Appropriate lymph node harvesting for patients with gastric cancer is fundamental for a correct staging and is strongly related to survival. In this study, we present a new protocol for on-site macroscopic evaluation and sampling of lymph nodes for gastric cancer patients. With the joint collaboration of surgeons and pathologists, our method aims to provide the largest possible number of analyzed lymph nodes per patient, allowing for a better staging. We are convinced that this approach is routinely feasible, and our preliminary results seem to confirm better patient stratification compared to other lymph node dissection methods.p = 0.001). Moreover, by applying the proposed technique, we sampled lymph nodes with a very small diameter, some of which were metastatic. Noticeably, no difference in terms of postoperative course was identified between the two groups, again supporting the feasibility of an extended lymphadenectomy. By comparing the prognosis of patients, a better overall survival (p = 0.03) was detected in the study group; however, to date, no long-term follow-up is available. Interestingly, patients with metastasis in node stations number 8, 9, 11 or with skip metastasis, experienced a worse outcome and died. Based on our preliminary results, the pathologist/surgeon team approach seems to be a reliable option, despite of a slight increase in sfaff workload and technical cost. It allows for the harvesting of a larger number of lymph nodes and improves the outcome of the patients thanks to more precise staging and therapy. Nevertheless, since a higher number of patients are necessary to confirm our findings and assess the impact of this technique on oncological outcome, our study could serve as a proof-of-concept for a larger, multicentric collaboration.The downstaging of gastric cancer has recently gained particular attention in the field of gastric cancer surgery. The phenomenon is mainly due to an inappropriate sampling of lymph nodes during standard lymphadenectomy. Hence, collection of the maximum number of lymph nodes is a critical factor affecting the outcome of patients. None of the techniques proposed so far have demonstrated a real efficiency in increasing the number of identified lymph nodes. To harvest the maximum number of lymph nodes, we designed a protocol for on-site macroscopic evaluation and sampling of lymph nodes according to the Japanese Gastric Cancer Association protocol. The procedure was carried out by a surgeon/pathologist team in the operating room. We enrolled one hundred patients, 50 of whom belonged to the study group and 50 to a control group. The study group included patients who underwent lymph node dissection following the proposed protocol; the control group encompassed patients undergoing standard procedures for sampling. We compared the number and maximum diameter of lymph nodes collected in both groups, as well as some postoperative variables, the 30-day mortality and the overall survival. In the study group, the mean number of lymph nodes harvested was higher than the control one ( Gastric cancer (GC) is the fourth most common malignancy worldwide; although its incidence is declining in the Western world, this disease still remains the second leading cause of cancer related death in both sexes . The JapThe mean number of harvested LNs in D2 lymphadenectomy varies between 25 and 52 in different published series ,18. InadTo overcome the standoff situation of two comprehensive but seemingly concurrent approaches, we proposed and standardized a procedure of LNs on-site evaluation involving both surgeon and pathologist in the OR. The primary endpoint of the study was to evaluate if such an approach increases the number of detected LNs, providing a precise LNR. The secondary endpoint regarded the impact of D2 lymphadenectomy on mortality, morbidity and OS of GC patients. Moreover, we aimed to demonstrate if our approach is feasible and routinely applicable and could hopefully set a frame for future studies and discussion.The study was carried out by means of a prospective phase and a retrospective review of a prospective maintained database for GC at Azienda Sanitaria Toscana Nord-Ovest, based on a pivotal communication by our group . This stThe prospective phase started soon after the initial wave of the SARS-CoV-2 pandemic. We enrolled the first 50 patients presenting to the Surgery Unit of Ospedale Unico della Versilia  and Nuovo Ospedale Apuane  with a diagnosis of GC according to the latest World Health Organization (WHO) classification . ExclusiAs a second step, in the retrospective phase of the study, we collected the last 50 patients who had undergone a subtotal /total gastrectomy plus D1 or D2 lymphadenectomy  before the beginning of the SARS-CoV-2 pandemic. The operative procedures in the control group were performed by the same surgeons but the JGCA protocol was applied only in 10 patients. The surgical specimens were represented by en bloc resection of stomach, omentum and lesser and greater curve adipose tissue, plus soft tissues surrounding common hepatic artery (station no. 8), celiac trunk (station no. 9), splenic hilus (station no 10), and splenic artery (station no. 11) in the D2 lymphadenectomy. The samples were immediately fixed and sent out to the pathology lab for examination. LNs were harvested by two different pathologists dedicated to gastrointestinal pathology following the classic manual method in the GR (not in the