent to obtain a common database on which to run data analysis. This information could, in turn, be useful for future air quality plans aimed at reducing the source of emission which impacts the health of exposed population more.One of the most discussed questions among toxicologists is the identification of relevant correlation between different PM components and reported adverse effects. The correlations reported are always rather general: organic compounds, mostly PAHs, are associated with AhR activation and consequent Cyp enzymes’ expression with possible DNA damage ,123; PM In recent years significant emphasis has been devoted to the effects of ultrafine carbon particles from primary combustion emission, namely vehicular emissions and, in part, biomass burning. Urban areas close to highways generally suffer from worse air quality, compared to inner city areas or low traffic zones ,128. Thithe evaluation of the toxicological effects of micro areas, which are mostly impacted by vehicular emission, should also be considered for a more rationale planning of urban spaces and transports and for providing, in the end, equal access to clean and safe air to all of the population residing in a city.In this respect, the fast moving transition of private mobility from internal combustion engine vehicles to full electric or hybrid vehicles requires a specific focus on the increasing incidence of non-exhaust vehicle emissions  on airborne PM ,130,131.Finally, new emerging pollutants/environments of exposure are calling for new toxicological data in order to provide evidence relevant for their correct risk assessment and management. In the future research specific effort should be devoted to defining the toxicological impact of particulates characteristic of indoor environments ,133, newAlthough the effects of indoor air pollution on health are of mounting interest, research should continue to investigate outdoor PM; especially considering that at regional and local levels, the rapid climate changes forecasted for the next decades are predicted to significantly affect air quality, including PM concentrations and chemistry, and the concentrations of other pollutants, like ozone and other gases, with expected strong impact on public health ,139. Aga


Background and objectives: Postburn pruritus is a common complication of scars in burn patients. In our previous study, we discovered increased expression of TRPV3, TRPV4, and TRPA1. Among them, TRPV3, in particular, is predominantly expressed in the epidermis of the tissue of pruritic burn scars. We sought to evaluate the correlation between the expression of TRPV3 activators and itching after application of TRPV3 activator carvacrol over burn scars. Materials and Methods: Design: This was a double-blind clinical trial with non-randomized distribution. Setting: This study was performed in a hospital setting. Patients or participants: Patients with itching of burn scars , <3; n = 8; Group 1 and NRS, ≥3; n = 16, Group 2) and healthy volunteers  were included. Interventions: The investigational drug carvacrol  and control drug (10% ethanol) were applied over the scars using IQ Ultimate™ 1 × 1 cm chamber sheets. Main variables of interest: The presence of pruritus, erythema, edema, and other skin lesions was evaluated. The verbal NRS scores were also compared. Results: Carvacrol induced more itching in Group 2 than in Groups 1 and 3. In Group 2, 100%, 75%, and 50% carvacrol caused significantly more itching  than that in Group 1  and Group 3 . Conclusions: The TRPV3 activator carvacrol can cause itching if applied over burn scars. This study can help understand the mechanism and prognosis of post-burn itching and contribute to the development of its treatment. Pruritus following a burn injury is a common and stressful complication of burn injury. Empirical antipruritic treatment often fails to achieve a satisfactory outcome, as the mechanism has not been fully elucidated. In our previous study, we evaluated the clinical and histopathological characteristics of post-burn pruritus and found an increased expression of the transient receptor potential vanilloid channel 3 (TRPV3), TRPV4, and transient receptor potential ankyrin channel 1 (TRPA1) in the skin of these patients . HoweverOriganum vulgare and Thymus vulgaris, such as carvacrol [2-methyl-5-(1-methylethyl) phenol]; it is a monoterpene phenol extract; and it possesses a variety of biological and pharmacological activities, such as hepatoprotective, analgesic, antitumor, antioxidant, and anti-inflammatory properties. It is also a potent activator of TRPV3, and is a known mediator [One of the TRP family, TRPV3, has been found in many tissues and cells, and it is related to calcium permeability. This has been known to increase TRPV3 in cardiac hypertrophic patients ,5,6,7,8.mediator . Chronicmediator ,6. The TCarvacrol was purchased from Sigma Aldrich  and was dissolved in ethanol.n = 8; Group 1 and NRS, ≥ 3; n = 16, Group 2) and healthy volunteers without any history of burn injury  were included. The patients visited Hangang Sacred Heart Hospital, Hallym University, Seoul, Korea.Patients with itching of burn scars , <3; Written informed consent was obtained from each patient before surgery. The study was approved by the Bioethics Committee of Hangang Sacred Heart Hospital of Hallym University . The Institutional Review Board of Hangang Sacred Heart Hospital approved the study protocol. Written informed consent was obtained from each patient or their parents/legal representatives and participants. The inclusion criteria were patients with burn injury who were ≥7 years of age at the time of examination, those who were ≥19 years of age, and those who provided written consent prior to participation in the study and who were able to follow the requirements of the clinical trial (Groups 1 and 2), as well as healthy individuals without any acute or chronic physical conditions, including skin disorders (Group 3). The exclusion criteria were those with infectious skin conditions, pregnant or breastfeeding women, patients with abnormal skin findings , and other individuals who were determined by the principal investigator to be unfit for the study.This was a double-blind study. Five microliters of the investigational drug ) and the control drug (10% ethanol) were loaded onto IQ Ultimate™ 1 × 1 cm chamber sheets  and subsequently applied over the skin . In GrouThe presence of pruritus, erythema, edema, and other skin lesions was evaluated by the investigator. The efficacy was evaluated immediately after removing the IQ Ultimate™ chamber sheets. The verbal NRS scores were recorded and classified from score 0 (no itching) to 10 (severe itching) . It was p-value of < 0.05 was considered statistically significant. All analyses were performed using SPSS version 12.0 for Windows .All data are represented as the mean ± SD. Although the initial plan was to use one-way analysis of variance, the variables were not normally distributed. Therefore, the Kruskal-Wallis test was used for statistical analysis. A p < 0.001) , site, and time after burn injury were compared among the three groups (< 0.001) . The 24 < 0.001) .In Group 1, the change in the NRS score after the application of ethanol and carvacrol was not significant. As compared to Group 2, the administration of carvacrol  caused less itching . Carvacrol induced more itching in Group 2 than in Groups 1 and 3. In Group 2, 100%, 75%, and 50% carvacrol caused significantly more itching  as compared to Groups 1  and 3 .In this study, we found that the TRPV3 activator carvacrol induced itching when applied over burn scars. TRPV3 on keratinocytes appeared mainly in the skin, esophagus, distal colon, and cornea ,10,11. IPruritus following a burn injury is a common and stressful complication . The repPost-burn pruritis may not improve with time, and may worsen. The incidence of post-burn hyperplastic scarring can be lowered through the use of skin grafts, and post-burn pain can be reduced by the use of various anti-inflammatory analgesics. In contrast, post-burn pruritis is often difficult to treat. Since it is difficult to manage post-burn pruritis by the use of general anti-histamines (H1 or H2 blockers), local anesthetics, anticonvulsants (gabapentin), antidepressants, transcutaneous electrical nerve stimulation, massage, or cognitive relaxation therapy have been attempted, but without satisfactory results. In some patients, pruritus improves only with intra-dermal steroid injections or long-term steroid use. However, patients report much more pain when drugs are injected into burn scars, as compared to that felt after injection into normal skin. Furthermore, chronic steroid use is likely to cause systemic adverse effects . DespiteIn a previous experimental study that isolated the keratinocytes from burn patients’ scars with pruritis, protein kinases A (PKA) and C (PKC) were inhibited, and TRPV3 function was decreased. Expressions of TSLP mRNA and protein, and TSLPR protein increased, and TRPV3 function increased. It also showed an increase in intracellular calcium levels .The present study aimed to evaluate the expression of various TRP channels in burn scars. The expression was compared to that in normal skin to investigate the correlation between TRP channel expression and pruritus. In our previous study, the expression of TRPV 1, 3, and 4 and TRPA 1 was quantified in post-burn pruritic skin, and its relationship with the severity of pruritis and burns was analyzed. Moreover, the expression of TRP channels was estimated at the protein and ribonucleic acid levels, and the correlation between each variable was investigated. The present study aimed to confirm the findings of previous reports that the expression of TRPV3 and TRPA1 highly correlates with post-burn pruritis. Research on the role of TRP channels in mediating pruritus in burn scars and skin lesions would be important in elucidating the mechanism of pruritus.Although keratinocytes express various receptors  in neurons, unlike neurons, they cannot produce action potentials. Therefore, the specific roles of these receptors should be investigated. In particular, how keratinocytes directly read and transmit the information about pruritus to the neurons requires further research. Interestingly, intra-epidermal nerve fibers are less dense in severe pruritic lesions than that in normal skin, raising the possibility that factors other than nerve fibers may play crucial roles in transmitting pruritis . Recent The TRP channels are an ion channel group located on the plasma membrane of various types of cells. It has been shown that certain thermosensitive TRP channels, especially TRPV1, TRPV3, TRPV4, and TRPA1, play important roles in the pathogenesis of pruritus and pain . In a feThe results of this study are subject to some limitations. First, the sample size was small. Second, since a patch-based drug delivery system was used, it could have affected the absorption of the test drug depending on the thickness and condition of the burn area. Although the prick test is more accurate, it is invasive. Third, since NRS is a subjective evaluation, it could have been affected by several factors.This study showed that carvacrol, a TRPV3 activator, induced pruritis when applied over burn scar lesions. Therefore, the modulation of TRPV3 could be a possible therapeutic target for pruritis in patients with burn injuries.


