We aimed to evaluate the effects of different exercise rehabilitation (ER) programs on LVEF and the incidence of restenosis in patients after percutaneous coronary intervention (PCI) through a systematic review and an integrated network meta-analysis (NMA) to provide a reference for the clinical formulation of ER programs for PCI patients.Meta-analyses of the effects of different types of ER programs on LVEF and the incidence of reinfarction in post-PCI patients were retrieved from 11 domestic and foreign databases. The methodological and reporting quality of the included systematic reviews were evaluated using the AMSTAR 2 and PRISMA statements. The GRADE scoring system was used to evaluate the quality of evidence found in the studies included in the meta-analysis, and studies with high and intermediate-quality evidence were qualitatively analyzed. Stata software (version 16.0) was used to conduct an integrated NMA of the original RCTs with moderate and low risk of bias.Sixteen meta-analyses were included in this evaluation. The reporting quality of the included meta-analyses was relatively complete, and the methodological quality was low. Seventy RCTs were included in the NMA. The results showed that all types of rehabilitative exercises were safe and effectively increased LVEF and reduced the incidence of coronary restenosis in patients. The comprehensive exercise program was the most likely to improve LVEF, and the comprehensive exercise program, early exercise program, and high-intensity interval exercise were better than aerobic exercise. Comprehensive exercise programs, early exercise programs, and aerobic exercise reduced the incidence of restenosis in patients. However, Chinese Qigong did not reduce the incidence of restenosis in patients, and there was a risk of bias and inconsistency in the quantitative analysis of restenosis incidence.Comprehensive exercise programs have the greatest therapeutic significance in improving cardiac output and reducing restenosis rates in post-PCI patients. The early exercise program has great potential but requires kinesiologists to work with physicians to structure the program and strengthen out-of-hospital management. Aerobic exercise has the least therapeutic significance, and Chinese Qigong is suitable for promotion based on its better efficacy than aerobic exercise and may be an alternative to aerobic exercise, but more experimental evidence is needed.https://www.crd.york.ac.uk/PROSPERO/, PROSPERO CRD42022374590. PercutaAs an essential part of modern cardiovascular disease management, cardiac rehabilitation (CR) with exercise rehabilitation (ER) as the core is recommended by the American Heart Association (AHA) and the American College of Cardiology (ACC); it is a reimbursable expense and a Grade A recommendation for treating coronary artery disease and chronic heart failure . CurrentMany systematic reviews and meta-analyses have been published on the effect of ER after PCI. Nevertheless, recommendations for post-PCI early rehabilitation regimens vary among different national or regional guidelines, and some studies have conflicting results. The ACC, American Cardiopulmonary Rehabilitation Association (AACVPR), and AHA support a progression from moderate to vigorous aerobic exercise combined with resistance training. In contrast, according to guidelines upheld throughout Asia, the UK, France, and Japan, lower-intensity aerobic exercise is recommended and resistance training can be reduced or even ignored . In ChinLeft ventricular ejection fraction (LVEF) refers to the output per beat as a percentage of the end-diastolic volume of the ventricle and is a common clinical indicator of the contractility of the myocardium and output function of the heart. Coronary angiography is the gold standard for diagnosing coronary artery disease, and its review in patients after PCI to determine restenosis occurrence is a direct assessment of prognosis. This study re-evaluated the meta-analysis discussing the efficacy of ER in patients after PCI and performed a mesh meta-analysis involving randomized controlled trials (RCTs) to compare the effect of different exercise regimens on LVEF and restenosis incidence to inform clinicians' decisions to find a suitable exercise mode for the general population and community healthcare services.2.https://www.crd.york.ac.uk/PROSPERO/.This scheme was registered in PROSPERO (registration number: CRD42022374590) and is available at 2.1.Inclusion criteria are based on the PICOS principle.2.1.1.Patients who had previously undergone PCI, with no restriction on race, age, or sex.2.1.2.ER refers to exercise with specific flow and intensity control performed for medical purposes under the assessment and guidance of physicians, including but not limited to aerobic exercise, resistance training, endurance training, high-intensity interval training, and traditional Chinese exercises, such as Baduanjin.2.1.3.Other non-ER therapies  or ER with a different protocol from the experimental group.2.1.4.LVEF: Left ventricular ejection fraction was assessed by two-dimensional echocardiography (modified biplanar Simpson method).Restenosis: Coronary angiography confirmed restenosis ≥50% after the original intervention, including intrastent stenosis.2.1.5.Systematic reviews and meta-analyses before 1 April 2023.2.1.6.① duplicate publications; ② conference studies, reviews, etc.; ③ studies with data that could not be extracted or for which the full text of the literature could not be obtained; ④ studies with incomplete meta-analyses; ⑤ studies with missing critical information; and ⑥ studies in which the evaluation results did not include outcome indicators.2.2.Two researchers independently searched the electronic databases of PubMed, Web of Science, Foreign Medical Literature Retrieval Service (FMRS), Cochrane Library, Embase, Springer, China National Knowledge Infrastructure (CNKI), Wanfang database, Chinese scientific periodical database of Information (VIP), and Chinese Medical Association using a combination of network words and free words. Additionally, references to the included literature and registered PROSPERO were traced. Using PubMed as an example, the retrieval strategy is illustrated in BOX 1#1 percutaneous coronary intervention[MeSH] OR percutaneous coronary intervention[Title/Abstract]#2 exercise rehabilitation [Title/Abstract] OR Exercise[Title/Abstract] OR Exercise therapy[Title/Abstract]OR training[Title/Abstract]OR Physical training[Title/Abstract]OR kinesis[Title/Abstract]OR motion[Title/Abstract]OR sports medicine[Title/Abstract]OR sport[Title/Abstract]#3 meta-analysis[MeSH] OR meta-analysis[Publication Type]#4 meta analysis[Title/Abstract] OR meta analyses[Title/Abstract] OR meta-analysis[Title/Abstract]OR meta-analyses[Title/Abstract] OR data pooling[Title/Abstract]OR data poolings[Title/Abstract] OR clinical study overview[Title/Abstract]OR clinical study overviews[Title/Abstract]#5#3 OR#4#6 systematic review[Title/Abstract] OR systematic reviews [Title/Abstract]#7#5 OR #6#8#1 AND #2 AND #72.3.Two reviewers independently screened the titles and abstracts of the retrieved results and selected all potentially relevant references according to the search strategy. Bibliographic management was performed using Endnote X9 software.We used standardized data collection forms to extract the characteristics of the reviews, including the number of original references included, participants, experimental group intervention measures of the control group, and outcome indicators. We contacted the original authors when data needed to be included or information was unclear and analyzed the available data when we could not obtain the required information after sending an email. Disagreements were resolved through discussion or consultation with a third reviewer.2.4.Two researchers assessed the methodological, reporting, and evidence quality of the included literature, and a third researcher arbitrated conflicts.2.4.1.The PRISMA statement was used to assess the report quality. There were 27 items in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement. A complete report of each item was scored 1 point, a partial report was scored 0.5 points, and an unreported item was scored 0 points. The report was considered somewhat deficient if it scored between 15 and 21 but relatively complete if it scored above 21. According to PRISMA, “items with less than 50% completeness are missing reports” , 21.2.4.2.The AMSTAR 2 scale was used to assess the methodological quality of the meta-analyses. The AMSTAR 2 scale contains 16 items, of which Items 2, 4, 7, 9, 11, 13, and 15 are key items. A study with l1 inconsistent non-key item was rated as “high”, a study with >1 inconsistent non-key item was rated as “medium”, a study with 1 inconsistent key item with or without inconsistent non-key items was rated as “low”, and a study with >1 inconsistent key item with or without inconsistent non-key items was rated as “very low”. The AMSTAR 2 score satisfaction and partial satisfaction ≥ 70% can be considered a relatively complete item report –24.2.5.Qualitative analysis of the meta-analysis of outcomes and safety, GRADE , was use2.6.I2 and P values, with I2 ≤ 50% and p ≥ 0.05 indicating no significant heterogeneity in the included studies. The cumulative surface area under the curve and mean rank were used as indicators to rank the treatment effects of interventions  and 95% confidence intervals (CIs), and continuous variables are expressed as standardized mean differences (SMDs) and 95% CIs. Heterogeneity in two-arm comparisons was determined by ventions –28, and ventions .3.3.1.A total of 799 studies and 76 registries were retrieved from 10 databases and PROSPERO. After eliminating duplicates, the titles and abstracts of 326 studies were read for initial screening, and 40 studies were finally selected for reading in full, resulting in the inclusion of 16 meta-analyses for qualitative analysis and 70 RCTs for quantitative analysis. The study retrieval process is illustrated in 3.2.A detailed list of studies and reasons for exclusion can be obtained by contacting the corresponding author of this article.3.3.(1)Participants: All participants were post-PCI patients included in the meta-analysis.(2)Intervention measure: The intervention measures in all studies were ER; five studies , 43, 45 (3)Control measure: The control measure in one study  included(4)Outcome index: Eleven meta-analyses evaluated LVEF and six evaluated restenosis.