OR) on FF specimens (not on fresh tissue).We analyzed the two groups in terms of number and maximum diameter of LNs dissected, as well as LNR. Since the number of LNs differs upon the site of dissection, LNs were grouped along the lymphatic compartment and the number of LNs belonging to each compartment were matched, if possible. Moreover, we compared the postoperative data to assess if an extended lymphadenectomy might affect the recovery and the short-term outcome. The following parameters were considered: need of and time spent in intensive care unit, postoperative complications according to Clavien–Dindo classification, length of hospitalization, 30-day mortality, OS.Finally, we correlated the staff workload (including the time required for mapping) and the technical costs. In the prospective group, time for mapping was determined based on the time needed in OR and GR for harvesting and picking up all LNs. In the retrospective group, we considered the standard average time usually spent by pathologists for LNs sampling .For both groups, representative specimens of tumor, omentum and normal gastric tissue as well as of resection margins were performed, and followed standard and automatic procedures for processing and pathological examination.After surgery, all the patients were discussed by a multidisciplinary team to define the postoperative approach according to the following guidelines . The evaWe predicted a compliance rate of 67% would be achieved in this study similar to what was reported in previous studies examining conventional D2 LN dissection. The sample size was based on the alpha error at 0.05 and a power of 90%. As we set the equivalence difference to be 30% between the conventional and the proposed model, based on our pivotal communication , the totp values were two-sided with p < 0.05 considered statistically significant.Statistical analysis was carried out by using commercially available statistical software  to calculate the association of epidemiologic and clinicopathological characteristics between the two groups. Chi-squared and Fisher’s exact test were performed for descriptive statistics. Quantitative variables were expressed as frequency count, minimum, maximum, mean, and standard deviation, while qualitative variables as frequency count and percentage. The Mann–Whitney-U test was used to compare continuous variables not normally distributed. All The following variables were included in the univariable analysis: gender , postoperative complications (yes vs. no), intensive care unit hospitalization (yes/no), days in care unit, length of hospitalization, 30-day mortality (yes/no). Survival function estimation and comparison between the two groups were performed using Kaplan–Meier estimates and log-rank test, respectively.n = 28, 56%) were located in the antral region, and were represented by low-grade  [n = 11, 22%) [n = 19, 38%) according to Lauren [n = 11, 22%) following JGCA classification [n = 25, 50%), with multiple nodal involvement and 10 metastatic cases. The most frequent stage according to the TNM VIII [The study group included 29 males (58%) and 21 females (42%), the mean age at diagnosis was 77 years (reference range: 65−87 years); six patients underwent urgency surgery for occlusive or bleeding disease; 15 patients were subjected to neoadjuvant chemotherapy following the FLOT  protocol . R0 reseo Lauren , poorly fication . InteresAn average of 79 LNs were collected, with 77 LNs in subtotal and 83 LNs in total gastrectomy, respectively . The comn = 30, 60%) were located in antral region, and were represented by low-grade  [n = 14, 28%) [n = 18, 36%) according to Lauren [n = 14, 28%) following JGCA classification [n = 30, 60%), with only 3 metastatic cases. The most frequent stage according to the TNM VIII [n = 10, 20%) ; 4 patients underwent urgency surgery for occlusive or bleeding disease; 5 patients were subjected to neoadjuvant chemotherapy according to the FLOT regimen. The majority of tumors 19, 61%) , tubular14, 28%) , intestio Lauren , tubularfication . Six pat1% , tubu10, 20%) .The mean number of collected LNs was 29 , with a compliance rate of 54%. Maximum diameter ranged between 5 and 22 mm. An average number of 7 positive LNs were identified with micrometastasis and isolated tumor cells in 2 and 1 patients, respectively. Three patients who followed the JGCA protocol showed CnLN involvement, and one had skip metastasis .p = 0.05); accordingly, the mean number of LNs identified by the surgeon/pathologist team in OR was greater than the standard procedure of dissection carried out by the pathologist alone, with p = 0.001. Moreover, the smallest LNs were identified and harvested (p = 0.005), allowing for recognition of micrometastasis and isolated tumor cells. Interestingly, there was also a statistically significant difference in the number of LNs detected in the two groups based on a comparison of the lymphatic compartments (p = 0.05).Our approach significantly increased the compliance rate for D2-lymphadenectomy . In the study group, complications were represented by bleeding  and duodenal stump leakage ; whereas in the control group, 3 bleedings, 1 volvolus, 1 duodenal stump leak and 1 abdominal abscess occurred. Univariate analysis confirmed that age (p = 0.7), gender (p = 0.2), type of gastrectomy (p = 0.6), site of tumor (p = 0.5), histology (p = 0.3) and number of retrieved LNs (p = 0.4) are not related to morbidity. Complications