Therefore, high and worrying levels of antimicrobial resistance have been found in enterobacteria, and a way to minimize the accelerated emergence of their resistance includes developing or improving techniques that generate diagnoses with high efficiency and speed. A serious emerging problem worldwide is increased antimicrobial resistance. Acquisition of coding genes for evasion methods of antimicrobial drug mechanisms characterizes acquired resistance. This phenomenon has been observed in Enterobacteriaceae family. Treatment for bacterial infections is performed with antibiotics, of which the most used are beta-lactams. The aim of this study was to correlate antimicrobial resistance profiles in Enterobacteriaceae by phenotypic methods and molecular identification of 14 beta-lactamase coding genes. In this study, 70 exclusive isolates from Brazil were used, half of which were collected in veterinary clinics or hospitals Phenotypic methodologies were used and real-time PCR was the molecular methodology used, through the Sybr Green system. Regargding the results found in the tests it was observed that 74.28% were resistant to ampicillin, 62.85% were resistant to amoxicillin associated with clavalunate. The mechanism of resistance that presented the highest expression was ESBL (17.14%). The genes studied that were detected in a greater number of species were  Regarding taxonomy, in relation to taxonomy, the Enterobacteriacea family has 53 genera of which more than 170 species have already been named. Among these, 26 bacterial genera have already been associated with bacterial infections in humans. Members of this family are Gram-negative, facultative anaerobic rods and most species are able to grow at 37 °C, although some grow more properly at 25 to 30 °C [These microorganisms are widely distributed in nature and are found in soil, water, vegetables, in humans and vertebrates gastrointestinal tract . EnterobResistance among clinically important organisms to antimicrobial agents is severely threatening the repertoire of treatment options for common infections. The challenge is intensified by the fact that several of these organisms are resistant to multiple antimicrobials . InfectiCurrently, antimicrobial resistance is one of the most important factors that threaten public health . TransmiAntibiotics play a key role in the success of some medical practices. Unfortunately, they tend to lose their efficacy over time due to the emergence and spread of resistance among bacterial pathogens . AccordiDrug resistance genes can be spread from one bacterium to another through various mechanisms such as plasmids, bacteriophages, naked DNA or transposons. Some transposons contain integrons—more complex transposons that contain a site for integrating different antibiotic resistance genes and other gene cassettes in tandem for expression from a single promoter . BacteriBeta-lactams are preferred because of their clinical efficacy and safety by virtue of their highly selective toxicity . ResistaAntimicrobial susceptibility testing methods are divided into types based on the principle applied in each system . The antMolecular diagnosis is another method of identifying bacterial resistance that can be applied. The molecular technique performed through nucleic acids, while requiring advancements, may allow the patients to obtain a fast examination result, within a four-hour period; thus, initiating the most appropriate antibiotic therapy. This can improve treatment outcomes for the patient and reduce empirical antimicrobial prescriptions, decreasing the duration and cost of antimicrobial treatment. Thus, technologies with the diagnosis of nucleic acids have the potential to reduce the selection of new resistances as well as to reduce the potential of existing resistances .blaSPM, blaSIM, blaVIM, blaKPC, blaSHV, blaCTX-M, blaGIM, blaOXA, blaIMP, blaNDM, blaSME, blaDHA, blaCMY and blaTEM. This study is justified because it is assumed that molecular methods improve accuracy and efficiency compared to the classical phenotyping method. In addition, it can be released in a short time; helping to improve the effectiveness of antibiotic therapy.The objectives of this study are to correlate the resistance profiles of Enterobacteria using phenotypic and genotypic methodologies. The genes encoding resistance to beta-lactams are: The enterobacteria from this study were isolated from samples collected in four different types of origin. Among the total bacterial isolates, 40% are from the human clinic, 20% from an animal clinic, 10% from a human hospital environment and 30% from the veterinary hospital environment.This antimicrobial resistance study in enterobacteria characterized a phenotypic profile of resistance to beta-lactam antibiotics, in which among the 70 bacterial samples studied, 52 (74.28%) were resistant to ampicillin, 44 (62.85%) were resistant to amoxicillin associated with the beta-lactamase inhibitor clavalunate, 38 (54.28%) were resistant to cefazolin, and 6 (8.57%) were resistant to cefuroxime. Enterobacter aerogenes, Enterobacter agglomerans and Cedecea neteri species stand out, which have the highest resistance rate, being resistant to 10 of the 11 tested antibiotics (90.9%). Phenotypically, using the antibiogram method, The sensible and resistance profiles, found phenotypically in this study, determined that among the enterobacteria studied here there was a predominance of 2.8% sensible and 97.2% resistance profiles. Among the resistance profiles, potential MDR profiles were also researched and ESBL, AmpC, MBL, Carbapenemases and CRE were quantified. These data can be observed in Phenotypic analyzes revealed that 28.5% of the total bacteria studied are MDR. Still on the phenotypic profiles, the penicillin group was the antibiotic for which there was the highest resistance rate. There was resistance in at least one of the studied penicillins, in approximately 85.71% of the bacteria. For cephalosporins, there was resistance to at least one of those tested in 77.14% of bacteria, a relatively high number of which shows that these drugs, from the first to the fourth generation, are also losing their effect on enterobacteria. In relation to β-lactams used as drugs of last resource–carbapenems–in this study, there was phenotypic resistance to imipenem in 35.71% of the isolated bacteria determining the CRE profile. The antibiotic that presented the lowest percentage of resistance was the monobactam aztreonam with a resistance rate of 34.28%, which corresponds to a rate close to that of carbapenems, showing that these antibiotics were the most effective against most bacterial samples studied.E. agglomerans showed phenotypic resistance data with profiles sensitive, MDR and CRE. This shows that within the same bacterial species the resistance possibilities are very variable. The species Yersinia ruckeri, was the one that presented more number of resistance mechanisms . The species that did not present any of the mechanisms were: Escherichia blattae, Hafnia alvei, Raoultella terrigena and Citrobacter freundii. The species blaGIM and blaSIM genes, 61.11% for the blaDHA and blaTEM genes, 55.55% for the blaCMY, blaCTX-M, blaNDM, blaOXA genes, 50% for the blaIMP gene, 44.44% for the blaSHV and blaSPM genes, 38.88% for the blaKPC gene, 33.33% for the blaSME gene and 16.66% for the blaVIM gene. The percentages of amplification of the beta-lactamase genes, through the qPCR method, found among Enterobacteriaceae were: 66.66% for the E. aerogenes, E. agglomerans, C. freundii and C. neteri, stood out showing the highest resistance rate (90.9%). For methodological reasons the resistance mechanisms ESBL, AmpC, MBL and Carbapenemases have not been studied molecularly, only CRE. The molecular profile of resistance to beta-lactam antimicrobials was determined in 18 bacterial species among enterobacteria: 94.44% showing resistance for aztreonam, ceftazidima, cefoxitin and piperacillin associated with beta-lactamase inhibitor tazobactam. Phenotypic and molecular data are compared in The fenotypic profile of resistance to beta-lactam antimicrobials was determined in 19 bacterial species among enterobactérias: regarding all the phenotypic resistances found, the species aerogenes, R. terrigena, Moganella morganii, Edwardsiella ictaluri, C. neteri, Salmonella paratyphi and Y. ruckeri., exhibiting resistance potential for all antibiotics (100%) tested. C. freundii, Klebsiella spp., E. coli, E blattae, Providencia rustigiani and Erwinia persicina were studied, showing potential resistance in 10 among 11 antibiotics (90.9%) tested.By analyzing the amplification rate of the genes that confer beta-lactam resistance and making an association of the same with the literature review carried out in this study, it was observed that the species that showed potential resistance to a greater number of antibiotics were: E The resistance information obtained in this study shows that 100% of the analyzed species present a high potential for resistance to several beta-lactams. Among the potential profiles suggested by the qPCR analyzes, the MDR and CRE data were, respectively, 