(5)Study type: All literature types were meta-analyses for RCTs; eight studies reported grant support, and all included meta-analyses were correctly assessed for quality. One study  used theSixteen meta-analyses published between 2013 and 2022 were included in the qualitative analysis, and their main characteristics are shown in 3.4.The methodological quality of 16 meta-analyses was “very low”. There were nine entries in which AMSTAR 2 scores met or partially met ≥70%. Among the seven key items in the AMSTAR 2 quality evaluation, Items 2 and 7 had significant deficiencies, as shown in 3.5.The PRISMA scores of the studies ranged from 21.5 to 24.5 . One stu3.6.(1)p = 0.23) when comparing HIIT with MICT. The combined effect size of early ER as an intervention in two pieces of evidence p = 0.1)  .(3)Safety: All 16 meta-analyses reported that ER was safe .3.7.3.7.1.A total of 70 compliant RCTs were included, with the basic characteristics shown in the 3.8.The included RCTs were all at risk of bias assessment level B or above, and RCTs with a high risk of bias were excluded. The main reasons for bias were that most RCTs mentioned allocation concealment methods and blinding but did not describe them, some RCTs did not report self-help sources, and other biases could not be determined .3.9.3.9.1.(1)p = 0.3848 (>0.05), with no significant heterogeneity.LVEF: The result of the global inconsistency test for LVEF was (2)p = 0.038 (<0.05), suggesting heterogeneity.Restenosis: The result of the global inconsistency test for restenosis was 3.9.2.(3A), namely, RT-MICT-HIIT, RT-RE-AE, RT-EEP-AE, and RT-AE-Qigong, with the largest number of studies comparing EEP with RT, followed by RT with CEP. (2) Restenosis: Fourteen studies evaluated restenosis and did not form a closed loop (3B), with most studies examining AE vs. RT and the fewest examining Qigong vs. RT.The network evidence plot is shown in 3.9.3.4A for LVEF and 4B for restenosis. The two-by-two comparison between interventions in the forest plot, with the intersection with the 0 axis indicates that the difference between this group is not significant, and the specific values of graphs A and B are shown in (1)LVEF: All six ER protocols, RE, Qigong, MICT, HIIT, EEP, and AE, were superior to RT, and the differences were all significant; CEP vs. AE, ERP vs. AE, and HIIT vs. AE were all significant; and CEP, ERP, and HIIT were superior to AE.(2)Restenosis: EEP, CEP, and AE were all superior to RT, with significant differences, while Qigong vs. RT was not significant.The results of the NMA are shown in the forest plot : 4A for 3.9.4.The surface area under the effective cumulative curve represents the effectiveness of a particular intervention in improving the outcome indicators, with larger surface areas being more effective. The curves for LVEF and restenosis are shown in Figures A and B, respectively. The ranking results showed that CEP was most likely to improve LVEF in patients after PCI (90.9%), followed by RE, CEP, EEP, HIIT, Qigong, AE, and RT . EEP mos3.9.5.There was little overlap between LVEF and restenosis in the reports of all RCTs included in this study; therefore, the two indicators were tested independently. Funnel plots are shown as LVEF: As shown in Restenosis: As shown in 4.4.1.All included studies had very low methodological quality. The main reason involves significant shortcomings in two important aspects : none of the meta-analyses were preregistered, and a list of excluded literature was not provided. Other common problems include the following: (1) the authors did not investigate funding and conflicts of interest in the literature, (2) failure to characterize the included studies according to the PICOS principle, and (3) failure to discuss publication bias in the quantitative analysis results.In terms of the report quality, we included only studies with relatively complete research reports. However, overall, there were serious deficiencies in the entry of “protocol and registration” and some deficiencies in the four items of “introduction rationale,” “eligibility criteria,” “search” and “limitations”: (1) all the studies were not registered in advance; (2) there was a lack of explanation of the review principle; (3) the status of the current research of the same type and the necessity of this research was lacking; (4) there was no reasonable explanation of the strategy; (5) only the search term is given, but the specific search strategy is not given; and (6) there was no statement of the limitations of the study.As noted above, the deficiencies may be related to factors such as unregistered and unreviewed research design schemes, lack of awareness of report quality assessment tools during writing, and layout of limitations, which are common problems in current non-Cochrane analyses. This also shows that the writing of the included studies was not done strictly according to the checklist.4.2.All meta-analyses evaluated ER as safe, and all high- and moderate-quality evidence levels of outcomes confirmed that ER is effective in improving cardiac output capacity and reducing the incidence of restenosis in patients after PCI, which is consistent with the current findings. A direct comparison of the amount of combined benefit found that early ER was significantly superior to other intervention options although this finding is not reliable due to possible overlap in the included literature between different meta-analyses.4.3.First, we excluded RCTs with a high risk of bias from the screening process to increase confidence in the final analysis. The main reason for the high and medium risk of bias in RCTs of interest was the implementation of blinding and allocation concealment. Most of the included articles only mentioned blinding without describing the process; similarly, most of the articles only mentioned the use of a computerized random sequence for allocation without describing the people who performed this grouping. The majority of the included articles only mentioned the use of computerized random sequences for allocation but did not describe whether the participants in this grouping were informed of each other. Notably, male participants comprised the majority of subjects in the included RCTs, even more than six times the number of female participants in the studies, which is relevant to the population with CHD but also suggests that the results of such studies in the past may have been biased by sex and that future studies targeting women or comparing men with women are necessary. RCTs focusing on women or comparing men and women are necessary in the future.Second, in the process of including research literature, we found that most researchers specified the type, frequency, and duration of exercise. However, except for protocols with defined exercise intensity, such as HIIT and MICT exercise and Chinese medicine Qigong, which has fixed exercise steps –48, otheThe results of the reticulated meta-analysis indicate that all seven ER protocols  are effective in improving LVEF in post-PCI patients, which is consistent with the qualitative analysis. The current results indicate that the CEP, ERP, and HIIT training protocols improve LVEF better than AE. It also shows that CEP has the highest potential to improve LVEF in post-PCI patients—which does not suggest that TCM Qigong reduces restenosis rates in post-PCI patients and which diverges from the previous qualitative analysis and may be a source of inconsistency in this set of analyses. However, the results of the publication bias analysis suggest the possibility of publication bias in restenosis studies, which may have an impact on the reliability of the results of this analysis.4.4.4.4.1.Critical CR organizations, such as AHA, AACVPR, CACR, and EACPR, all recommend a combined training approach. However, the implementation of CEP is mostly carried out in rehabilitation centers (relying on equipment and doctors), is more expensive, and may not be suitable for less developed countries and regions. Strictly speaking, EEP is similar to CEP in that it includes aerobic, resistance, and flexibility training, and the intervention is delivered earlier than CEP.4.4.2.HIIT training and MICT training both have clear definitions and training modalities, and similar to RE, the implementation of HIIT and MICT is mostly device-dependent and physician-supervised. In the quantitative analysis of this study, we did not observe direct evidence that HIIT, MICT, and RE can reduce the incidence of reinfarction, but all three can improve LVEF, and more relevant high-quality future studies are needed. Compared to CEP, HIIT requires similar equipment, medical and nursing conditions, and less time to train, so it may be possible to participate in optimizing CEP protocols in the future.4.4.3.AE and Qigong have the common advantages of being simple and easy to implement, not limited by space or equipment, and not imposing an additional financial burden on the patient, making them more suitable for community CR. AE has a low likelihood of improving both indicators, and CEP, ERP, and HIIT were superior to AE and were more time-consuming and less therapeutically meaningful. Although Qigong was superior to AE in improving LVEF and reducing the incidence of reinfarction in the likelihood comparison, the results of Qigong in reducing the incidence of reinfarction were not meaningful and could not be evaluated in this study. Thus, more studies need to be conducted in the future.5.The methodological quality of the current meta-analysis examining the impact of ER on LVEF and coronary restenosis incidence in patients after PCI is low. This is mainly because there was no preregistration, no list of excluded literature was provided, and the writing of the included studies did not strictly follow the requirements of the checklist. The RCTs included were mostly unclear in their descriptions of blinding and allocation concealment and had a much larger proportion of male participants than female participants. The intervention methods included a comprehensive exercise program, early exercise program, high-intensity interval exercise, medium-intensity continuous training, resistance exercise, aerobic exercise, and Chinese Qigong. The results of the quantitative analysis showed that while all seven programs could improve LVEF, the comprehensive exercise program was the most likely to improve LVEF, and the comprehensive exercise program, early exercise program, and high-intensity interval exercise were better than aerobic exercise. Comprehensive exercise programs, early exercise programs, and aerobic exercise reduced the incidence of restenosis in patients. However, Chinese Qigong did not reduce the incidence of restenosis in patients, there was a risk of bias and inconsistency in the quantitative analysis of restenosis incidence, and more experimental evidence is needed in the future.