mainly affected patients treated under emergency regimen (p = 0.001). Nine patients in the study group and 6 patients in the control group needed intensive care with at least one postoperative day (p = 0.3). Median hospital stay was 9 days in the study group and 11 days in the control one (p 0 0.5). The 30-day postoperative mortality was 8% (n = 4) in the study group and 14% (n = 7) in the control one (p = 0.4). Causes of death were: acute myocardial infarction (n = 1), disease progression (n = 1) following peritoneal carcinosis, uncontrolled bleeding (n = 2) despite embolization. In the control group, 1 patient diedfor acute pulmonary embolism, 1 due to intestinal volvolus leading to intestinal obstruction, 2 following acute myocardial infarction, and 3 for uncontrolled bleeding despite additional treatment. Univariate analysis showed that high 30-day mortality was related to age >75 years (p = 0.04), emergency regimen (p = 0.01), advanced stage (p = 0.02) and higher LNR (p = 0.02). These findings again support the feasibility of D2 lymphadenectomy, as overlap those reported for D1 lymphadenectomy [No intraoperative mortality was registered in the two groups. The overall postoperative complication rate requiring a second operation was not significantly different between the two groups , urgency surgery (p = 0.01), advanced stage (p = 0.001), LN metastasis (p = 0.002), multiple LNs and CnLN involvement (p = 0.01 and p = 0.04) respectively), and skip metastasis (p = 0.03). Although no long-period follow-up is available to date to draw definite conslusions, this finding shows a better outcome in the study group patients (p = 0.03)  , again cAs far as the feasibility of our approach is concerned, the proposed protocol by increasing the number of collected LNs obviously affects the technician and medical workload as well as the technical cost. Staff cost was not affected, as in Italy it is fixed. The time for sampling was shorter in the control than in the study group (30 vs. 45 min). Moreover, we observed an average increase of 20 blocks and 30 hematoxylin and eosin (H&E) slides per case to be prepared and examined. No streghthening in timing was detected for block processing, as in our lab all the procedures are fully automatized. However, technicians spent 40 additional minutes to section a case, and pathologist 15 additional minutes to examine the slides. In terms of technical costs, since EUR 3 is needed to produce an additional H&E slide, we observed an increase of 60 EUR per patient.LN stage is absolutely one of the leading prognostic factors influencing the OS after curative resection of GC . Accordip = 0.005), also when grouped by stations. “On site” evaluation of fresh tissue by the pathologist and surgeon allows for easier identification and grouping of the complex three-dimensional and multidirectional lymphatic system involved in GC [Gastrectomy and LNs harvesting and sampling are strictly operator-dependent. Although surgical procedures had been standardized, differences in the applied technique and in surgeon skills remain and may impact LNs removal. Moreover, in contrast to Eastern countries , D1-lymphadenectomy is still very common in Western countries, mainly because of lower rate of proximal stomach involvement and poorly cohesive histology . The paled in GC ,34,35. Ip = 0.03). This finding might be explained by the higher number of LNs removed, that not only reduced the mechanism of metastasis but also allowed for a precise staging and therapy [Thanks to the technique we proposed, we picked up the smallest LNs and identified 2 SLM, 5 micrometastasis and two patients with isolated tumor cells , thus co therapy .The major issues we identified were the need for at least two dedicated pathologists who, in turn, should be present in the OR, and the increase in medical and technician workload and in technical costs. However, if considering the number and dimension of harvested LNs, the positive LNs identified and the better OS in the study group, we would not regard these tools as a drawback.This study is not a randomized clinical trial and obviously suffers from some clear limitations, such as the low number of patients, the short-term follow-up, and the partial retrospective nature. Moreover, the advanced stage of patients belonging to the study group may, at least in part, accounts for the very high number of detected LNs if compared to the already published studies. However, our findings are statistically significant and strongly support the feasibility of the proposed technique, pending precise organization, skilled personnel and moderate-to-high volume centers. It is niteworthy that as we observed progressive improvement in medical skills during the study, we are aware that such an approach is also applicable in non-high-volume centers after a learning curve period for both the surgeon and pathologist.It is fair to reiterate that suboptimal LN harvesting contributes to stage migration, inadequate treatment planning and poor outcome ,41,42,43We are aware that we cannot draw definite conclusions on the impact of this technique on oncological outcome; however, our findings support the notion that D2 lymphadenectomy is safe and achieves a better survival.The technique we proposed seems to also be feasible in non-high-volume centers after accurate training of dedicated personnel; therefore, our study could serve as a proof-of-concept for a larger, multicentric collaboration.