100% and 88.88% among the studied species. These data can be observed in The Pearson coefficient was calculated to linearly correlate two variables. The Pearson correlation coefficient varies between −1 and 1. The signal indicates the direction of correlation (negative or positive) while the value indicates the magnitude. The closer to 1 the stronger the level of linear association between variables 3. In this study, the detection rate of antimicrobial resistance by molecular methodology was generally higher than the detection rate by phenotypic methodology. This study showed that the presence of the resistance gene in the bacterial genome does not necessarily imply its expression, therefore it is necessary to develop the phenotypic methodology.EcoR I and Hind III restriction enzymes. After these results, the authors decided that the best experiment to observe the restriction plasmid profile must be the sequencing experiments that will be done in another study.An experiment was carried out to verify the plasmid profile and from plasmid DNA digested were no identified sites to E. aerogenes, E. agglomerans and C. neteri are related among those that present resistance to multiple drugs [The phenotypical results of the present study are in agreement with a retrospective study that was carried out in a laboratory of clinical analyzes of Goiânia, Goiás, which evaluated the prevalence and antimicrobial susceptibility profile of the isolated microorganisms from 432 samples, in which the species le drugs . C. neteri, which is a strain of animal origin, these bacterial species are in accordance with epidemiological data indicated by ANVISA [Except for y ANVISA , as theyRegarding multidrug resistance (MDR) and carbapenem-resistant Enterobacteriaceae (CRE) profiles have recently been updated by the Center for Disease and Control and European Center for Diseases Control and Prevention, promoted aiming at the international standardization of these terminologies, as published by Magiorakus et al, , in whicAccording to the authors, Enterobacteria resistant to carbapenems infections are associated with high mortality rates (up to 70%), making them particularly challenging from a clinical standpoint .Logan and Weinsteins  showed aIn a study of CRE isolated from patients who received medical care at Stanford Health Care and Lucille Packard Children’s Health, California, USA, between January 2013 and December 2016, carbapenem minimum inhibitory concentration (MICs) for the CRE card ranged from ≤1 to 265 >8 μg/mL for imipenem, which also demonstrated higher resistance to this antimicrobial . A study carried out in northeastern Brazil showed that in 672 positive urocultures for urinary tract infection, the etiological agent belonged to the Enterobacteriaceae family in 86.9%, and among them 29 (4.8%) were ESBL .A literature review was carried out to determine the potential of resistance by molecular methodology found in this study, which has genes described in the literature that encode the β-lactamase enzyme. The result of the literary survey is shown in blaOXA, blaTEM, blaKPC, blaCTX-M and blaSHV. However, among the total genes found in this study, the ones with the highest percentage were blaGIM and blaSIM, both with 66.66%. In the literature review, the blaGIM gene was correlated only with beta-lactam imipenem, while blaSIM was correlated with ampicillin, aztreonam, ceftazidime, cefepime, imipenem and piperacillin + tazobactam. Resistance to beta-lactam imipenem gives the bacteria the CRE profile, and although phenotypic analyzes show a low detection rate of imipenem (35.71%), the molecular detection rate of imipenem was the second-highest detection rate with a value of 88.88%.The data from the bibliographic review carried out here show that the beta-lactamases most frequently correlated with beta-lactams are blaKPC, blaOXA-48, blaNDM, blaSPM, blaIMP, blaVIM and blaGIM genes were investigated by PCR and multiplex PCR. About the number of studied microorganisms, the genotypic tests evidenced that blaKPC was the most prevalent gene, in 31% (n = 10) of the samples, followed by blaIMP, in 12.5% (n = 4) [In a study carried out with clinical isolates of carbapenem-resistant Enterobacteriaceae collected at the University Hospital of Santa Maria, Rio Grande do Sul, Brazil, the  (n = 4) .Klebsilla pneumoamiae and 1 Klebsilla oxytoca. All isolates showed blaDHA gene and (4/12) 33.33% blaTEM gene [In a study conducted in eight hospitals in Paris surroundings, France, twelve isolates were collected in twelve patients, 11 TEM gene .E. coli, K. pneumoniae and Salmonella spp. were isolated. All the isolates were tested for the presence of blaCTX-M-1 and/or blaSHV genes by PCR assay. A total of 54 (13.04%) isolates carried at least one ESBLs genes tested under this study, of which 41 (9.90%) E. coli, 11 (2.66%) K. pneumoniae and 2 (0.48%) Salmonella were found to be positive for blaCTX-M-1/blaSHV gene. A total of 4 (10.14%) and 9 (2.17%) isolates were positive for blaCTX-M-1 and blaSHV genes, respectively, whereas, 3 (0.72%) K. pneumoniae isolates were positive for both the genes. On the other hand, only 2 (0.48%) Salmonella isolates for blaCTX-M-1 gene [In another study, 88 phenotypically ESBLs positive isolates samples collected from hospitals located in Mizoram, India, enterobacteria such as M-1 gene .In our study, both rates are lower than those found in the phenotypic profile, however, it should be considered that the molecular analysis was performed in only 18 representatives of the studied species, thus presenting a smaller sample than the phenotypic tests. This fact explains why the data from the molecular analyzes are denominated only as potential and also, in addition, the presence of genes in the genome does not necessarily imply phenotypic expression of them .Even molecular analyzes genes are not expressed as host carriers and the only fact of being present in circulating strains is already a high risk, since the onset and spread of the microorganism with drug resistance shows the problem of the interaction of several factors such as an exchange of genetic information between microorganisms, through the transfer of genes to new hosts . The presence of more resistance profiles in molecular analyzes than in phenotypic analyzes testify the greater sensitivity of the molecular methodology. qPCR provides a high advantage of fast transferring detection rate and quantification of target DNA sequences in different matrices. The low amplification time is facilitated by the simultaneous amplification and visualization of the new amplicons formed. However, the mere presence of genes responsible for components of antimicrobial resistance or toxin production does not automatically signify their expression or production . Thus, a2 = 0.0015 or r = 0.038), it should be known as a comparative analysis of the efficiency of the two methodologies for the detection of antimicrobial resistance. The molecular methodology, PCR, is appreciated due to its high capacity of sensitivity and specificity [The statistical method was performed , and acccificity . The lowHowever, it should be bear in mind the conditions offered in the growth medium diverge from the actual conditions of a host organism. Since the growth medium is a favorable environment for bacterial growth, it offers optimal conditions for bacterial metabolism, a fact that does not occur in the host organism. This variation of conditions may be determinant for gene regulation, generally leading to the expression of a greater number of genes in the environment of metabolic stress or gene suppression in an environment with favorable growth conditions. This explains why the molecular data found here is compatible with epidemiological data .Klebsiella pneumoniae, Proteus mirabilis, Citrobacter freundii, Morganella morgani, Providencia spp., Enterobacter aerogenes, Enterobacter agglomerans, Raoultella terrigenes, Escherichia coli, Escherichia blatteae, Edwarsiella ictaluri, Cedecea neteri, Erwinia persicina, Providencia rustigiani, Salmonella paratyphia, Salmonella typhi, Yersinia ruckeri, Serratia marcecens and Hafnia alvei , according to the bacterial genus were used the antimicrobials ampicillin 30 μg, amoxiline-clavulanate 20/10 μg, aztreonam 30 μg, cefazolin 30 μg, cefepime 30 μg, cefoxitin 30 μg, cefuroxime 30 μg, ceftazidime 30 μg; ceftriaxone 30, imipen 10 μg and piperacillin-tazobactam 100/10 μg. The quality control procedure was followed, strains ctamases . For the phenotypic detection of extended spectrum beta-lactamases (ESBL) production, from enterobacteria of this study, the statistic method was performed  and accoFor the AmpC-type beta-lactamase phenotypic detection, the induction test was performed using antimicrobial susceptibility testing, performed by the disk diffusion assay (Kirby–Bauer technique) according to the 2015 European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations .Imipenem and meropenem discs were used for the carbapenemases phenotypic investigation and interpretation of the sensitivity following the criteria established by CLSI . At leasE. coli ATCC 25922® on the entire surface of a Müller-Hintos agar plate. A meropenem disk was placed in the center of the plate and around this disk streaks were made with the suspected samples, as recommended by CLSI [For enterobacteria, in addition to the EDTA test, the modified Hodge test (MHT) was also performed. MHT consists of the inoculation of  by CLSI .