To analyze the microbiological and microstructural part of indwelling urinary catheters and their association with urinary tract infection prevention.This is a cross-sectional study, from June to December 2020, in which 42 indwelling urinary catheter tips and sterile urine samples were collected for analysis of crystals in optical microscopy and biofilms in scanning electron microscopy. Culture analysis and specification of the type of bacteria were performed.Proteus mirabilis, Enterococcus faecalis, Staphylococcus epidermidis, Enterococcus faecium and Enterobacter cloacae stood out. The presence of magnesium-ammonium-phosphate crystal was associated with the presence of urinary tract infection and with Proteus mirabilis. There was a significant association (p = 0.001) between the use of prophylactic antibiotics versus urine culture >105 CFU/mL.It was found that 35.71% of the samples had mature biofilm adhered to the catheter tip. Biofilms of The analyzes contributed to clinical practice, as it reinforces the development of effective and monitored strategies on cultures and urinary tract infection prevention associated with indwelling urinary catheters. In Brazil, CA-UTI are responsible for 35 to 45% of infections acquired in hospitals. CA-UTI may be associated, mainly, with inadequate aseptic handling, prolonged catheter use, inadequate indication and bacterial colonization due to the formation of biofilms.Urinary tract infection (UTI) is one of the major healthcare-associated infections (HAI). In the USA, about 80% of UTIs are associated with indwelling bladder catheter (IBD) (CA-UTI) and approximately 15% occur due to inadequate indications–5. An experimental study indicated that, regardless of the type of polymeric material of IBD, there was growth of viable bacteria, such as Escherichia coli, Pseudomonas aeruginosa and Proteus mirabilis, for a period ranging from 6 to 72 hours post-insertion, adhered to the EPS matrix on the device wall. After 72 hours, signs of biofilm detachment and the presence of new areas of recolonization were observed under microscopy. In clinical practice, in addition to CA-UTI, it can lead to the formation of fouling and occlusions of catheters, favoring the precipitation of calcium, magnesium, ammonium and phosphate crystals, causing urinary stasis in the bladder or delaying emptying, contributing to increased proliferation of microorganisms.Biofilms can promote IBD blockage, adhesion via strong covalent bonds between the exopolysaccharide matrix (EPS) and the polymeric device wall, consequently leading to polycolonic formation with co-colonization of different types of bacteria adhered to the catheter wall,7,8,9 that develop on the IBD wall show remarkable progress in science, truly effective and widely applicable strategies to manage biofilm-related complications faced by catheterized patients are still a gap in scientific knowledge and a challenge for clinical practice.Although studies on biofilm. Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa were identified as the main multidrug-resistant bacteria that may be linked to biofilms. Other bacteria, such as Enterococcus fecalis, to Stahphiloccus aureus and Proteus mirabilis, also showed resistance, but to a limited spectrum of antibiotics. On the other hand, all of them were identified as biofilms on the IBD wall.In another study, the authors assessed, by culture, biofilm detection tests, biochemical identification of bacteria, and antibiotic sensitivity tests of UTI pathogens in patients with IBD–9. Thus, the formation of biofilms, fouling and the emergence of antibiotic-resistant microorganisms are still the main challenges in CA-UTI management, and must be actively monitored, so that public health policies can be taken to guide, not only health professionals, but also patients, in order to obtain effective surveillance in managing IBD-related adverse events.In this sense, scientific evidence reinforces the need to adopt effective measures to prevent CA-UTI, especially during the device insertion and maintenance, aiming at patient safety and clearly indicating to health professionals, the ease of infection and dispersion of bacteria in the catheter from the moment it is inserted and maintained in patients. Catheterization can lead to serious complications, such as pyelonephritis, bacteremia, septicemia, and sepsis, which are responsible for increased morbidity and mortality in patients with IBDFaced with uncertainties and gaps both in clinical practice and in scientific knowledge regarding UTI prevention associated with IBD, the question is: how can the microstructural and microbiological analysis of IBD be associated with UTI prevention?This is a cross-sectional observational study. The present study used the STROBE protocol for cross-sectional studies.Neurological, surgical, medical clinic and intensive care center in a philanthropic health institution in the countryside of Minas Gerais.Participants older than 18 years who were using IBD for more than 48 hours and patients without a diagnosis of UTI at admission were included. Unconscious patients and those with neurogenic bladder, urethral stenosis, urinary incontinence, bladder dysfunction and prostate hyperplasia were excluded..Participants were selected, by convenience sample, and 42 samples were collected between June and December 2020, considering the statistically recommended minimum number of 30 individualsIt is noteworthy that, although there was a sample larger than the minimum necessary for statistical analysis, due to the COVID-19 pandemic in 2020 and changes in the institution’s protocol, especially not authorizing the presence of researchers in the sectors, the sample was concluded in 42 participants.For urine culture, immediately before IBD removal, about 10 mL of sterile urine was collected in a sterile collection flask. The sample was collected after a medical prescription that there was no longer indication and IBD use.°C until observation. The IBD segment, stored in 0.9% saline solution, was analyzed up to a maximum of one hour after collection.Aseptically, 5 cm from the IBD tip was also removed. This was cut with sterile scissors to divide the tip into two parts: a) one part for culture, being inserted in a sterile collector with 0.9% saline solution; b) another part being inserted in a sterile collector, which contained 70% alcoholic solution, being used for visualization in scanning electron microscopy (SEM). This tip was conserved at 4Universidade Federal dos Vales do Jequitinhonha e Mucuri (UFVJM), to perform the culture and isolate the bacteria found, using the gram staining method to identify gram-positive and gram-negative bacteria.The samples were transported to the Bioprocess laboratory of the Department of Pharmacy of the For culture, both in the urine sample and in the IBD tip, the surface scattering technique was performed. Müeller-Hinton agar supplemented with 5% sheep blood and a differential rich medium (Cled BD agar) were used to analyze growth, quantification and isolation. After sown, samples were incubated for 24 hours at 37°C.°C until the time of gram analysis.After that, colony counting was performed manually with the counting of the total load (CFU/mL). Isolation was performed with the aid of a sterile inoculation loop, in which different types of colonies were delicately collected, if this was the case for that sample. Each isolated colony was deposited in different Eppendorf microtubes, properly identified, containing 0.9% saline solution and 2 drops of sterile glycerol conserved at +4After identification of gram+ and gram− bacteria, colonies were inserted and inoculated into Microscan Walkaway automated equipment (Siemens-Sacramento-CA-USA). Through this method, it was possible to identify the different types of bacteria and fungi that could have grown in the sterile sample from both IBD tip culture and urine.®, vortexed for 1 minute. Next, 1 μL was sowed on Müeller-Hinton agar supplemented with 5% sheep blood and a differential rich medium (Cled BD agar), being incubated for 24 hours. After 24 hours, the colonies were counted and the strains were identified by the automated method mentioned above.For the catheter tip, the same method was also used; however, the tip was cultured on CLED agar by the scattering technique, and a second IBD fragment was also cultured in a medium containing a broth BBL Trypticase Soy Broth-BD°C), suprapubic tenderness, costovertebral angle pain or tenderness, when there was no other recognized cause, and positive urine culture with a maximum of two species of microorganisms with growth ≥105 units of colony formation per mL of urine (CFU/mL) were standardized. To assess the presence of pain, a professional nurse, part of this research, carried out a physical assessment with the patient after the IBD removal, checking on fever.As a cut-off criterion of CA-UTI, patients undergoing catheterization for more than 48 hours or after catheter removal within 48 hours and who had fever  with the aid of a Neubauer camera for counting and typology of the crystals. Crystal analysis aimed to compare the presence of specific crystals that could be associated with CA-UTI.For analysis of urine crystals, 5 mL of urine was inserted into Falcon tubes, centrifuging at 1,500 revolutions per minute (RPM) for 5 min at a temperature of 20In SEM, the degree of biofilm development and maturation was observed. The IBD segments were cut longitudinally, about 1 cm, to expose both the endoluminal and external surfaces. The cut was performed under a biological safety cabinet using a sterile scalpel. The sample was delicately manipulated, so that it did not allow the destruction of biofilm, microorganisms or crystalline precipitates that were visualized.After removing the samples of 70% alcohol, they were washed with sterile water and dehydrated, in ascending order, with alcoholic solutions: 70% alcohol for 10 minutes, 90% alcohol for 10 minutes and 100% ethanol for 10 minutes. Next, the sample remained in the biosafety cabinet for 15 minutes for drying.Scanning electron microscope  was used. The samples were placed on a metal support, and then gold coating , with 15 mA, was made for 2.5 minutes. High-definition micrographs were obtained with a magnification of 500 to 4,000x.In participants’ medical records, the following variables were collected: sex; age; patient diagnosis; risk factors ; and urine culture. Data collection in the medical records occurred from June to December 2020.For descriptive analysis, mean ± standard deviation and minimum and maximum values were used. The Shapiro-Wilk test was used to analyze the normality of the quantitative variables. For analysis of means, Student’s t-test was performed. For the comparison of three groups, the ANOVA with Tukey post hoc (parametric) was used. Pearson’s and Fisher’s chi-square test was used to analyze the categorical variables. The level of 0.05 was used in a fixed manner.Conselho Nacional de Saúde). The participants who agreed to take part in the study signed the Informed Consent Form (ICF).This study was approved by the Institutional Review Board (IRB) of UFVJM, under Opinion 3,523,335. This study was approved in 2019 and is in accordance with Resolution 466/12 of the Brazilian National Health Council  had no growth either in the urine culture or in IBC tip culture.proteus sp, UC9: Escherichia coli, UC15: Enterococcus faecium/Enterococcus cloacae, UC19: Enterococcus faecium/Escherichia coli, UC28: Proteus sp/Escherichia coli, UC32 and UC36: Escherichia coli and UC37: Enterococcus cloacae/Escherichia coli. All had >105CFU/mL. However, the signs and symptoms of UTI in these patients had not been described.As for urine culture, there were results in the medical records of only 8 patients. UC8: It was found that 14 (33.3%) samples showed magnesium- ammonium-phosphate in the urine when viewed under optical microscopy; in 8 (19.04%), no crystals were observed; in 9 (21.42%), uric acid was observed; in 8 (19.04%), amorphous urate was observed; and in 3 (7.14%), uric acid and calcium oxalate were observed. 