Mechanistic studies that will identify the nature of this effect and the clinical relevance of ALT and AST levels to the post-bariatric liver function are warranted.Patients that undergo bariatric surgery experience weight loss and a reduction in the plasma levels of the hepatic enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST). We used the Israeli national bariatric registry, which includes demographic, clinical, and biochemical data on 19,403 patients, of which 1335 patients had two-year follow-up data on ALT, AST, A1C, and BMI, to test the dependence of the reduction in the levels of ALT and AST on weight loss. The data were analyzed using regression models, retrospective matching, and time course analyses. Changes in liver enzymes did not correlate with change in BMI, and linear regression models did not demonstrate that the change in ALT and AST values were dependent on pre-operative levels of BMI or the extent of weight loss. ALT and AST levels were reduced two years after surgery compared with a cohort of retrospectively matched patients for ethnicity, sex, age, BMI, and A1C. Finally, patients who regained weight displayed a reduction in levels of liver enzymes. Our results suggest that bariatric surgery affects AST and ALT levels via weight loss dependent and independent mechanisms Bariatric surgeries lead to weight loss and improvement in obesity-associated comorbidities, such as type 2 diabetes (T2D) ,2,3,4. CObesity is associated with non-alcoholic fatty liver disease (NAFLD), and it has been shown that weight loss of 10% can improve NAFLD in many cases ,24,25. BMolecular studies show that hepatic DNA methylation, gene expression, and several signaling and metabolic pathways are affected by bariatric surgery, also independent of weight loss. These studies indicate that the post-bariatric liver assumes a new molecular state that is different than the state of the healthy liver or of a liver affected by NAFLD or NASH ,22,35,36The mechanistic studies and clinical outcomes indicate that bariatric surgery has weight loss independent effects on the liver. In this study, we sought evidence supporting this claim through analysis of the Israeli Bariatric Registry ,41,42.We analyzed data from the Israeli Bariatric Surgery registry from January 2014 to December 2018. The variables we used were sex, ethnicity , surgery type ), pre-operative smoking (self-reported), alcohol consumption (self-reported), and diagnosis of hypertension. Numerical values were age, BMI (body mass index), glycated hemoglobin (A1C) percentage, plasma triglycerides (TG) levels, and levels of ALT and AST. Pre- and two-year post-surgery data of BMI, A1C, ALT, and AST were available from 1335 patients. Data on revision surgery were excluded. Data on alcohol consumption during the follow-up period were not available.Abnormal ALT levels were defined as higher than 25 IU/L for females and higher than 33 IU/L for males, according to the American College of Gastroenterology . Weight 2 test was used for categorical variables. Bonferroni correction was used to correct for multiple hypothesis testing in each table or figure.Continuous variables were reported as the median and interquartile range. The Mann–Whitney U test or Kruskal–Wallis H tests, followed by a post hoc Mann–Whitney U test, were used for continuous variables. The χRegression models: Backward variable elimination linear regression models were used in multivariate analyses. We incorporated demographic characteristics, age, surgery type, pre-surgical ALT or AST levels, TG, BMI, and A1C, or the difference in BMI and A1C, to estimate the change in ALT or AST levels two years after surgery. Pre-surgical levels of AST and TG were not included in the models for the change in ALT, and ALT and TG were not included in the model for AST due to the high correlation between ALT, AST, and TG levels.Matching: Pairs were matched within the same categorical subgroups of sex and ethnicity. Matched populations were obtained by choosing pairs with the minimal Euclidean distance in age, BMI, and A1C, ensuring similarity in each continuous variable included in the matching algorithm. https://scikit-learn.org), statsmodels (www.statsmodels.org), and SciPy packages (www.scipy.org/scipylib) . A. An = 152). The final levels of ALT in these patients were significantly lower two years after surgery. In particular, 17 of the 47 patients had abnormally high ALT levels before surgery, while only nine had abnormally high levels two years later (We identified 47 patients who did not lose weight two years after surgery (∆BMI: 0.87 (0.35–1.49) kg/mrs later D–F. AST Bariatric surgery often causes weight loss and a reduction in the levels of A1C, ALT, and AST in patients with obesity ,48,49,50There was no correlation between the changes in ALT or AST and changes in BMI. This is unexpected, considering previous studies. For example, Burza et al. analyzed the Swedish Obesity Study database  and haveWe constructed a multivariate regression model to gain better insight into the role of changes in BMI and A1C in alterations in the levels of plasma liver enzymes. This model includes demographic parameters, surgery type, preoperative comorbidities, smoking and alcohol consumption, ALT or AST levels, A1C or change in A1C levels, and BMI or change in BMI two years after surgery. Neither change in BMI and A1C nor pre-surgical levels of BMI and A1C were associated with the change in ALT or AST levels. SG surgery type, younger age, and high pre-surgical levels of ALT or AST significantly contributed to a greater reduction in liver enzymes. These results also apply to patients who had abnormal pre-surgical ALT levels. There is a complex relationship between age and outcomes of bariatric surgeries. Younger age was associated with a higher risk for weight regain ,53 and aHigh ALT levels are associated with hyperglycemia, insulin resistance, and the risk of developing T2D . We founThe finding that SG contributed to a greater reduction in ALT in the regression model despite lesser or equal weight loss and