®, according to the manufacturer’s instructions. For the qPCR assays, specific primers were designed based on the sequences deposited in GenBank  real-time PCR kit, following the methodology suggested by the manufacturer. For the positive and endogenous control of the reaction the primers were used to amplify the 16S RNA, for the negative control, water was added in place of the DNA. Fisher’s test was used to compare the techniques considering isolated samples.EcoR I and Buffer V2 for Hind III); 1 μL of EcoR I or Hind III enzyme (10 UI/μL) (Ludwigbiotec), 15 μL H2O; 2 μL template DNA (~300 ng/μL). The tubes were placed in thermo-blocks at 37 °C overnight and were then incubated at −20 °C for 15 minutes. From these preparations agarose gel electrophoresis was performed, as controls were used the preparation without the enzyme and a non-incubated preparation.Purified plasmid DNA preparations were digested with restriction enzymes for identification and characterization of the genes of that study according to the preparation: in microcentrifuge tubes were added: 2 μL of 10× Buffer (Ludwigbiotec), , while the molecular profile was demonstrated from the Molecular Resistance Potential analyzes, which associates data from the literature review to the amplification by quantitative PCR. MDR and CRE profiles were found. In this characterization, the detection rate by molecular methodology was higher, demonstrating the greater sensitivity of this technique.According to the results obtained here, it can be determined that, given the need for faster diagnosis in emergencies or not, the molecular method, because its more sensitive, faster and less laborious process, can be considered superior to the phenotypic method in which has some limitations such as dependence on specific conditions of reproduction for the ideal growth of bacteria, detection of only cultivable organisms, previous preparation of the material, greater manipulation and risk of contamination, and longer time for the final diagnosis.


Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter sp.) and the biosynthesis pathway for pyridoxal 5′-phosphate and its vitamers (vitamin B6), found in S. aureus. Using current genomic data, we discuss the possibilities of inhibition of enzymes in the pathway and review the current state of the art in the scientific literature.Antibiotic resistance is a worldwide concern that requires a concerted action from physicians, patients, governmental agencies, and academia to prevent infections and the spread of resistance, track resistant bacteria, improve the use of current antibiotics, and develop new antibiotics. Despite the efforts spent so far, the current antibiotics in the market are restricted to only five general targets/pathways highlighting the need for basic research focusing on the discovery and evaluation of new potential targets. Here we interrogate two biosynthetic pathways as potentially druggable pathways in bacteria. The biosynthesis pathway for thiamine (vitamin B1), absent in humans, but found in many bacteria, including organisms in the group of the ESKAPE pathogens ( Antibiotic resistance is an urgent threat to human health and requires urgent actions from physicians, patients, industries, governmental agencies, and the academic community worldwide. According to the last document from the Centers for Disease Control and Prevention (CDC) regarding antibiotic resistance in the United States, from 2013, the number of people with serious infections caused by resistant bacteria reaches 2 million every year, with at least 23,000 deaths per year directly caused by these infections  theAntibiotic resistance, as a threat to human health, should be addressed in multiple and simultaneous ways. For the academia, an interesting way to address antibiotic resistance is the discovery and validation of new targets/pathways that could be specifically targeted by new antibiotic candidates. It is worthy of note that, according to Kelly and Davies , no new Escherichia coli genome, 303 genes, were shown to be composed of essential genes , the activity of several carbohydrate metabolism enzymes is impaired, including pyruvate dehydrogenase, which connects glycolysis and the citric acid cycle . Other tsubtilis .Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter sp.)? (ii) What enzymes are present, and what do we know about them?The pathways involved in the synthesis of vitamins, in particular, vitamin B1 (thiamin) and B6 , seem to be of great relevance, since they are involved in central processes in the metabolism of carbohydrates and amino acids, and the corresponding enzymes are found in most bacteria, fungi, and plants but not in humans , favorinPlasmodium falciparum , a KEGG search for the maps of thiamin and pyridoxal metabolism was done, looking for the existing genes in commercial strains for each of the ESKAPE organisms. For the sake of comparison, some additional resistant strains were listed, with no differences in the observed genes for the pathways under study. A similar search was carried out for vitamin B6 metabolism (map 00750) in KEGG, interrogating the existing enzymes for the ESKAPE pathogens in comparison with humans. The analysis was focused in the enzymes in the biosynthesis of B1 and B6 vitamins in an attempt to identify the most promising targets for future medicinal chemistry studies. Finally, a table listing the existing genes for each pathogen was compiled and is presented in the following sections.The analyses provided here are the result of the interrogation of the enzymes in the biosynthesis pathways for thiamin and pyridoxine phosphate or pyridoxal phosphate using the KEGG database , 15. Fordruggability of the identified promising targets.Using the existing literature, we sought to identify whenever possible the cases of enzymes with functional redundancy with another enzyme for the same organism. The identified cases were discussed as less promising targets for medicinal chemistry campaigns. Additionally, the existing data for enzyme inhibition using natural or synthetic compounds was compiled to provide initial proof-of-concept clues about the When necessary, the sequence of an enzyme of the pathway was used to search for homologs using BLAST  searchesThe biosynthesis pathway for thiamin involves two branches, as shown in Q9H3S4. Other than that, the remaining enzymes in the biosynthetic pathway for thiamin are missing in humans, making them very attractive for the design of chemical probes that could be used as proof-of-concept compounds.According to the KEGG pathway database, humans lack many of the enzymes of this pathway, but not all of them. In humans, a TPK (TPK1) enzyme is found, with UNIPROT ID E. faecium (ATCC 8459) in the KEGG database did not identify genes for ThiE, ThiM, and TenA. For this species, only ThiD and TPK were positively identified. However, a BLAST search for TenA homologous within the non-redundant database restricted to E. faecium (TAXID 1352) identified a single result for thiaminase II (GenBank: SAM74984.1), four results for ThiE , and two results for ThiM . The small number of hits in the BLAST search may suggest some issues in the annotation.The analysis of the thiamin pathway for Enterococcus faecalis thiamin pathway is compared with the pathway observed for E. faecium, many differences are found. The KEGG pathway for E. faecalis (ATCC 29212) shows that the entire pathway, as described in E. faecium. Additionally, there is no change in the repertoire of enzymes in the pathway for the Vancomycin-resistant strain V583 compared to the ATCC 29121 strain.Interestingly, when the S. aureus (NCTC 8325) and K. pneumoniae (subsp. pneumoniae ATCC 43816 KPPR1), the KEGG pathway indicates that all the enzymes in the pathway are observed, with no changes to an S. aureus-resistant strain such as COL (MRSA) compared to the NCTC 8325. For A. baumannii (ATCC 14978), all enzymes are observed but TPK. Instead, thiamin phosphate may be converted directly to thiamin diphosphate by a thiamin-monophosphate kinase, and then converted to thiamin triphosphate by an adenylate kinase. In the case of P. aeruginosa (NCGM 1900), some enzymes are missing: ThiD, TenA, TPK. Finally, Enterobacter sp. (638) has ThiD, ThiE, ThiM and misses TenA and TPK.For The overall panel of enzymes for the thiamin pathway for the ESKAPE pathogens is summarized in Bacillus subtilis, the deaminase activity is somewhat redundant with the ThiC (ThiA) activity, since both activities provide HMP or HMP phosphate, which can be further phosphorylated by ThiD , and ThiM function can be dispensable since thiazole phosphate can be synthesized P. aeruginosa, which lacks ThiD, and E. faecium, which lacks ThiE, according to the KEGG data. For ThiE, in particular, it was reported that 