5 CFU. Comparing Of the 42 IBC tips analyzed, 15 (35.71%) presented mature biofilm at the IBC tip, namely: UC 4, 5, 9,12, 14, 15, 16, 19, 22, 30, 34, 37, 38, 39 and 42. All these patients also had suprapubic sensitivity and pain costovertebral angle soon after IBC removal.Proteus mirabilis, and the others formed by Enterococcus faecalis (46.66%), Staphylococcus epidermidis (33.33%), Enterococcus faecium (13.33%), and Enterobacter cloacae (13.33%). Of the 42 samples, in 17 (40.47%) there was EPS production adhered to the IBC wall. Moreover, of the total of samples, in 10 (23.80%) there was a conditioning phase of cell adhesion to the surface, with adhered cocci and presence of protein residues. Of the 15 samples, 9 (60.00%) were biofilms of 5 CFU/mL). The IBD model was the two-way Foley for all participants. Regarding the type of IBD material collected, 35 (83.30%) were latex and 7 (16.70%) were silicone coated latex. There was no significant association (p > 0.05) between the type of IBD and presenting or not mature biofilm when observed in SEM.As for the data that were taken from the medical records, the mean IBD length of stay was 9.43 ± 5.68 days, with a maximum time of 24 days and a minimum of 4 days. There was no significant association (p = 0.236) between length of stay and positive urine culture  were female and 25 (59.53%) were male.5 CFU. However, 6 (14.28%) of participants who had previous UTI, 6 (14.28%) who had chronic kidney disease and 3 (7.14%) who had both previous UTI and chronic kidney disease had catheter tip and urine culture values greater than 105 CFU/mL. As for the other participants, 7 (16.66%) were diabetic and 4 (9.52%) were alcoholics. Of the participants who did not have bacterial growth, 6 (60%) were diabetic and 4 (40%) were alcoholics.Regarding the risk factors for CA-UTI, 16 (38.09%) did not have any risk factor, however, all of them had positive urine culture and catheter tip culture. Thus, having or not a risk factor for CA-UTI was not significantly associated (p > 0.05) in this study with having or not urine culture >105 CFU/mL. Of these, 11.8 (72.72%) had already started antibiotic therapy prior to the date of registration verified in the medical record.Regarding the use of antibiotics, it was found that 11 (26.19%) patients used therapeutic antibiotics due to their own diagnoses of the disease recorded in the medical records, such as ruptured ectopic pregnancy, sepsis of pulmonary and abdominal focus, septic shock and bacterial endocarditis, pelviperitonitis, spondylodiscitis and osteomyelitis. Of these, 1 (9.09%) sample presented urine culture and catheter tip culture >105 CFU/mL.Of the total participants, 17 (40.47%) used prophylactic antibiotics and 14 (33.33%) did not use antibiotics. Among those who did not use antibiotics during hospitalization, 10 (85.71%) had urine culture and catheter tip >105 CFU/mL. Of these, 11 had a catheter tip culture >105 CFU/mL. Furthermore, patients used prophylactic antibiotics without previous urine culture. When antibiotic use (yes or no) was associated with urine culture >105 CFU/mL, there was a significant association (p = 0.001).Of those participants who used prophylactic antibiotics, due to their own condition of hospitalization, and use of invasive procedures, 15 (88.23%) had urine culture >10Escherichia coli, Pseudomonas aeruginosa, Staphylococcus spp, Proteaus mirabilis, Klebsiella pneumoniae, Proteus vulgaris, Enterococcus spp and Candida albicans.According to the results, it was found that, even though urine was collected in a sterile way, there was growth of microorganisms both in urine culture and in IBD tip culture  and 2. T. The most prevalent microorganisms and common to the two selected sites in the IBD were Escherichia coli and Enterococcus faecalis. In this study, there was also a high prevalence at the IBD tip in Enterococcus faecalis 12 (28.57%) and Escherichia coli 8 (19.04%). Despite a lower prevalence, the presence of Proteus mirabilis, Pseudomonas aeruginosa, Candida albicans, Staphylococcus epidermidis and Enterococcus faecium was also verified when compared to the present study.In a study conducted in the United Kingdom, the authors collected 58 IBD from patients admitted to a health institution to verify the presence of bacteria in the catheter balloon and lumenIn this study, the number of colonies identified was not statistically associated (p > 0.05) with the IBD length of stay. It is noteworthy that, even in a short IBD stay (<28 days), it was possible to visualize mature biofilms, which suggests a reflection on the probable risk in long-term patients (>28 days), especially in nursing homes, which can lead to detachment of biofilm cells.5 CFU. These results support an experimental study in which the authors did not verify an association between length of stay and bacteria growth, and they also verified that antibiotics did not significantly reduce (p > 0.05) the colonization in the lumens of IBD removed from patients. It can be inferred that the inappropriate use of prophylactic antibiotics induces a response of microbial populations that accelerate the natural process of bacterial resistance. Another fact that drew attention is that patients used antibiotics prior to urine culture, which hinders the growth of microorganisms and provides false-negative results.Another factor that did not influence colony growth was the use of antibiotics. Most patients on prophylactic antibiotics presented urine culture values >10Also, mechanisms of bacterial resistance expression such as production of inactivating enzymes, alteration of the receptor for drug action, alternative metabolic pathways and even the formation of biofilms accelerate the maintenance process of an infectious process such as CA-UTI. In this study, it was possible to observe several stages of biofilm development  and 3, gProteus mirabilis, which, in turn, develops a crystalline biofilm that encrusts on the IBD surface and blocks urinary flow (Proteus mirabilis is a flagellate bacterium that has the ability to render urine alkaline, producing magnesium and calcium phosphate microcrystals that accumulate on the catheter surface forming a mineralized biofilm structure,9. Microcrystals adhered to the bacterium may remain viable after catheter removal and trigger chronic urinary infections, recurrent catheter lumen blockage, acting as re-infection reservoirs after UTI treatment or device replacement. Due to its high migration capacity and because it contains elongated cells, Proteus mirabilis is able to easily displace from the periurethral region into the bladder which can often lead to an eventual reflux of infected urine into the upper urinary tract and kidneys,9. In this study, 33.3% of participants showed magnesium-ammonium-phosphate in urine when viewed under optical microscopy (Proteus mirabilis at the IBD tip.Another factor that may contribute to a specificity of the type of mature biofilm visualized in this study was that 60% were formed by ary flow . Proteuscroscopy  and the Enterococcus faecalis, and it can be stated, according to another experimental study, that the factors secreted by Enterococcus faecalis increase the pathogenic potential of Proteus mirabilis, promoting a high urease production and increased cytotoxicity. In this study, two samples had the co-infection of the two bacteria at the IBD tip (Another bacteria prevalent at the IBD tip was  IBD tip , which c.In this sense, the structural analyses of the IBD visualized in SEM and of microorganisms in urine culture made it possible to infer that, for patients in this study who removed IBD, there was a possibility of presenting CA-UTI, since, associated with a positive urine culture, all of them reported minor suprapubic tenderness and costovertebral angle pain on physical examination. In addition to this, nursing must perform an active and daily surveillance of the catheter length of stay, indication and length of stay of patients with IBD, indicators established and associated with UTI-AC prevalence maintenanceThis research sought to broaden and give a new meaning to the view nursing team’ view on CA-UTI, since knowing the formation of biofilms in the IBD and the results of urine culture of all patients analyzed, it is understood the need for a collective assessment of the entire multidisciplinary team for a decision on strict criteria for physical and clinical examinations of all patients expected to remove the IBD and confirmation of the need to request a urine culture before discharge.This study also contributed to generate quality indicators that contribute to the development and implementation of CA-UTI prevention and management methods, in order to avoid adverse events in patients, often unknown by the multidisciplinary team.As already evidenced in the results, it is necessary to be attentive; therefore, the use of antibiotics, which for the most part was started entirely empirically, the growth of pathogenic microorganisms in the urine, the formation of crystalline precipitates, the development of bacterial biofilm occur and, together with this, length of hospital stay and morbidity and mortality can increase in the institution.Dulce Aparecida Barbosa


Arabidopsis thaliana. Initially, we utilized CollaborativeNet, also known as TF-Cluster, to construct a collaborative network of all TFs, which was subsequently decomposed into many subnetworks using the Triple-Link and Compound Spring Embedder (CoSE) algorithms. Functional analysis of these subnetworks led to the identification of nine subnetworks closely associated with regeneration. We further applied principal component analysis and gene ontology (GO) enrichment analysis to reduce the subnetworks from nine to three, namely subnetworks 1, 12, and 17. Searching for TF-binding sites in the promoters of the co-expressed and co-regulated (CCGs) genes of all TFs in these three subnetworks and Triple-Gene Mutual Interaction analysis of TFs in these three subnetworks with the CCGs involved in regeneration enabled us to rank the TFs in each subnetwork. Finally, six potential candidate TFs—WOX9A, LEC2, PGA37, WIP5, PEI1, and AIL1 from subnetwork 1—were identified, and their roles in somatic embryogenesis (GO:0010262) and regeneration (GO:0031099) were discussed, so were the TFs in Subnetwork 12 and 17 associated with regeneration. The TFs identified were also assessed using the CIS-BP database and Expression Atlas. Our analyses suggest some novel TFs that may have regulatory roles in regeneration and embryogenesis and provide valuable data and insights into the regulatory mechanisms related to regeneration. The tools and the procedures used here are instrumental for analyzing high-throughput transcriptomic data and advancing our understanding of the regulation of various biological processes of interest.We employed several algorithms with high efficacy to analyze the public transcriptomic data, aiming to identify key transcription factors (TFs) that regulate regeneration in The online version contains supplementary material available at 10.1007/s42994-023-00121-9. Plants have a remarkable capacity for regeneration, which is defined as the physiological renewal, repair, or replacement of tissues or organs , root apical meristems (RAM), and lateral meristems contain stem cell masses that are responsible for continuous growth and development by differentiating into various specialized cell types. When plants are wounded or specific tissues or organs are cut and cultured on callus induction medium (CIM), certain combinations of autogenous or externally applied plant hormones, such as auxins and cytokinins, can induce de-differentiation at wound sites. These hormones help initiate cellular reprogramming and enable specific differentiated cells to revert to an undifferentiated state, including those in callus and meristematic tissues. In an undifferentiated state, cells gain regenerative capability and can differentiate into new tissues, organs, or even whole plants  and root induction medium (RIM) to facilitate the regeneration of the shoots and roots, respectively. In general, roots, shoots, leaves, whole explants, and hypocotyls are initially cultured in an auxin-rich CIM to stimulate callus formation. Subsequently, these calli are transferred to a medium rich in cytokinins, known as SIM, to induce shoot regeneration. In addition, callus cells or shoot explants can be transferred to a medium with a higher concentration of auxins relative to cytokinins, called RIM, for root generation , Horstman et al. performed ChIP-seq analysis to identify the binding sites of BBM transcription factor in somatic embryo tissue  construction of a Shared Co-expression Connectivity Matrix (SCCM) . We queried the SRA database by combing these words with and tissues. Finally, manual curation was performed to obtain the data that met our requirements.Considering the aim of the study, we collected transcriptomic data (RNA-seq) from the Sequence Read Archive (SRA) database, National Center for Biotechnology Information (NCBI). In our research, we utilized NCBI’s user interface to query transcriptome data (RNA-seq) generated from de-differentiation and re-differentiation-related processes, for example, callus induction from various tissues and differentiation of callus and explant tissues (stems or leaves) into SAM and RAM . A Python wrap was developed and used to call fastq-dump to download all data sets recursively.Metadata was obtained via NCBI’s specialized user interface functions. We evaluated sample properties including cell and tissue types, and experimental methods for ultimate inclusion in the study. We used the Unix operating system to download and preprocess the data. Fastq-dump, a utility bioinformatics tool in NCBI’s SRA toolkit, was employed to download transcriptome data with the SRA accession identifiers of RNA-seq samples (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Given that some datasets exhibited quality issues, we employed Trimmomatic to remove adaptor sequences and low-quality reads from high-throughput sequencing data , of A. thaliana TAIR10 were downloaded from Phytozome . Then, the alignment of the sample’s reads was performed by supplying the index files and obtaining output files in BAM format using STAR. Finally, Cufflinks was used to assemble the individual transcripts from the reads aligned to the genome and to calculate expression levels for all splicing variants of each gene   within each subnetwork by leveraging existing annotations.