reduction in A1C compared with bypass surgeries supports the hypothesis that factors related to bariatric surgery ,57 and nTo test whether a history of surgery affects ALT or AST levels, we matched BMI, A1C, and demographic parameters between patients before surgery and patients two years after surgery. The post-operative patients had lower levels of AST and ALT, and the fraction of patients with abnormal ALT levels was three-fold smaller: 40% of the pre-operative patients had abnormal ALT levels, and 13% of the matched post-operative patients had abnormal ALT levels. Therefore, having bariatric surgery can reduce the occurrence of abnormal ALT levels by 67%. This finding supports the impression that bariatric surgery has an effect on liver enzymes. It is important to note that the pre-operative patients had obesity, but were leaner and had lower glycemia than most patients that undergo surgery, while the post-operative patients had obesity after surgery. Moreover, the post-operative patients had already experienced weight loss, and therefore, it is possible that significant weight loss, rather than current BMI, is the important factor in reducing ALT and AST levels after bariatric surgery ,24,25. If indeed a reduction in ALT and AST is mostly a result of weight loss, then we would expect that weight regain  would inWe identified 47 patients with obesity who did not display any weight loss two years after bariatric surgery, likely due to a complete regain of weight. Even in this population, the median ALT level was significantly lower two years after surgery, and 47% (8/17) of the patients who had abnormal ALT levels before surgery normalized their ALT levels with no weight loss. It will be important to study whether the effect of bariatric surgery on ALT levels after weight regain is temporary. AST levels did not change in this group of patients, yet AST levels were not high pre-operatively. Altogether, our results provide evidence that factors induced by bariatric surgery, other than weight loss and improvement in glycemia, contribute to the reduction in the plasma levels of ALT and AST. Notably, ALT levels were reduced to their post-surgical levels just six months after surgery, even in patients with high pre-surgical ALT. Weight loss is usually a longer process, and the reduction in BMI six months after surgery was not reflected in changes in ALT levels, supporting a lack of correlation between changes in weight one year after surgery and changes in ALT .2 from weight regain that was experienced by patients between one and five years after RYGB surgery did not affect NAS score [2 five years after bariatric surgery experienced resolution of NASH without worsening of fibrosis according to histological scoring [In a recent clinical study, it was shown that RYGB has a greater effect on the histological NAFLD activity score (NAS) than gastric banding five years after surgery, and that this effect cannot be explained by weight loss alone . In the AS score . In anot scoring . Fibrosi scoring . A smallSeveral mechanisms may be responsible for driving a reduction in the levels of ALT and AST following bariatric surgery. The level of circulating bile acids is increased after bariatric surgery and their composition is modified, possibly following a change in the gut microbiome. Upregulation in bile acid signaling in the liver may underlie some of the weight loss independent effects of surgery ,10,12. THistological studies show that the extent of improvement in NAS score following bariatric surgeries in patients with NAFLD is greater in patients that experienced a larger weight loss ,32,33,34An important limitation of our study is that we do not have information on the status of NAFLD before or after surgery. Furthermore, we do not have information on the physical activity, dietary habits, medications, or levels of insulin and bilirubin before and after surgery. Alcohol consumption is self-reported only before surgery. All of these factors may affect the outcome of bariatric surgery and levels of liver enzymes ,67,68. AThis two-year study based on data from the Israeli Bariatric Surgery registry suggests that a reduction in AST and ALT levels following bariatric surgery is in part independent of weight loss and reduction in A1C levels. Mechanistic studies ,19,20,22


The purpose of this article was to systematically review titanium allergy manifestations in patients treated with dental implants and report a clinical case; (2) Methods: A systematic electronic search was performed for articles published in the English language until July 2021. The following eligibility criteria were adopted: (1) Population: individuals undergoing titanium and/or titanium alloy implant-supported rehabilitations; (2) Exposure: peri-implant soft tissue reactions attributable to implant insertion; (3) Outcome: evidence of titanium allergy, diagnostic methods, and forms of resolution; (3) Results: The included studies, in summary, presented evidence that titanium should not be considered an inert material, being able to trigger allergic reactions, and may be responsible for implant failure. A 55-year-old male patient received 3 implants in the posterior region of the left mandible and presented an epulis-like lesion developed from the peri-implant mucosa. The immunohistochemical analysis of the biopsy specimen confirmed the initial diagnosis of allergic reaction to titanium; (4) Conclusions: Although the evidence is weak, and titanium allergy has a low incidence, hypersensitivity reactions should not be underestimated. A rapid and conclusive diagnosis is mandatory to prevent further complications. Titanium (Ti) is a widely used material in the manufacturing of dental implants. Ti plays a pivotal role in the osseointegration process by supporting the physicochemical attraction of osteoblast lineage cells. This leads to bone deposition that fills the space between the recipient bone and the implant surface . The useIn regards to Ti implant failures, they are generally linked to biologically related complications, more specifically