4-amino-2-trifluoromethyl-5-hydromethylpyrimidine (CF3-HMP), an HMP analog, can be converted by ThiD to CF3-HMP pyrophosphate, which in turn inhibits ThiE , B. subtilis [PDB ID 2I5B (Thermus thermophilus (PDB ID 1UB0), A. baumannii (PDB ID 4YL5), Bacteroides thetaiotaomicron (PDB ID 3MBH), and the bifunctional enzyme from Saccharomyces cerevisiae (PDB ID 3RM5). Typically, ThiDs are folded as typical ribokinases, with a central eight-stranded β-sheet surrounded by eight α-helices, and structural studies suggest that some surface loops may have a structural change based on the presence of the nucleotide  has a dual role, phosphorylating pyridoxal and pyridoxine in the pyridoxal de novo biosynthesis pathway as well as HMP in the thiamin biosynthesis pathway, with a KM almost 20 times greater for HMP than for pyridoxal and kcat values three times faster for pyridoxal (S. aureus ThiD (SaThiD), with less efficiency, though.Interestingly, the yridoxal . So, SaPSaThiD was shown to be potentially inhibited by Rugulactone (Ru0), a natural product, as well as by its derivatives Ru1 and Ru2, with IC50 values ranging from 14 to 32 μM , B. subtilis [PDB IDs 1G4T, 3O15, 3O16, 1G69, 1G4E, 1G67, 1G4P, 1YAD (Mycobacterium tuberculosis (3O63), and for the bifunctional enzyme from Candida glabrata [PDB IDs 3NL2 and 3NM1  is a close homolog of Bacillus subtilis Pdx1 [81% identity, PDB ID 2NV1 . S. aureus Pdx2 has Geobacillus kaustophilus [PDB ID 4WXY (G.s stearothermophilus (PDB ID 1Q7R), and B. subtillis [PDB IDs 2NV0 and 2NV2  interact with 12 Pdx2 enzymes. However, Pdx2 molecules do not interact with each other in this complex -helices . The mostopology . Curious complex .in vitro inhibitory effects on the enzyme Pdx1 from P. falciparum  and B6  may be interesting molecular targets for the development of new chemical probes aiming to inhibit the synthesis of these essential cofactors in pathogenic organisms. Model compounds, mainly based on substrate analogs showed that inhibition of the vitamin B1 biosynthetic enzymes, ThiD and ThiE, as well as Pdx1, Pdx2, and PdxK in the pyridoxal 5′-phosphate biosynthesis pathway have shown an initial proof of concept for the model, and it is up to the scientific community and/or researcher in the industry to explore these targets further.The datasets generated for this study are available on request to the corresponding authors.CW, IP, and AN conceived the project. LM and RG analyzed the thiamine pathway, while AB and LD analyzed the pyridoxal pathway. All authors wrote and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The current study investigated the effects of barbell placement on kinematics and muscle activity during the sticking region of back squats. Ten healthy medium- to well-trained male powerlifters  with at least 3 years of resistance-training experience were recruited. In a single session, participants performed 5RM movements using high bar and low bar squats, where absolute load, descent depth, and stance width were matched between squat conditions. The final repetition was analyzed using 3D kinematics and electromyography (EMG) around the sticking region. No differences in barbell and joint kinematics were observed in any phase, between both barbell modalities. Increased muscle activity in the rectus femoris, vastus medialis, and lower part of the erector spinae with the high bar, when compared with low bar conditions, was recorded. Furthermore, the gluteus maximus and medius had increased muscle activity over the three regions (pre-sticking > sticking > post-sticking), while the erector spinae, soleus, vastus lateralis, and rectus femoris experienced decreased muscle activity during the ascending phase. When depth and stance width were matched, the low bar technique was associated with lower erector spinae and quadriceps activity than the high bar technique. Thus, when the goal is to maximally activate knee extensors and the external load is matched, high bar placement would appear preferable. Barbell back squats are a commonly used exercise within general fitness programs, and as an event within the sport of powerlifting, they are often included in rehabilitation programs for the lower body  with at least 3 years of resistance training in squats were recruited. Participants were familiar with both squat techniques as they use both techniques in their training. Inclusion criteria included that they be capable of lifting 1.5 times their own body mass in 1RM squat , with good technique determined by the test leader who is an experienced powerlifting trainer. Participants had no injuries that could impact their performance. No participants performed leg resistance training 24 h before testing. Participants were informed verbally and by writing of possible study risks. All provided written consent before inclusion. The study complied with current ethical regulations for research and was approved by the National Center for Research Data, in accordance with the latest revision of the Declaration of Helsinki.A 5RM test was used to investigate kinematics and muscle patterns around the sticking region, during a high bar and low bar squat. 5RM repetition range is a typical training load used to increase maximal strength  and used for subsequent attempts to prevent differences in width stances between the two conditions from influencing the moment arms around the different joints. All participants wore weightlifting shoes during all attempts. A minimum depth requirement was that the hip joint had to be lower than the knee joint in accordance with the IPF   in a weightlifting rack . Participants performed repetitions in a self-paced tempo environment, from full knee extension until the hamstring touched the rubber band, and returned to the starting position.V0), first maximal barbell velocity (Vmax1), the lowest barbell velocity (Vmin), and the second maximal barbell velocity (Vmax2) of the concentric phase  attached to the inside of the barbell measured barbell distance and velocity over time, with a resolution of 0.019 mm and a 200-Hz sampling rate. Barbell velocity was calculated using a five-point differential filter, using Musclelab v10.190 software . From the last repetition of each condition, the vertical displacement and velocity of the following events—lowest barbell point (ic phase —were meaV0) to the first maximal barbell velocity (Vmax1) event was called pre-sticking. The second region from the first maximal barbell velocity (Vmax1) to the lowest barbell velocity (Vmin) event was called the sticking region, while the last region, post-sticking, was from the lowest barbell velocity (Vmin) to the second maximal barbell velocity (Vmax2).Wireless electromyography (EMG) was recorded with a sampling frequency of 1,000 Hz using a Musclelab 6000 system and analyzed by Musclelab v10.190 software . EMG activity was measured for 11 muscles: (1) lower and (2) upper part of the erector spinae, (3) gluteus maximus, (4) gluteus medius, (5) vastus lateralis, (6) vastus medialis, (7) semitendinosus, (8) rectus femoris, (9) biceps femoris, (10) soleus, and (11) gastrocnemius. Before testing, the skin was shaved, abraded, and washed with alcohol before gel-coated, self-adhesive electrodes were placed . The electrodes (11 mm contact diameter and 2 cm center-to-center distance) were placed on the dominant leg, along the presumed direction of the underlying muscle fiber, according to the recommendations by SENIAM or similar studies  motion capture system , with eight cameras sampling at a frequency of 500 Hz, was used to track reflective markers, creating a 3D positional measurement. The 3D motion capture system was synchronized with the linear encoder and EMG recordings, using the Musclelab 6000 system . The markers were placed, one on each side of the body, on the lateral tip of the acromion, the iliac crest, greater trochanter, the lateral and medial condyle of the knee, the lateral and medial malleolus, and the distal ends of the first and fifth metatarsals. Two markers were also placed on the middle of the barbell between the hands and shoulders 80 cm apart, to track barbell displacement. Segments of the feet, lower and upper leg, pelvis, and trunk were made in Visual 3D v5 software . Barbell position and velocity, joint angles of hip extension, knee extension, and plantar flexion were calculated using Visual 3D software. Joint angles were estimates of the anatomical angles calculated from lines formed between the center of reflective markers. The joint angles of hip extension/flexion, knee extension/flexion, and ankle plantar/dorsal flexion  at the fpost-hoc test was performed. In cases where the sphericity assumption was violated, a Greenhouse–Geisser adjustment for p-values was reported. The level of significance was set at p ≤ 0.05. For statistical analysis, SPSS version 25.0  was used. All results are presented as the mean ± standard deviation (SD) and effect sizes were calculated using η2 (Eta squared), where 0.01 < η2 < 0.06 constituted a small effect, 0.06 < η2 <0.14 denoted a medium effect, and η2 > 0.14 indicated large effect  analysis of variance (ANOVA), with repeated measures was used. To