−2 for the corrected P values. The enriched GO terms representing the various biological processes related to pluripotency or multipotency, for example, regeneration, somatic embryogenesis, callus formation, and embryo development, were studied. The enriched GO terms were used to construct GO trees with ‘Ancestor Chart’ tool available at from EMBL-EBI (https://www.ebi.ac.uk/) to view the hierarchical locations of nodes of interest, especially, the parent and child nodes, so that their relevance to pluripotency or multipotency can be determined.We used ShinyGO V0.76  in a set of CCGs. We used the data within CIS-BP Database, where TFBSs were obtained from experimental validation using techniques such as Protein-Binding Microarray experiment or chromatin immunoprecipitation (ChIP). Once a binding site of a TF was identified, a PWM was constructed based on the alignment of known instances of the binding site. This database includes the DNA sequence motifs and the corresponding transcription factors that recognize them  algorithm  and metadata associated with RNA-seq samples from A. thaliana. From this initial dataset, we carefully selected samples that captured crucial moments in de-differentiation or re-differentiation processes. We identified 78 samples to be included in our data after manually curating and filtering them based on several criteria . Among them, twenty-two and six samples were taken from stem cell mass regions called SAM and RAM, respectively. Seven samples were taken directly from calli. In addition, we included four leaves, four root, thirteen whole explant, eleven shoot, and eleven hypocotyl samples, all of which were cultured in CIM. These samples covered the entire processes, starting from de-differentiation into calli from a specific tissue and re-differentiation into shoots or roots from calli in cytokinin-rich or auxin-rich media, respectively.Arabidopsis TAIR 10 reference genome, and transcripts were assembled and quantified. The result is a set of FPKM values (Fragments Per Kilobase of Transcript per Million mapped reads) for each transcript, which provides normalized expression levels considering both sequencing depth and gene length.RNA-seq data files in FASTQ format were downloaded from the SRA database, NCBI, and were subjected to quality control and trimming procedures on the sequencing reads. Next, the cleaned reads were aligned to the A. thaliana are shown in Supplemental Table 2. This information is important for understanding the tissue specificity and functions of the identified TFs and non-TFs, as well as their roles in various developmental stages during the regeneration process.Then, we conducted an extensive literature review to collect information on known transcription factors (TFs) that regulate pluripotency, multipotency, and regeneration with an assumption that pluripotency and multipotency are essential for regeneration. We identified a total of 145 genes, including 78 TFs and 67 non-TFs, from literature . These genes are known to play a role in pluripotency and multipotency maintenance and regeneration process. To visualize the expression patterns of these 145 pluripotent genes across the 78 samples, we generated a heatmap . The details on the origin of these 78 samples collected from A. thaliana is the presence of specific TFs that control the expression of genes involved in cell division and differentiation. An example is the WUSCHEL (WUS) gene, a pivotal regulator of stem cell maintenance in A. thaliana, which is a key regulator of stem cell maintenance in A. thaliana and is required for the regeneration of whole plants from somatic embryos . Initially, all 1622 known TFs were used to build SCCM, which held all collaborative regulatory gene pairs. The SCCM was then decomposed into 280 non-overlap subnetworks  using a Triple-Link algorithm in the CollaborativeNet package. It's important to observe that subnetworks created earlier exhibited a higher number of high collaborative edges compared to those generated later in the decomposition process, and that the subnetworks generated after the initial 100 were generally smaller and featured weaker collaborative strengths in comparison to their predecessors . This is because the Triple-Link algorithm always chooses two TFs with the highest concordance of collaboration in the SCCM to decompose it. We studied the functional annotation of TFs in each subnetwork with respect to the regeneration. Thus, we mapped the 78 known pluripotent TFs  to facilitate the identification of crucial subnetworks. We also incorporated existing gene annotations in In Fig. P-values to identify the most significant GO Terms. 20, 131, 313, and 141 enriched GO terms were obtained for subnetworks 1, 12, 17, and 74, respectively. The most significant GOs associated with regeneration are displayed in Fig. P values of 0.007 and 0.02, respectively . Subnetworks 12 was dominated by the GO terms that represent root system development , while subnetwork 17 was enriched with the GO terms of root/shoot/floral meristem growth and callus formation . We also implemented GO analysis to subnetwork 74’s CCGs but failed to identify GO terms relevant to regeneration . But there were some tissue-specific regeneration processes like leaf formation, seedling development, regionalization, and meristem structural organization. Subnetworks 1, 12, and 17 are obviously more relevant to the regeneration process, as evidenced by the prominent GO terms shown in Fig. Since most TFs in a subnetwork are not well characterized and annotated in functions, we merged the top 100 CCGs of each TF for each given subnetwork and then removed redundant ones for inferring the enriched functions terms that can present the primary function of the subnetwork. This can be accomplished by GO enrichment analysis. For example, after merging the CCGs of 39 TFs in subnetwork 1 and removing the redundant ones, 980 unique CCGs were obtained and used for GO enrichment analysis. The importance of GO analysis lies in its ability to provide the enriched biological processes. By analyzing the enrichment of GO terms representing biological processes and their hierarchical relationships, researchers can gain a better understanding of the biological processes that are active in a particular tissue. We conducted GO analysis with the unique CCGs associated with each of the three subnetworks using ShinyGO V0.76  the CCGs that are not a TF, defined as non-TF CCGs. Due to the discrepancies in functions of subnetwork 1, 12, and 17, the non-TF CCGs that are usually not shared by the three subnetworks, especially between Subnetwork 1 and the other two subnetworks. Relatively speaking, subnetwork 12 and 17 shared more common non-TF CCGs than any of them with Subnetwork 1. Based on the gene ontology  and gene functional annotation, the CCGs of Subnetwork 1, are primarily involved seed and embryo development; the CCGs of Subnetwork 12 are primarily involved in organ, root, root cap, tissue development, meristem growth and development as well as shoot development, and the CCGs of Subnetwork 17 are primarily involved in root and leaf development, meristem development, and callus formation. The nonTF CCGs account for 88.6% of all CCGs of Subnetworks 1, 12, or 17; (2) The CCGs themselves are a TF but not the one in the Subnetwork 1, 12, and 17; only 2% CCGs of Subnetworks 1,12, and 17 are of this type. (3) The CCGs themselves are a TF in Subnetwork 1, 12, or 17 and belong to another TF in the three subnetworks. About 9.4% CCGs of Subnetworks 1,12, and 17 are of this type. Interested readers may look up Supplemental Table 8 for CCGs that may potentially contribute to the regeneration.LEC2 and PGA37 of Subnetwork 1. Likewise, three motifs were found for PLT1 and ARF16 in Subnetwork 12  and the regeneration GO:0031099) that were enriched in CCGs of Subnetwork 1. For the TGMI analysis, we merged the biological process genes involved in both the somatic embryogenesis and regeneration, and then combined each TF in Subnetwork 1 with two biological process genes from the merged somatic embryogenesis and regeneration processes as a trio for evaluation. When all combinations of the possible trios were assessed, and the number of times each TF interfered with each biological gene present in various trios was calculated  in Supplemental File 1.We also used TGMI to evaluate TFs in Subnetwork 12 on their interference frequencies with the biological process genes involved in embryo development (GO:0009790) , and TFs in Subnetwork 17 on their interference frequencies with embryo development (GO:0009790) and callus formation (GO:1990110) . For Subnetwork 12, the TFs that interfered with the embryo development included TRO7, PLT1, PLT2 BBM, PLT3, and WIP4. For Subnetwork 17, the TFs that interfered with the embryo development included LBD31, PLT3, LBD16, LBD18, MYB56, WOX5B, etc., while the TFs that interfered with the callus formation included MYB56, LBD16, PLT3, WOX5B, and LBD29. Column 4 in Table WUS, for example, promotes stem cell identity in SAM for mass production and proliferation in differentiation phase, and CLAVATA restricts WUS expression to manage the stem cell population , and somatic cells induced from callus induced differentiated somatic cells  . Next, the samples were preprocessed by our pipelines, which includes acquiring FASTQ files, conducting quality control and read trimming, and aligning the processed clean reads to the A. thaliana reference genome, as well as quantifying transcripts to gain gene expression values in the form of FPKM. The construction of a collaborative network, followed by its decomposition using the Triple-Link algorithm led to the generation of 280 non-overlapping subnetworks. CollaborativeNet prioritizeed subnetworks based on coordination strengths, ranking those with high coordination strength first. The algorithm's preference for TF pairs demonstrating the highest coordination strength within SCCM for a subnetwork decomposition led to this prioritization. Typically, the top 100 subnetworks are more biologically meaningful, while those beyond 100 tend to be smaller, and often containing just a few TFs as shown in boxplot . Through the alignment of known pluripotent TFs  with subnetworks TFs and employing gene annotation-based selection, we narrowed down the initially identified subnetworks to just nine  within their associated CCGs. Our analysis revealed an enrichment of the binding motifs of the TFs from Subnetwork 1 in their CCGs, as confirmed by the hypergeometric test Table A. Howeveure Fig. , consideWOX9A, LEC2 and PGA37 in regeneration and/or embryogenesis have been previously been demonstrated by both in vivo and in vitro level experiments /MYB118 in the regulation of somatic embryogenesis and the transition from vegetative to embryonic states  trigger damage repair and regeneration. During the globular stage of embryonic development, WIP5, along with NTT and WIP4, is expressed in the hypophysis cells. This expression continues as the hypophysis produces the apical and basal daughter cells, redundantly facilitating the initiation of the root meristem . In Subnetwork 12, the TFs that interfered with the embryo development included TRO7, PLT1, PLT2 BBM, PLT3, and WIP4. For Subnetwork 17, the TFs that interfered with the embryo development included LBD31, PLT3, LBD16, LBD18, MYB56, and WOX5B, and the TFs that interfered with the callus formation included MYB56, LBD16, PLT3, WOX5B and LBD29. Many of these are known TFs that regulate the regeneration-related biological processes. For example, PLT1-3 and BBM are involved in early embryo development Supplementary file2 (DOCX 410 kb)Supplementary file3 (DOCX 253 kb)Supplementary file4 (DOCX 43 kb)Supplementary file5 (XLSX 14 kb)Supplementary file6 (XLSX 340 kb)Supplementary file7 (XLSX 12 kb)Supplementary file8 (XLSX 13 kb)Supplementary file9 (XLSX 13 kb)Supplementary file10 (XLSX 23 kb)Supplementary file11 (XLSX 132 kb)Supplementary file12 (XLSX 18 kb)Supplementary file13 (XLSX 19 kb)Supplementary file14 (XLSX 16 kb)Supplementary file15 (XLSX 16 kb)Below is the link to the electronic supplementary material.