recognized as peri-implant mucositis and peri-implantitis ,11,12,13In view of the above, the present study aimed to perform a comprehensive systematic review of the literature focused on Ti allergy related to dental implants. Moreover, a case of allergic reaction to Ti was also presented to better understand the clinical and histological manifestations of this adverse event.The present study has been designed as a comprehensive systematic review performed according to the guidelines listed in The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement . A modifThe selection of all relevant studies was performed independently by three reviewers  and in three different databases: MEDLINE (PubMed), EMBASE, and The Cochrane Library. All human studies published in the English language from 1990 reporting on dental implant allergy were considered eligible for inclusion. The latest electronic search was undertaken in July 2021. The following combinations of Medical Subject Headings (MeSH) and entry terms were used in the electronic search: “)”. A fourth author (L.P.F.) reviewed the workflow of the search strategy. The articles were selected by title and abstract and in accordance with the inclusion and exclusion criteria.In the study protocol, inclusion and exclusion criteria for study eligibility were defined. Due to a reduced number of randomized controlled clinical trials (RCTs), all clinical study types on human subjects were included in the present review, including RCTs, controlled clinical trials, prospective and retrospective studies, case series, and case reports. Conversely, animal or in vitro studies, as well as review articles, were excluded. During the collection of data, any disagreement was discussed and resolved. At the end of the screening process, no disagreement among the authors regarding the results was found. The interexaminer Cohen’s kappa coefficient (κ) was used to evaluate the inter-rater agreement between the authors in the selection of the articles for each database. After study selection, a high level of agreement among independent examiners was observed in all databases: MEDLINE (PubMed): (κ = 0.94); EMBASE: (κ = 0.9); The Cochrane Library: (κ = 1).The PRISMA flow diagram is illustrated in The search strategy in the selected databases yielded a total of 288 articles. Overall, 223 articles were excluded after the evaluation of the title and abstract according to the inclusion and exclusion criteria. There were 41 duplicate references, and 8 articles were neither accessible from MEDLINE (PubMed) nor retrievable from the corresponding authors. Thus, 16 articles were finally available for the full-text examination ,32,33,34Of the seven studies selected for the qualitative analysis ,32,33,34Three of the included studies ,29,33 prClinical manifestations have been reported in six studies ,32,33,34Only 3 studies reported radiographic manifestations ,29,34. IThe only study that reported the use of antibiotics following detection of clinical manifestations was the case reported by du Preez et al. , who preOverall, five studies reported details on the treatment that had been performed ,32,33,34Histopathological analyses were described in three studies ,29,34. I® test and patch test. The results reported by du Preez et al. [®, 21 (37.5%) were positive, 16 (28.6%) ambiguous, and 19 (33.9%) negative to Ti. The authors concluded that Ti can induce hypersensitivity and should not be considered an inert material. In both studies published by Hosoki et al. [In the study by Olmedo et al. , the idez et al. , showed z et al. , the firz et al. , 25.7% oi et al. ,33, patci et al. .®, Arujá, SP, Brazil: 3.75 × 11 mm (region of the first premolar); 4 × 7 mm; 4 × 7 mm (region of the second premolar and first molar respectively)) were placed with a submerged approach. After 5 months from implants placement, the re-entry procedure was performed to uncover the implants and connect the Ti healing abutments. After a healing period of seven days, an epulis-like, reddish lesion was observed, with oedematous soft tissues covering the healing abutments connected to the most distal implants  biopsy of the peri-implant soft tissues including the lesion; (2) avoid contacts between the peri-implant soft tissues and Ti by means of metal-free prosthetic components; (3) perform skin and enzyme immunoassays to verify allergies to Ti and other metals; (4) consider peri-implant plastic surgery techniques including subepithelial connective tissue grafts or free-gingival grafts to improve the peri-implant soft tissues, particularly in those situations presenting with a lack of keratinized mucosa.Despite the low level of evidence, hypersensitivity to Ti and its alloys can occur and should not be underestimated. Clinical manifestations and histological features are typical of allergic reaction and may help the clinician in the differential diagnosis. Lesions attributable to Ti allergy should therefore be recognized and diagnosed early in order to enhance both treatment and prognosis.


Escherichia coli as a whole have a higher incidence rate in adenocarcinoma patients compared to controls, whereas Bacteroides fragilis toxin does not seem to be associated with pre-cancerous nor with cancerous lesions. These results have been obtained irrespectively of the presence of CRC-risk loci.Colorectal cancer (CRC) is a leading cause of cancer death worldwide, and its incidence is correlated with infections, chronic inflammation, diet, and genetic factors. An emerging aspect is that microbial dysbiosis and chronic infections triggered by certain bacteria can be risk factors for tumor progression. Recent data suggest that certain bacterial toxins implicated in DNA attack or in proliferation, replication, and death can be risk factors for insurgence and progression of CRC. In this study, we recruited more than 300 biopsy specimens from people undergoing colonoscopy, and we analyzed to determine whether a correlation exists between the presence of bacterial genes coding for toxins possibly involved in CRC onset and progression and