evaluate differences in EMG activity between different conditions and regions, a 2 (conditions) × 3 (regions) ANOVA with repeated measures was performed for each muscle. If significant differences were found, a Holm–Bonferroni t Cohen, .p ≥ 0.18, Vmax1) and ended at 39.7 ± 9.7% (Vmin) of the total upwards barbell displacement. Furthermore, no significant differences were observed in ankle, knee, and hip flexion angles at any events, between high and low barbell placements , with higher muscle activity in high bar conditions, when compared with low bar. Furthermore, an effect of region was observed for the gluteus maximus and medius, all three quadriceps, soleus, and both parts of the erector spinae , with no significant placement*region interaction effects  coincided with the timing of the first peak angular velocity of the knee extension and plantar flexion, while the Vmin event occurred with the minimal plantar flexion and knee extension angular velocity  is essential to eliminate unnecessary planar motion, thus ensuring a stable trunk during the ascent. Since there is a lumbar–pelvis relationship, and the spinal angle increases when the hips are flexed (Schoenfeld, Our findings provide further evidence that supports Van Den Tillaar , who sugNo differences in barbell and joint kinematics were observed around the sticking region between high bar and low bar back squats when absolute load, depth, and stance width were controlled. Increased muscle activation of the rectus femoris, vastus medialis, and lower part of the erector spinae was observed with high bar conditions, when compared with low bar. Therefore, if the goal is to maximally activate knee extensors and the external load, minimum depth and stance width are matched, and high bar placement appears preferable. Otherwise, it is recommended to coaches and athletes that when external load, minimum depth, and stance width are matched, a low bar approach may be an advantageous choice of squat technique, due to less stress on the erector spinae, especially at the start of the ascending phase.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by National Center for Research Data. The patients/participants provided their written informed consent to participate in this study.RT conducted the data collection, supervised the study, wrote the article, and performed partly the analysis and interpretation. TK conducted the data collection, performed the initial data analysis, interpretation, and wrote parts of the article. SL performed additional data analysis, interpretation, and also wrote parts of the article. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Shape of the aneurysm) score obtained from an individual-based meta-analysis. The series consists of 87 patients with 111 UIAs and 1669 person-years of follow-up between aneurysm size measurements . These were initially diagnosed between 1956 and 1978, when UIAs were not treated in our country. ELAPSS scores at baseline did not differ between those with and those without aneurysm growth. The area under the curve (AUC) for the receiver operating curve (ROC) of the ELAPSS score for predicting long-term growth was fail , and the optimal cut-off point was obtained at ≥7 vs. <7 points for sensitivity (0.829) and specificity (0.217). In the present series UIA growth was best predicted by female sex (4 points), smoking at baseline (3 points), and age <40 years (2 points). The AUC for the ROC of the new scoring was fair , which was significantly better than that of ELAPSS score (p < 0.05). The optimal cut-off point was obtained at ≥4 vs. <4 points for sensitivity (0.971) and specificity (0.304). A new simple scoring consisting of only female sex, cigarette smoking and age <40 years predicted growth of an intracranial aneurysm in long-term follow-up, significantly better than the ELAPSS score.The purpose was to obtain a reliable scoring for growth of unruptured intracranial aneurysms (UIAs) in a long-term follow-up study from variables known at baseline and to compare it with the ELAPSS (Earlier subarachnoid hemorrhage, Location of the aneurysm, Age > 60 years, Population, Size of the aneurysm, and  Subarachnoid hemorrhage (SAH) is a serious disease with a high rate of unfavorable outcomes ,2,3,4. TSince the prevalence of aneurysms is not decreasing , the risAccording to two recent meta-analyses of aneurysm growth, female sex, cigarette smoking at baseline, aneurysm size, posterior circulation aneurysms, and possibly age, hypertension, and aneurysm multiplicity may increase the risk of UIA growth, while prior SAH and a family history of aneurysms do not ,14. AneuEarlier SAH (decreased risk), Location of the aneurysm, Age >60 years, Population, and Sze and Shape of the aneurysm (ELAPSS score). The 3-year growth rate ranged between <5% and >42% and the 5-year growth rate between <9% and >60%, depending on the risk factors. The results between ELAPSS score and previous meta-analyses differed between each other for sex, age, previous SAH, populations, and some aneurysm locations. Cigarette smoking was not studied in the ELAPSS study. Most of the cohorts were from selected populations where the patients with UIAs were those left for conservative follow-up because of an estimated low rupture risk, or a limited life expectancy or high treatment risks [A large meta-analysis of individual data from 1507 patients in 10 cohorts showed UIA growth in 257 cases 17%) during 5782 patient-years of follow-up . Predictnt risks . UIA may% during nt risks .An almost lifelong follow-up cohort of UIAs ,15 seen Approval for the surveys and follow-up data collection was obtained from the local ethics committee  and all aspects of the study were in compliance with national legislation and the Declaration of Helsinki [The series comprised of 87 patients with UIAs diagnosed at the Department of Neurosurgery of Helsinki University Central Hospital between 1956 and 1978, when UIAs were not operated on in Finland and when this hospital was responsible for neurosurgical services for almost the whole Finnish population ,15,17,18Of the 142 patients with UIAs diagnosed before 1979, 56 patients were studied in the outpatient department during 1996–1998 ,17. Onlyn = 33) or at autopsy (n = 10) between 1959 and 2008 because of an aneurysm rupture or a need to check the aneurysm status. The last follow-up was performed in 2012.In addition, the aneurysm sizes of 43 patients were monitored with conventional or digital angiography (p < 0.001).Since the cohort included patients who were admitted during a time period of 22 years (1956–1978), there were a further 51 patients with UIAs who had already died without a control angiography, vast majority of them from unrelated causes ,17,18. ADetailed follow-up protocols have been reported previously ,19,20,21Additional information on all the patients was obtained from medical records supplied by other hospitals and general practitioners, and the accuracy of the medical data including blood pressure (BP) values was recorded ,15,17,19Core and most highly recommended supplementary variables for the study of UIA were available for the present purpose , and theAll angiographies and CTAs were examined by 2 experienced neuroradiologists who had no knowledge of the patients’ case histories ,19,20,21t-tests and the Mann–Whitney U-test.The data were analyzed with IBM SPSS Statistics, version 25.0, for Windows . The ELAPSS score variables at baseline were compared according to the UIA’s growth status using Fisher’s exact test or the Pearson Chi-square test. Sum scores (expressed as mean ± standard deviation (SD) and median with interquartile range (IQR)) were compared by means of p < 0.15). The new integer risk score points for aneurysm growth were based on the regression coefficients in the final multivariable logistic regression model. The areas under the curve (AUC) for the receiver operating curves (ROC) were calculated for both the ELAPSS and the new growth scores. The C-statistic with 95% CIs was used to test how the total scores matched the observed rupture rates, the AUC between the scores being analyzed using the technique of Hanley and McNeil [p value < 0.05 was considered statistically significant.Odds ratios (ORs) with 95% confidence intervals (CIs) of the risk factors for growth of UIA were analyzed by multivariable unconditional logistic regression. A maximum-likelihood stepwise forward elimination procedure was used, with selection of the variables to be added into the model on the basis of the magnitudes of their probability values . Twenty-seven patients (31%) suffered from a SAH and of them, 10 were fatal. The mean measurement follow-up time in these 27 patients was 12.0 years (range 1.2 to 24.1 years), and that for the 60 patients without a rupture was 22.4 years (1.3 to 51.0 years). All 27 UIAs which later ruptured had increased in size by at least 1 mm and out of the largest UIAs of 60 patients without an aneurysm rupture, 13 increased by ≥1 mm.p = 0.35).Factors of ELAPSS score stratified by aneurysm growth ≥1 mm are shown in p = 0.019), smoking at baseline , age <40 years  predicted best long-term UIA growth. Smoking