Inbreeding depression is expected to be more pronounced in fitness-related traits, such as pig litter size. Recent studies have suggested that the genetic determinism of inbreeding depression may be heterogeneous across the genome. Therefore, the objective of this study was to conduct a genomic scan of the whole pig autosomal genome to detect the genomic regions that control inbreeding depression for litter size in two varieties of Iberian pigs (Entrepelado and Retinto). The datasets consisted of 2069 (338 sows) and 2028 (327 sows) records of litter size  for the Entrepelado and Retinto varieties. All sows were genotyped using the Geneseek GGP PorcineHD 70 K chip. We employed the Unfavorable Haplotype Finder software to extract runs of homozygosity (ROHs) and conducted a mixed-model analysis to identify highly significant differences between homozygous and heterozygous sows for each specific ROH. A total of eight genomic regions located on SSC2, SSC5, SSC7, SSC8, and SSC13 were significantly associated with inbreeding depression, housing some relevant genes such as FSHR, LHCGR, CORIN, AQP6, and CEP120. The most significant consequence of inbreeding in the phenotypic performance of livestock populations is the occurrence of inbreeding depression . TheoretOne method widely used to detect identical-by-descent (IDB) genomic segments is to conduct runs of homozygosity (ROH) . ROH areIn the context of Iberian pig populations, non-uniform effects of inbreeding depression across the genome were observed in a closed experimental flock of the Guadyerbas variety . HoweverThe dataset utilized comprises 2069 records (pertaining to 338 sows) for the Entrepleado variety and 2028 records (related to 327 sows) for the Retinto for both TNB  and NBA (Number Born Alive). In conjunction with this, a pedigree that contains genetically interconnected individuals was incorporated, with a total of 581 individuals for Entrepelado and 541 individuals for Retinto. The mean phenotypic performance values for TNB and NBA are summarized in Each sow was genotyped using the Geneseek GGP PorcineHD 70 K  chip. Subsequent to genotyping, the genotypic data underwent filtration using PLINK 1.9 . FiltersFirstly, we formulated a mixed linear model to assess the variance components and calculate the inbreeding depression through the gradient of a covariate associated with the percentage of individual heterozygosity, measured as the number of heterozygous SNPs per individual × 100 divided by the total number of SNPs.y represents the vector comprising phenotypic records , f is a vector encompassing individual heterozygosity, and b is the vector of systematic effects, which incorporates order of parity at 5 levels . Additionally, h is a vector of random herd–year–season, with 96 levels for Entrepelado and 113 for Retinto, u denotes the vector of additive genetic random effects, p is the permanent environmental sow effect and e stands for the vector of residuals. Moreover, d serves as a covariate of the relationship between individual heterozygosity and phenotypic performance. The matrices X, T, Z and W are the corresponding incidence matrices. The genomic relationships (G) among the additive genetic effects (u) were calculated using the single-step approach [http://nce.ads.uga.edu/wiki/doku.php?id=application_programs, accessed on 8 October 2023) [The model we postulated for both varieties is as follows:approach ,17. For approach  was adoper 2023) .https://github.com/jeremyhoward/Unfavorable-Haplotype-Finder, accessed on 8 October 2023) [Secondly, the Unfavorable Haplotype Finder software (er 2023)  was empler 2023) .t-test.Concluding this step, the blupf90+ software  was utilThe results of the variance component estimation are presented in The estimates of the (co)variance components were similar to the ones provided by Srihi et al. , and thep = 0.017) and 0.057 ± 0.028 (p = 0.021) for NBA and TNB in the case of Entrepelado and 0.077 ± 0.051 (p = 0.065) and 0.067 ± 0.050 (p = 0.090) for NBA and TNB in the case of Retinto. In all traits and populations, there was an increase in the litter size as the percentage of heterozygosity increased, leading to significant results for the Entrepelado population.Given the estimates of the variance components, the estimates of the covariate with the percentage of heterozygosity were 0.055 ± 0.026  consisting of more than 15 SNPs within the Entrepelado and the Retinto varieties, respectively. The ROH with the minimum length had 145,783 base pairs, and the larger ROH comprised 15,162,018 base pairs. These distributions highlight the prevalence of short ROHs in the Entrepelado population, in contrast to the right-skewed distribution observed in the Retinto population. This observation may suggest more recent inbreeding within the Retinto population. The average size of the ROHs in each population was 25.79 SNPs (±18.00) for Entrepelado and 35.96 SNPs (±24.30) for Retinto. Furthermore, the average percentage of an individual’s genome covered by ROHs, considering overlapping regions between ROHs, was 26.87% (±3.78%) for Entrepelado and 40.74% (±3.20%) for Retinto.Among all the detected ROHs, we were able to identify 20,143 (Entrepelado) and 26,771 (Retinto) ROHs shared by at least 5% and at most 95% of individuals, composed of more than 15 SNPs, in which we expected to find that most of the variance was due to the ROH effect. Therefore, we solved 20,143 and 26,771 mixed-model equations for Entrepelado and Retinto, respectively. The objective was to obtain estimates of the effects related to the presence or absence of each specific ROH. The distributions of these effect estimates, pertaining to TNB and NBA, are illustrated in p < 0.05) encompassed 1123 and 1533 runs of homozygosity (ROH) for NBA in Entrepelado and Retinto, respectively, while for TNB, they numbered 1197 and 1453 regions. These findings represent a proportion of significant ROHs that ranged from 5.4% (for RR and TNB) to 5.9% (for EE and TNB), slightly higher than what would be expected at random.The average estimate of the effects was consistently close to zero across all scenarios, indicating that most of the ROHs were not associated with inbreeding depression. The genomic regions associated with inbreeding depression  gene, whose encoded protein plays an essential role in embryo development and implantation [BAG Cochaperone 2) and RAB23 . The former is potentially linked to infertility, as the mediated inhibition of CHIP expression contributes to endometriosis [The regions identified in the NBA are also observed in the TNB results and are located on SSC7, having the lowest p-values. These regions span between 20,074,761 and 20,586,603 base pairs (bp), wherein proximity to QTLs associated with pig litter size ,28 is noantation . Additioetriosis , and theetriosis , and witetriosis . Lastly,Centrosomal Protein 120) gene, which has been associated with maternally derived aneuploidy [Activating Transcription Factor 1) gene, known to be involved in the estrogenic signaling pathway [Aquaporin 5 and Aquaporin 6), which have been suggested as markers for male infertility in livestock [Rac GTPase-Activating Protein 1), the inhibition of which is required in vitro for human embryonic trophoblast invasion into endometrial stromal cells [Calsyntenin 2), which has been proposed as a potential candidate gene in Erhualian pigs [The remaining regions with p-values lower than 0.001 in the TNB are distributed across SSC2 (7 ROHs), SSC5 (2 ROHs), and SSC13 (2 ROHs) in a contiguous manner. Within SSC2, spanning from 126,506,354 to 127,890,446 bp, we identified the CEP120 , which plays a role in inhibiting GnRH neurons. This inhibition is essential for the production of the GnRH hormone, which, in turn, is crucial for the synthesis of LH (luteinizing hormone) and FSH (follicle-stimulating hormone), both of which are crucial for reproduction [Corin, Serine Peptidase) is up-regulated in the decidua of the pregnant uterus, which suggests a potential role during pregnancy [follicle-stimulating hormone receptor) and LHCGR (luteinizing hormone receptor), both critical in regulating female reproductive processes. Additionally, GTF2A1L  may play an important role in testis biology and male infertility [Ubiquitin-Specific Peptidase 16) gene, responsible for regulating embryonic stem cell gene expression [Cilia- and Flagella-Associated Protein 298) has been described, with a mutation known to cause infertility in human patients [In the case of the RR population, we identified 17 ROHs with p-values lower than 0.001 in the NBA, 10 of which were shared with the TNB, as detailed in oduction ,42,43. Cregnancy , and it regnancy . Furtherertility . On SSC1pression . In thispatients . Lastly,The results of this study indicate the presence of inbreeding depression in the litter size traits of two strains of Iberian pigs. Furthermore, the distribution of the inbreeding depression effects is heterogeneous along the genome, and the architecture of inbreeding depression differs between populations. Additionally, we were able to identify eight genomic regions significantly associated with inbreeding depression that contain several relevant genes, such as FSHR, LHCGR, CORIN, AQP6 and CEP120.