the different stages of CRC. We also analyzed to determine whether CRC-predisposing genetic factors could contribute to bacterial toxins response. Our results showed that CIF toxin is associated with polyps or adenomas, whereas pks+ seems to be a predisposing factor for CRC. Toxins from  Colorectal cancer (CRC) is among the major threatening diseases of the present times, as in both sexes, it is the second deadliest of all cancers, responsible for more than 935,000 deaths in 2020 and the third most common cancer, with about 1,900,000 new cases diagnosed. A high mortality rate is associated with CRC and is mainly due to the delay in diagnosis, as symptoms appear late [Decreasing the mortality rate by CRC is a clear, unmet need today, and the medical community agrees that the high number of CRC-related deaths could be prevented by adequate screening programs for early detection and also in order to reduce patient-care-associated costs. In the last years, it has been already proven that CRC insurgence can be considered as a multifactorial process . Many stFusobacterium in human CRC compared to controls, suggesting an association with the later stages of CRC [Helicobacter pylori CagA, a protein that activates the pro-oncogenic catenin and induces an invasive phenotype [Bacteroides fragilis (ETBF), known to produce the B. fragilis toxin (BFT), whose long-term colonization may increase CRC risk [Escherichia coli strains and positive for the genomic island polyketide synthetase (pks), or the cytolethal distending toxin (CDT), also produced by E. coli. The other route is an engagement of signaling pathways that modulate cell proliferation, replication, and death, ultimately resulting in transformation, as for BFT and the E. coli cytotoxic necrotizing factor 1 (CNF1) or the cycle-inhibiting factor (CIF)  and the different stages of CRC. Moreover, we aimed analyze the possibility that known CRC-risk loci contribute to the response to bacterial toxins. For this purpose, we created a database with patients’ information, and we explored the association of the bacterial toxins with hyperplastic polyps (HP), pre-cancerous polyps/adenomas (PA), and adenocarcinomas (ADK).Colorectal biopsies from 330 eligible individuals were analyzed. We excluded samples from five subjects because of vials A and B inversion. Samples from the remaining 325 subjects, 162 men and 163 women, included healthy tissues  or tissues with hyperplastic polyps , pre-cancerous polyps or adenomas , or adenocarcinomas , as shown in ADK patients were more frequent in Regina Elena National Cancer Institute  (83%), while HP patients were mostly collected by Policlinico Umberto I Sapienza University  (82%). Female cases were 45% (13) in the ADK group and 59% (96) in controls.The median age of enrolled subjects was 60 years, with higher values for ADK patients (68 years) compared to PA (60 years), HP (62 years), and controls (59 years). BMI ranged from 13.8 to 40.4 (median at 24.9). Information on alcohol (spirits) and wine consumption, type of diet, smoking condition, and physical activity was highly complete (>95%). Insufficient data quality and high proportion of missing values were observed for meat consumption . Previous removal of polyps was reported for 130 subjects (40%) and was the prevalent condition of patients with HP (60%). Overall, 89% of the biopsies were sampled from the colon and 11% from the rectum. Sigma was the prevalent location for controls (75%), HP (40%), and PA (27%), while ADK was more frequently diagnosed in the rectum (45%).p-value for trend: 0.0124).As depicted in The overall and outcome-specific distribution of the bacterial toxins is shown in cnf1 (29%), cdt (13%), and cif (10%).Among aerobic bacteria, the most frequent trait in the whole population was the positivity for the pks island (pks+) , followed by cif (16% vs. 7%) and cdt (20% vs. 12%) were detected more frequently compared to controls, while pks+ and cnf1 incidence rate was higher in ADK patients, 52% and 41%, respectively, compared to controls (35% and 27%). cdt incidence was similar in the ADK e PA group, 20% vs. 19%, respectively.In the PA group, bft toxin gene emerged in only 43 samples (13%). bft-positivity rate was similar in all outcome types, ranging from 11% in HP cases to 14% in both controls and ADK groups.Overall, the p-value < 0.1) with ADK in univariate logistic regression models were represented by the presence of pks+, sum of aerobic bacterial toxins, PRS, and age, as shown in p-value: 0.055).Factors associated within 10% tolerance  and sex  were the only risk factors that retained statistical significance.In the multivariate logistic regression model for PA condition, after adjusting for all parameters with univariate association, p-value: 0.026), non-drinker condition , and previous benign vs. malignant removal of polyps  resulted to be independent risk factors for HP condition in the multivariate logistic regression analysis applied to models, including risk factors proven to be significant in the univariate analysis. Factors no more significant after adjustment in multivariate analysis were sex, alcohol consumption, and smoking , CIF, and CNF1. CIF acts by deamidating NEDD8 proteins [Waf1/Cip1 and p27kip2 proteins, thus arresting the cell cycle at the G2/M or at the G1/S transition [Our results clearly indicate that the oworkers , who demproteins , ultimatproteins ,21,22. Bproteins  and mediproteins ,25. In aansition ,22,26 deansition .E. coli toxins genes as a whole seem to have a higher incidence rate in ADK patients, are in agreement with previous studies. In particular, E. coli are commonly isolated from both CRC patients and healthy controls. However, in CRC patients, more pathogenic E. coli strains are found [E. coli in biopsies of CRC. The presence of the genomic island pks+ in particular seems to be a predisposing factor for CRC. pks+ is one of the most studied factors related to CRC development, and several groups reported that mucosa samples from CRC patients more frequently harbour pks+ E. coli strains with respect to healthy