at the end of follow-up associated better with growth  than smoking at baseline but it cannot be used in a new score since it is not a predictive variable but represents better a long-term habit (p = 0.003). A new system for scoring of UIA growth was constructed from the regression coefficients of the three variables: women 4 points, smoking 3 points, and age <40 years 2 points (p = 0.006).All the ELAPSS factors with additional UIA locations, sex, hypertension, smoking, alcohol consumption, and family history were tested with logistic regression analysis to identify the variables which best predicted a future aneurysm growth. Women , and the optimal cut-off point was obtained at ≥4 vs. <4 points for sensitivity (0.971) and specificity (0.304) (p < 0.015). The optimal cut-off point was obtained with the sum score at ≥2 vs. <2 points with a sensitivity (0.771) and specificity (0.457). The AUC of the ELAPSS score was fail , and the optimal cut-off point was obtained at ≥5 vs. <5 points for sensitivity (0.829) and specificity (0.217) (p < 0.05). The AUC of ELAPSS, which also included patients with missing values for smoking was 0.450  and the cut-off point at ≥5 vs. <5 points yielded sensitivity 0.800 and specificity 0.213  . The AUC (0.217) . So, falty 0.213 .The growth score of UIAs obtained from this prospective study with an almost lifelong follow-up and a very low treatment selection bias suggests that growth of UIAs among patients of working age can be estimated quite reliably with only three variables at baseline. Female sex, patient age (<40 years), and cigarette smoking determine the risk of UIA growth significantly better than does the ELAPSS score.Simultaneously with the discovery of increasing numbers of UIAs, it is likely that the indications for treatment will decrease, because of the decline in the prevalence of smoking and the incidence of SAH ,8 and beAccording to the results of two recent meta-analyses of aneurysm growth, female sex, cigarette smoking at baseline, aneurysm size, posterior circulation aneurysms, and possibly age, hypertension, and aneurysm multiplicity may increase the risk of UIA growth, while prior SAH and a family history of aneurysms do not ,14. TherThe present study with an almost lifelong follow-up of UIA patients also included patients with a high UIA rupture risk as was seen in the relatively high UIA growth rate among patients of working age. Expectedly, UIA growth score obtained from the present score included partly same factors as treatment score based on risk factors of UIA rupture . The treEarlier subarachnoid hemorrhage (decrease risk!), Location of the aneurysm, Age > 60 years, Population, Size of the aneurysm, and Shape of the aneurysm (ELAPSS). Of the total 1507 patients with UIAs only 18 patients (1.2%) had an aneurysm rupture, and of 257 growing UIAs only 8 (3.1%) ruptured. Cigarette smoking which has been most significant risk factor for UIA growth and SAH [Previously published results from this cohort ,17,18 ha and SAH ,25 was n and SAH ,15. MeanSelection criteria for aneurysm occlusion treatment and conservative follow-up with/without control angiograms in participating centers in the ELAPSS score study were heterogeneous. In some countries, small UIAs in elderly are routinely followed radiologically. In Finland only those patients with uncertainty for treatment are followed up on clinical-based criteria. This was seen also in the ELAPSS score study where patients with angiographic follow-up in Finland were significantly younger than elsewhere and these patients are known to be more likely smokers and have higher UIA growth risk ,15,17,18The strengths of this study lie in the complete and almost lifelong follow-up of patient of working age and the very limited treatment selection bias ,15,18. CA limitation of this study is the relatively small sample size, despite a long total follow-up time as compared with other large prospective study populations, which on the other hand, had shorter follow-ups and a high treatment selection bias. Patients with a prior history of SAH have not been shown to have a significantly higher risk of aneurysm rupture or growth than others when confounding factors are taken into account . In thisThe present simple, rapid scoring system for UIA growth is significantly better and easier to use than the ELAPSS score. This new growth score contains also the same factors than the treatment score obtained from this cohort. The latter score is directed to evaluate rupture risk of UIAs and its value for prediction is somewhat better than that for growth score. The treatment score is preferable in clinical practice for treatment decision and it likely reduces need of follow-up angiographies.


Sodium/salt consumption is a risk factor for cardiovascular diseases. Although global targets to reduce salt intake have been established, current levels and trends of sodium consumption in Latin America and the Caribbean (LAC) are unknown. We conducted a systematic review and meta-analysis of population-based studies in which sodium consumption was analyzed based on urine samples (24 h samples or otherwise). The search was conducted in Medline, Embase, Global Health, Scopus and LILACS. From 2350 results, 53 were studied in detail, of which 15 reports were included, providing evidence for 18 studies. Most studies were from Brazil (7/18) and six collected 24 h urine samples. In the random effects meta-analysis, 12 studies  were analyzed since 2010. The pooled mean 24 h estimated sodium consumption was 4.13 g/day . When only national surveys were analyzed, the pooled mean was 3.43 g/day ; when only community studies were analyzed the pooled mean was 4.39 g/day . Studies had low risk of bias. The estimated 24 h sodium consumption is more than twice the World Health Organization recommendations since 2010. Regional organizations and governments should strengthen policies and interventions to measure and reduce sodium consumption in LAC. High sodium and sodium chloride  consumption is a risk factor for raised blood pressure  and cardGlobal efforts have aimed to quantify sodium consumption in all countries and territories, and estimates were made for 2010 . ConsequTherefore, to improve the evidence for Latin America and the Caribbean so that it can inform local policies and surveillance systems, we aimed to synthesize the available information on sodium consumption at the general population level in these countries. For this, a systematic review was conducted to provide recent population-based estimates on sodium consumption using urine samples ; we also highlighted research needs and opportunities. Following the PRISMA guidelines We sought for population-based studies  following a random sampling technique; that is, we did not include studies with convenience samples or in which patients  or high-risk individuals  were studied. Studies should have measured sodium in urine samples, regardless of laboratory method or collection time period ; however, we did not include studies which assessed sodium consumption based on questionnaires  or following any other self-reported method. We focused on adult men and women from Latin American and the Caribbean countries; therefore, we did not include studies which sampled Latino populations in other countries, or which focused on foreigners living in Latin American and the Caribbean countries. We sought five search engines: Medline, Embase and Global Health through Ovid, as well as Scopus and LILACS. No language restrictions were set, and we restricted the search to studies from 1990 onwards. We made this decision because studies with urine samples were uncommon before 1990, and because we aimed to summarize recent trends rather than historic trends. The search was conducted on 20 December 2019. The search terms we used are available in Following the eligibility criteria, titles and abstracts were screened by two reviewers independently (RMC-L and AB-O); discrepancies were solved by consensus. After the screening phase, selected reports were studied in detail by two reviewers independently (RMC-L and AB-O); if there were discrepancies, these were solved by consensus. Finally, when two or more reports used the same data , we selected the oldest report or the one which presented the most information.The authors developed a data extraction form including the following information: publication year, data collection year, country, study scope , sample size, mean age, proportion of male subjects, proportion of people with hypertension, sodium assessment method (whether a formula was used such as the Kawasaki or Tanaka equations), urine sample collection , and estimated 24 h sodium consumption. The data extraction form was not modified during data collation. Data extraction was conducted by one reviewer (RMC-L) and independently verified by another reviewer (AB-O); discrepancies were solved by consensus. The data collation form is available in Risk of bias of independent studies was conducted following the Newcastle–Ottawa quality assessment scale for cohort studies . One revResults are presented qualitatively and