The presence of residual antibiotics in food is increasingly emerging as a worrying risk for human health both for the possible direct toxicity and for the development of antibiotic-resistant bacteria. In the context of food safety, new methods based on microfluidics could offer better performance, providing improved rapidity, portability and sustainability, being more cost effective and easy to use. Here, a microfluidic method based on the use of magnetic microbeads specifically functionalized and inserted in polymeric microchambers is proposed. The microbeads are functionalized either with aptamers, antibodies or small functional groups able to interact with specific antibiotics. The setup of these different strategies as well as the performance of the different functionalizations are carefully evaluated and compared. The most promising results are obtained employing the functionalization with aptamers, which are able not only to capture and release almost all tetracycline present in the initial sample but also to deliver an enriched and simplified solution of antibiotic. These solutions of purified antibiotics are particularly suitable for further analyses, for example, with innovative methods, such as label-free detection. On the contrary, the on-chip process based on antibodies could capture only partially the antibiotics, as well as the protocol based on beads functionalized with small groups specific for sulfonamides. Therefore, the on-chip purification with aptamers combined with new portable detection systems opens new possibilities for the development of sensors in the field of food safety. Antibiotics are widely used both to treat and prevent infections in humans and animals, and to obtain favorable effects on animal growth. This widespread use has unwanted side effects, i.e., the possible contamination of the environment and of the food chain ,2, with,In this scenario, the development of automated purification methods may lead to the establishment of in-field usable instruments ,12, whicThe ideal biosensor for the on-site detection of food contaminations should be able to process raw food and precisely quantify the possible presence of antibiotics. These characteristics are difficult to obtain in a single system since modern detectors are often based on label-free methods, such as SPR and electrochemical biosensors. SPR is highly sensitive, but the equipment is costly and not portable. On the contrary, electrochemical biosensors are not sensitive enough to apply them to real samples, due to the problem of interface effect on the electrode surface, which produces an extremely high background that impedes the proper quantification of biomolecules ,22. ManyIn this work, we focus indeed on the evaluation and comparison of the performance in terms of the efficiency of antibiotics’ capture and release, and of several binding strategies, namely aptamers, antibodies and small functional groups, using functionalized magnetic microbeads as a common platform. Two different kind of microbeads are evaluated, namely polystyrene based : methanol, acetonitrile, oxalic acid, ethanolamine, glycine, dithiothreitol (DTT), sulfanilic acid, hydrochloric acid, ammonium hydroxide, ammonium sulphate and all powders for buffer solutions.The following microbeads were used: DynabeadsAll aptamers were obtained from IDT Integrated DNA Technologies . Aptamers are listed in Anti-antibiotics antibodies were obtained from Fitzgerald : sheep polyclonal anti-tetracycline antibody (a-TC Ab); sheep polyclonal anti-chloramphenicol antibody (a-CAF Ab); and rabbit polyclonal anti-sulfamethazine antibody (a-SMZ Ab). The fluorescent antibody Ab-A568 (rabbit anti-goat IgG (H+L), Cross-Adsorbed Secondary Antibody, AlexaFluorThe following buffers were used:2, 5 mM KCl, 1 mM CaCl2, 20 mM Tris/HCl pH 7.6Binding Buffer : 100 mM NaCl, 2 mM MgCl2, 20 mM potassium phosphate buffer pH 7.5Binding Buffer Muller (BBM): 100 mM NaCl, 10 mM MgClBinding and Washing Buffer (B&W): 1 M NaCl, 0.5 mM EDTA, 5 mM Tris/HCl pH 7.5Coupling Buffer I (CBI): 150 mM NaCl, 100 mM sodium phosphate pH 7.4Phosphate Buffered Saline (PBS): 138 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate pH 7.42, 20 mM Tris/HCl pH 8Binding Buffer SDM (BBSDM): 50 mM NaCl, 5 mM KCl, 5 mM MgClThe Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta (IZSPLV), provided raw milk and honey, all tested for the absence of antibiotics. IZSPLV also provided pure tetracycline, sulfonamides and chloramphenicol for spiking solutions.Microfluidic chips, with four chambers of 100 µL volume each , were purchased from microfluidic ChipShop GmbH .The specific recognition of aptamers and antibodies  with their targets was evaluated in solution, using two different methods: the effect of binding on the intrinsic fluorescence of tetracycline was exploited for testing the a-TC aptamers  Section, while a2, 5 mM KCl, 1 mM CaCl2, 20 mM sodium phosphate pH 7.6 for a-TC8, a-TC40 and a-TC76 or BBM for a-TCmu and specific aptamers were added at increasing concentrations. The aptamers were titrated in such a way as to not exceed a total volume increase of 5%. The solution was stirred during each titration step and allowed to equilibrate for 5 min before data collection . Fluorescence spectra were acquired for each titration point with a SPEX FluorMax spectrofluorimeter  at 25 °C. An excitation wavelength of 370 nm was used to acquire the emission spectrum from 380 to 610 nm. The fluorescence emission signal from 500 to 535 nm was then integrated and plotted against the aptamer concentration.Different concentrations of TC were tested for their intrinsic fluorescence signal in order to optimize the concentration to be used as starting amount. Therefore, TC was dissolved at fixed nanomolar concentration in a total volume of 1 mL of buffer 100 mM NaCl, 2 mM MgClg, allowing around 50–60 µL of solution to flow to the lower filter compartment. The antibiotic concentration found in the flow through is a good estimate of the free antibiotic present in the original reaction vial. On the other hand, it is expected that the antibiotic bound to the aptamer is  confined in the upper filter compartment.Vials with 100 µL solutions of macromolecules:antibiotic (1:1 volumes) were prepared at a fixed antibiotic concentration and increasing macromolecule concentration and left to reach equilibrium for 30 min. Afterwards, the solutions were transferred in the upper compartment of filters with a nominal cutoff of 10.000 Dalton , or 3.000 Dalton , depending on the MW of the tested aptamer. Then, the solutions were centrifuged for 8 min at 12.000× k is obtained in both models, while the fluorescence yields for the free . The paon 15-3, , with thon 15-3,  as the gk) the filter throughput losses Concerning the fit of equilibrium filtration experiments, the fitted parameters are , equipped with a helium/neon laser (543 nm), was employed for imaging the beads, acquiring data with a 40× objective in air. Images were also acquired using the additional channel of the transmitted light photomultiplier to image all the microbeads, independently of their functionalization. Spectral data of functionalized and untreated microbeads were acquired to confirm the correspondence of the detected light with the fluorescence spectra of the used fluorophores.The XPS analysis is particularly suitable for the chemical characterization of samples carrying biomolecule layers, as already shown . Samples Canteri .260 ssDNA = 33 µg/mL) and antibodies  to check the amount of aptamer/antibody present. Spectra from 190 to 340 nm were acquired and the absorbance at 260 (aptamers) or 280 nm (antibodies) was used to quantify the amount of aptamers , and the microbeads were washed for at least three times with the suitable binding buffer. Then, different conditions of ionic strength, pH, and temperature were tested for the elution of the antibiotics. A final washing step was then performed. All fractions were collected for quantification in HPLC.The binding test performed on the microfluidic chip was adapted from Lunelli et al.  and is sg for 10 min, were manually injected through a Rheodyne® model 7725i injector connected with a 50 µL loop and passed through a precolumn Security GuardThe antibiotic present in the fractions collected during the binding/elution test  was quantified by means of a Shimadzu  HPLC instrument. The instrument was equipped with the UV/Vis Detector SPD-20A, the Shimadzu prominence Communications Bus Module CBM-20A, the binary pump system Liquid Chromatograph LC- 20AB and with a degasser Fluorescence testk, and an increase of ≃ 12 times for the tetracycline fluorescence yield, when bound. When a-TC8 (data not shown), a-TC40 and a-TC76 are employed, instead, no measurable changes in fluorescence intensity are detected. Correspondingly, in these cases, k values can be obtained for these aptamers with this method.The binding affinity of anti-TC aptamers was tested in solution, exploiting the intrinsic fluorescence of TC , which m(b)Equilibrium filtration testk obtained for the a-TCmu aptamer compares very well with the value obtained with the fluorescence method. For the a-TC40 aptamer, a k of the same order of magnitude is obtained, while for the a-TC76 aptamer, a much lower binding is measured. Note that when the a-TC40 aptamer is employed, a noticeable filter leakage is detected, as expected considering the MW of this aptamer (13 kD), not far from the filter cutoff of 10 kD.The anti-TC aptamers were then tested with the equilibrium filtration method d–f. Becak value of a-SMZ results far higher than the range of explored aptamer concentration, i.e., larger than 2.5 µM. Therefore, in the tested conditions, no apparent interaction of SDM neither of SMZ with their specific aptamers was observed. To be noted, the filtration test with the a-SDM aptamer was performed with filters of 3 kD nominal cutoff since the MW of the a-SDM aptamer is approximately 7.4 kD, i.e., lower than the cutoff of the standard filters used for all other aptamers.The same test was performed on SMZ and SDM sulfonamides, using a-SMZ and a-SDM aptamers, respectively. Unfortunately, as shown by the plots reported in The ideal conditions for the interaction aptamer target could be very different when this couple is present in a solution with respect to having the aptamer bound to the microbeads surface. For this reason, aptamers were coupled to beads different for the dimensions, material and exposed chemical group . Amino-Upon assessing the conjugation of aptamers to the microbeads, a binding test was setup, firstly in vial and then on-chip. The different microbeads functionalized with anti-antibiotic aptamers were incubated with the corresponding antibiotic and solutions were collected after each step of the test . All the collected fractions were quantified via HPLC in order to estimate the amount of antibiotic captured and released from aptamer-functionalized beads. All the a-TC aptamers bound to DynaEpoxy beads captured a good amount of the target, significantly higher than the nonsense (NS) aptamer , panel aMoving on to a-TC aptamers conjugated via thiol chemistry to PureCube Maleimide beads, they seem to capture a quite good amount of TC and also the eluted TC seems in good percentage , panel bThe anti-sulfonamides aptamers were also tested in similar conditions, obtaining a small capture of SMZ (around 2%) by the a-SMZ-b aptamer bound to DynaSA, while no capture of SDM could be measured with the same chemistry, i.e., a-SMZ-b and DynaSA (data not shown). Analogously, as expected, no elution was observed for both the tested sulfonamides. To be noted, both aptamers are conjugated to the beads, as verified with the spectrophotometric analysis. Nevertheless, these results are in good agreement with the data of filtration test .The binding test performed in vials allowed to select the most promising aptamer for the implementation of the on-chip purification of antibiotics. The a-TCmu aptamer was selected for the purification of tetracycline, binding its biotinylated form to MagarNA or DynaSA beads. The functionalized beads were inserted in the chip chamber and TC was fluxed at the lowest TC concentration allowed in milk , and Given the good results obtained starting from TC spiked in buffer , the on-Besides aptamers, antibodies recognizing antibiotics have been developed despite the difficulty given by the small size of the antibiotic molecules ,47. Antik are reported in As reported for aptamers in Since the interaction in solution or on the microbeads surface could give different results, the binding test was performed for all the three antibodies conjugated to the respective microbeads.Before testing the binding of antibiotics to the functionalized beads, the successful conjugation of antibodies to the surface of both DynaEpoxy and PureCubeNHS was verified with the confocal analysis. A fluorescent antibody (Ab-A568) was conjugated following the same protocol developed for DynaEpoxy beads , giving as a result a broad peak comprising both phenomena. a-TC Ab was selected for the characterization of the binding to the DynaEpoxy beads, finding a percentage of bound antibody of (33 ± 15) % and (1.2 ± 0.3) The successful functionalization with antibody was checked for DynaEpoxy beads and a-TC Ab also with XPS measurements, acquiring the N 1s signal. DynaEpoxy indeed presents a small amount of nitrogen on its surface before functionalization , was able to concentrate almost two times the initial amount of antibiotics. Unfortunately, all the strategies evaluated in this paper showed some weak points when applied to the purification of antibiotics directly from raw food, leaving room for improvement in the interactions between the target and its ligand in complex food matrices, such as milk or honey.


The active vitamin D metabolites have anticancer effects on many human neoplasms. The vitamin D receptors have been detected in salivary glands tissue. This study aimed to evaluate the serum level of vitamin D in patients with malignant and benign salivary gland tumors.In this retrospective and cross-sectional study, 151 participants, including 42 patients with benign, 42 malignant salivary gland tumors, and 67 healthy subjects, participated. The serum level of vitamin D was measured using an enzyme-linked immunosorbent assay (ELISA).The mean serum level of vitamin D was 42.7 ng/mL in patients with benign tumors, 40 ng/mL in malignant tumors, and 36.7 ng/mL in the control group. There was no significant difference between the mean vitamin D level and vitamin D status in patients with salivary gland tumors and normal controls (P=0.2). There was a significant positive correlation between vitamin D level and age in the control group (P=0.04).The results showed a high prevalence rate of vitamin D deficiency/insufficiency in salivary gland tumors and normal subjects, with no significant difference. Therefore, the serum level of vitamin D might not play a significant role in the pathogenesis of these tumors, similar to many human cancers. However, further prospective studies are recommended focusing on specific tumors and considering other interventional factors.  Salivary gland tumors (SGT) account for approximately 3-5% of head and neck tumors. They usually arise from the parotid gland, and a high percentage of them are benign. Pleomorphic adenoma (PA) is the most common benign SGT, and mucoepidermoid carcinoma (MEC) and adenoid cystic carcinoma (ADCC) are the most common malignancies in this area . DespitePreclinical studies suggested that the active metabolite of 1,25-dihydroxyvitamin D3 2D3), known as calcitriol, or vitamin D analogs, has anticancer properties due to their anti-proliferative, anti-angiogenic, and apoptotic activities . The actVitamin D3 increases the expression of the Transforming Growth Factor-β1 and β2 (TGF- β1 and β2) genes and downregulates the expression of the prostaglandin E2 (PGE2) gene, leading to a decrease in the risk of breast cancer incidence . A study showed that the higher vitamin D serum levels were associated with enhanced the incidence of prostate cancer . EpidemiAlthough different studies have investigated this issue, there is little information available about the serum level of vitamin D in patients with SGTs. Previous studies detected VDR in salivary glands . MoreoveThis research was approved by the Medical Ethics Committee of Shiraz University of Medical Sciences (IR.SUMS.REC.1397.249). In addition, written informed consent forms were obtained from all the participants. In this cross-sectional study, 84 patients with benign and malignant SGTs referred to Khalili hospital affiliated to Shiraz University of Medical Sciences, and 67 healthy volunteers participated, from November 2018 to November 2019. SGTs included 38 subjects with PA and 4 other types of benign tumors, 42 with malignant tumors , and 8 other types of carcinomas).Patients with a history of previous anticancer treatments, disease recurrence, or those who had recently (the last two months) taken complementary and vitamin D medication, as well as the presence of other inflammatory or systemic diseases and also diseases those alter vitamin D serum levels such as thyroid or parathyroid diseases and hyperparathyroidism, were excluded from the study. The control group was age and gender-matched healthy subjects with cancer patients.From each patient and the normal control group, a 2 cc serum sample was prepared and stored at -70°C until further analysis. Vitamin D concentration was measured using a sandwich ELISA test with a Monobind ELISA kit. The serum levels <20, 20-29, 30-100, and > 100 ng/mL were considered as vitamin D deficiency, insufficiency, normal, and toxic, respectively .Statistical Analysis:Data were analyzed using SPSS software. The Kruskal-Wallis, Mann-Whitney, ANOVA, Chi-square, and Spearman’s rank correlation tests were used to compare the mean level of vitamin D and vitamin D status between groups. P <0.05 values were considered to be statistically significant.Baseline data regarding patients’ age, gender, and tumor stage are shown in The mean of vitamin D levels was 36.7 ng/mL in the control group, 42.7 ng/mL in patients with benign, and 40 ng/mL in patients with malignant tumors. The Kruskal–Wallis test showed no significant difference in the mean serum level of vitamin D among the groups (P=0.2). The correlation between patients’ age with vitamin D levels was evaluated using Spearman’s rank correlation test. It showed that the vitamin D level was not affected by the patients’ age with benign tumors  and malignant tumors . However, there was a significant direct correlation between vitamin D level and age in the control group . A higher level of vitamin D was observed in older individuals. Mann-Whitney test did not show any significant difference between vitamin D levels in males and females. (P=0.43). Details are shown in   Present study results showed that vitamin D deficiency/insufficiency was frequently found in the patients with benign or malignant SGTs and the age and gender-matched healthy subjects, with no significant difference.Several studies investigated the correlation between vitamin D concentration and the risk of malignancies, obtaining different and even controversial results. It seems that the normal or abnormal level of vitamin D in patients is more important than the mean concentration of vitamin in the sera. In this study, 31% of patients with benign tumors, 43% with malignant tumors, and 49% of the control subjects showed vitamin D deficiency/insufficiency, with no statistically significant difference. Similarly, in another study, 55% of non-Hodgkin’s lymphoma patients had a normal vitamin D status, and 44% of them were detected to have vitamin D deficiency . However, in one study, 65% of patients with head and neck cancer were revealed to have vitamin D deficiency and insufficiency . In a stOur result also showed a high prevalence rate of vitamin D deficiency in the control group. Vitamin D insufficiency is quite common amongst people due to increased use of sunblock, increased indoor activities, and skin covered by clothing . TabriziIt seems that cultural factors, type of clothing, and skin full coverage outside, especially in women, and also nutritional factors, such as low consumption of seafood in the Iranians, play a role in reducing the vitamin D level. This matter should be considered in general health programs, duo to the significant roles of vitamin D in life. This article was a primary study to evaluate the serum levels of vitamin D in patients with SGTs. The current study did not reveal a significant difference between tumor patients and normal subjects. These findings are in line with a study by Hannah Arem et al. who did not observe any relationship between the serum 25(OH)D concentration and the risk of larynx and oropharynx cancers. In the aforementioned study, the mean vitamin D concentration, as well as the normal or abnormal level of vitamin D in patients and control group, were the same, and the findings did not support the hypothesis that the higher serum level of vitamin D reduces the risk of head and neck SCC . McCulloBased on the results, it seems that vitamin D might not have a significant role in SGT pathogenesis. Huang et al. showed that vitamin D could reduce proliferation rate and cisplatin resistance in the ADCC cell line. Active metabolites of vitamin inhibited the tumor invasion and migration in vivo. They found that NF-κB and glutathione peroxidase 1 (GPX1) were reduced after cell treatment with 1,25 (OH) 2D3 . DiffereIf patients take vitamin D supplements in two months, it can interfere with the results. Moreover, patients might change their diet or lifestyle after realizing the disease symptoms. Some researchers have stated that a lower post- diagnostic vitamin D levels might be the consequence of the disease rather than the reason for cancer, which might be due to some changes in lifestyle, such as less physical activity, sun exposure, food intake, or systemic effects of cancer itself .The result of a cohort study on the patients with pancreatic cancer did not support the hypothesis that 25(OH)2D) concentration plays a protective role against pancreatic cancer . Xue et Many in vitro and in vivo studies have shown that vitamin D has anti-proliferative, pro-apoptotic, and anti-angiogenic properties. Calcitriol treatments and many cytotoxic medications have synergic or at least coordinating effects . Vitamin D3 upregulates the expression of TGF-β1 and TGF-β2 genes and reduces the PGE2 expression and, eventually, the risk of breast cancer development . Also, iGrimm et al. found that the serum level of vitamin D in patients with oral cancer was low; however, VDR was significantly more in premalignant lesions and oral SCC. They concluded that due to the increase in VDR in the patients, natural or therapeutic synthetic vitamin D can be used to induce an apoptotic effect in cancerous cells . Some stIn addition to the limitations mentioned earlier, one of the unexpected limitation in this study was Covid 19 pandemy and a significant increase in vitamin D intake and life style changes among all people,which made further sampling impossible. After this period, vitamin D levels cannot be compared with the previous data. In conclusion, the results of this preliminary study showed a high prevalence rate of vitamin D deficiency/insufficiency in patients with malignant SGTs and normal subjects. The serum level of vitamin D might not play a significant role in the pathogenesis of these tumors, similar to many human cancers. However, further prospective studies are recommended focusing on specific tumors and considering other factors, such as BMI, past long-term exposure, and long-term history of supplementary intake, exercise, skin complexion, sampling season, smoking habits, and parathyroid hormone levels.