quantitatively. First, we summarized study characteristics as well as estimated 24 h sodium consumption, as reported in the original studies. However, for consistency, sodium consumption results were transformed from the original units  to grams per day (g/day). To translate sodium consumption into (sodium chloride) salt consumption, sodium consumption (in g/day) was multiplied by 2.54. Second, following a random effects meta-analysis approach, we estimated the pooled mean 24 h sodium consumption, yet we restricted this analysis to studies published since 2010. We made this decision to summarize current estimates, thereby providing timely results; notably, there were few results before 2010 and including these in the analysis did not substantially change the results . The pooNo ethical approval was sought as this is a systematic literature review in which no human subjects were studied. The funder had no role in the study design, data collation, and results analysis or results presentation. The authors alone are responsible for the opinions in this work. All authors had access to the data, are responsible for its accuracy and approved the submitted work. The search yielded 2350 results, and 2297 were excluded at the screening stage; thus, 53 were studied in detail. Finally, 15 reports  were included in the systematic review providing results for 18 studies  9,10,11,21,22,23Brazil contributed to the review with seven studies ,18,19,20Six studies collected 24 h urine samples ,19,22,23The meta-analysis included 12 studies published since 2010 , ,21,22,23None of the summarized studies reported mean estimated sodium consumption below the World Health Organization recommendations (2 g/day) . The est2: 99.7%) [The random effects meta-analysis including studies published since 2010 revealed a pooled mean estimates 24 h sodium consumption of 4.13 g/day  ,21,22,232: 99.0%) [2: 97.8%) [When these studies were further divided, those based on a national sample exhibited a pooled mean estimated sodium consumption of 3.43 g/day  ,21; thos: 97.8%) ,19,22,232: 97.9%) [2: 99.8%) [The pooled mean estimated sodium consumption amongst studies with urine samples collected after 24 h was 3.82 g/day  ,19,22,23: 99.8%) ,15,21. T2: 98.0%) and 4.75 , respectively. We further compared estimated sodium consumption per 24 h urine samples ,19,22,232: 97.5%); for studies published in 2018 the pooled mean was 3.67 g/day ; finally, for those published in 2019, the pooled mean was 3.78 g/day . Although there was a limited number of studies per year, there seemed to be an increasing trend, which has reverted recently. The mean estimated 24 h sodium consumption in the study published in 2015 was 4.06 g/day ; for studies published in 2016 the pooled estimated mean was 4.67 g/day . In addition, in comparison to 24 h urine samples, a meta-regression model adjusting for study scope  revealed that the Tanaka formula retrieved slightly overestimated results , yet the Kawasaki formula showed a stronger association . In meta-regression analysis including only studies published since 2010, one predictor was strongly associated with mean estimated 24 h sodium consumption . CommuniAll reviewed studies had a low risk of bias . In this systematic review to study sodium consumption in Latin America and the Caribbean, the pooled mean estimated 24 h sodium consumption since 2010 was 4.13 g/day , more than twice the World Health Organization recommendations. Interestingly, these estimates varied depending on the study scope. The pooled mean estimated 24 h sodium consumption from national samples was notoriously lower (3.43 g/day of sodium) compared to the pooled mean from community samples (4.39 g/day of sodium). We also observed some overestimation when studies relied on the Kawasaki or Tanaka formula. Although the meta-analytic results need to be interpreted in light of the heterogeneity of populations and methods, these preliminary findings could suggest that national studies are most needed, along with research to estimate 24 h sodium consumption with easier, but accurate, algorithms/formulae. While acknowledging the limitations of this review along with the limitations of the original reports, the results suggest that sodium consumption in several countries in Latin America and the Caribbean is above the international recommendations ,6. AlthoBesides the INTERSALT study conducted in the late 1980s and not included in this review , and theA global pooling endeavor which provided estimates until 2010 reported a daily sodium intake in Latin America and the Caribbean raging between 2.6 and 4.1 g/day . The resWe found that most recent estimates are slightly smaller than those of previous years. A similar finding was pinpointed by a recent global work , in whicDue to the limited/null number of studies for several countries, it seemed impossible to compare countries or sub-regions . The results only show that any estimate provided for South America is larger than the result for the Caribbean; this is consistent with the findings by Powel and colleagues . DiffereWe followed a strong methodology using five search engines, summarizing studies with reliable methods to ascertain 24 h sodium consumption. We have summarized more results for Latin America and the Caribbean than previous and most recent global reviews ,8.Limitations must be acknowledged as well. First, we did not search the grey literature. Although this could have potentially provided additional reports, it is highly unlikely that these would have substantially changed the results or conclusions. These would be most likely community studies, probably without urine samples. Second, we did not retrieve enough results to compute strong meta-regressions for all potential predictors. However, the findings from the meta-regression analysis are still informative and could guide new studies or recommendations for future research. Third, we retrieved very few results from Central America and the Caribbean, which highlights the urgent need for this evidence in these sub-regions. Fourth, because few studies reported the years of data collection, we conducted the analysis based on publication year. Results about trends should be interpreted in line with this potential limitation. Fifth, we acknowledge that the overall mean sodium/salt consumption based on the meta-analytical approach may not be the best metric to provide country- or regional-specific summary measures, because few studies were national samples  and many countries did not have data available. However, this summary metric aimed to report on the regional current global landscape of (high) sodium consumption as a starting point for stronger studies to build on.A common limitation amongst the selected studies was the inconsistent methodology they followed to ascertain 24 h sodium consumption, i.e., some authors collected 24 h urine samples while others used spot samples, or they relied on different equations to compute 24 h sodium consumption. The results showed some potential differences in sodium consumption estimates based on 24 h urine samples versus estimates based on formulas; thus, for surveillance and in large national surveys, the results do not strongly support the use of any formula in comparison to 24 h urine samples. However, in order to have consistent and comparable results across countries and time, and to reflect the most up-to-date evidence, we suggest that regional organizations  or professional bodies  should update standard guidelines  for rese30% relative reduction in mean population intake of salt/sodium [The World Health Organization, as well as the Pan-American Health Organization, has issued the SHAKE package for salt reduction ,32. Thist/sodium . This syA 24 h urine sample is the gold standard to ascertain sodium consumption; this approach is also recommended by the Pan-American Health Organization ,23,25. HIn light of the global conflicting evidence and the results herein presented, and because the Pan-American Health Organization guidelines were published 10 years ago , this guFrom a research perspective, large studies could be conducted in Latin America and the Caribbean to provide local and regional evidence about the accuracy of available formulae and, when relevant, calibrate them. Data pooling studies, for example, those herein summarized, could be useful to adapt available formulas for populations in Latin America and the Caribbean. New technologies and prediction methodologies, for example, machine learning algorithms, could be tested for this purpose. Sodium consumption evaluation across Latin America and the Caribbean still seems to be limited. This calls for urgently strengthening research and national surveys to also study sodium consumption in a consistent and comparable fashion. Nonetheless, available evidence already suggests that 24 h our sodium consumption is twice the international recommendations. When stronger studies become available to improve regional evidence on this matter, regional organizations and governments should strengthen policies and interventions to reduce sodium consumption in Latin America and the Caribbean.