patients [Although not statistically significant, our results, showing that re found ,30. In tre found  reportedpatients ,31,32.E. coli expressing pks+, the same mutational signature detected in a subset of human cancer genomes was found [E. coli toxins genes herein studied, cnf1 and cdt, did not reach any significant association with HP or PA alone. However, due to the low number of ADK, we cannot rule out the possibility that they can be associated with cancerous lesions. Both of them, in fact, are closely studied for their activities strongly reminiscent of carcinogenesis [The fact that the product of the genomic island pks+  can be considered a predisposing factor for CRC is also supported by its activity in eukaryotic cells since it induces DNA damage, cell-cycle arrest, mutations, and chromosomal instability ,34,35,36as found , furtherogenesis ,37,38.B. fragilis, our results revealed no association between the presence of bft and HP and PA and ADK, highlighting a discrepancy with previous reports from other groups. B. fragilis is a normal inhabitant of the gastrointestinal tract, but the enterotoxigenic form producing BFT is present in approximately 20% of the healthy population [B. fragilis strains producing BFT have been shown to contribute to carcinogenesis [bft gene and pre-cancerous and cancerous lesions in human [bft are not in agreement with the current literature. We have to underline, however, that studies revealing an association between bft and CRC strongly differ from our study in terms of sample dimension, exclusion criteria, and statistical analyses. In fact, no more than 60 cases and 60 controls were analysed in a single study; univariate analysis was only applied, and stool samples instead of mucosa were used in some cases. Moreover, due to the low number of ADK samples in our study, we cannot exclude the existence of an association between bft and cancerous lesions.As regards pulation . Currentpulation ,41, and pulation ,43. In aogenesis ,45,46, ain human ,48,49 ThE. coli toxins and BFT with cancerous lesions, with the aim of exploring the possibility of incorporating bacterial toxins as biomarkers into CRC screening protocols or as tools for risk stratification.Our results indicate for the first time a role for the CIF toxin in the early stages of carcinogenesis and pave the way for further insights into the association of An observational case-control study was designed to evaluate if the presence of bacterial toxins (one or more) in colorectal tissue is positively associated with pre-cancerous (PA) or cancerous lesions (ADK). Colorectal biopsies with polyps/cancer (cases) were compared to disease-free colorectal tissues (controls). Moreover, the study evaluated whether known CRC-predisposing genetic factors contribute to the response to bacterial toxins.The research was conceived as a collaborative study among Istituto Superiore di Sanità , Policlinico Umberto I Sapienza University, Regina Elena National Cancer Institute, and Karolinska Institutet. It was approved by the ISS Ethical Committee (reference number: PRE 564/16) and carried out in accordance with the recommendations, with written informed consent from all subjects. All subjects involved were adult and gave written, informed consent in accordance with the Declaration of Helsinki.The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines were followed in processing this study .Intestinal biopsy samples were obtained from 330 Caucasian adult subjects that underwent colonoscopy between September 2016 and July 2018.Before the procedures, a questionnaire was administered to collect information on subjects’ anamnesis and lifestyle. Exclusion criteria included the use of probiotics and antibiotics in the previous 15 days; prophylaxis for diverticulosis; type II diabetes; familiar hereditary CRC and inflammatory bowel diseases. A database of the subjects enrolled was created where age, sex, clinical history, date of sampling, histopathology examination, information on the nature of the polyp (benign or transformed), as well as the presence of the bacterial toxins gene and the genetic risk factors obtained were specified. The experimental design as well as exclusion criteria are reported in All the patients received polyethylene glycol as sole bowel preparation for colonoscopy. During the procedure, biopsies were taken from the normal-appearing colic mucosa as well as from HP, PA, ADK, or portions of the colon immediately adjacent to these lesions. For each patient, two biopsies were taken, and each one was put in a different vial: the first one at or near the lesion  and the second one far from the lesion . For controls, the two biopsies were taken in the same place. The biopsy in Vial A was used for bacteria enrichment and real-time PCR to detect the presence of bacterial toxins genes. The biopsy in Vial B was used for genotyping of CRC-risk loci and was taken far from the lesion to avoid mutations due to the lesion itself. Collected biopsies were rapidly frozen in dry ice and kept frozen until subsequent use.B. fragilis, biopsy specimens were incubated at 35 °C in anaerobic conditions  in 10 mL of Brain Heart Infusion broth (Oxoid) supplemented with 1 g/L of cysteine, 5 mg/L of hemin solution, 20 mL/L of 10% NaHCO3 solution, and 100 mg/L of gentamycin. After 48 h of growth, 1 mL of culture broth was centrifuged at 10,000× rpm for 3 min, and pellets were kept frozen until subsequent use. To extract DNA, pellets from aerobic or anaerobic cultures were lysed by adding 200 μL of sterile water, vortexing, and heating at 100 °C for 10 min. Following centrifugation at 14,000× rpm for 5 min, DNA-containing supernatants were transferred to sterile tubes and kept frozen until subsequent analyses.Biopsies were grown on appropriate media to enrich aerobic and anaerobic bacteria. In particular, for aerobic bacteria, each bioptic fragment was placed in 3 mL Trypticase Soy Broth  tube. After overnight incubation at 37 °C under aerobic condition, 1 mL of TSB culture was centrifuged at 10,000× rpm for 10 min, and the bacterial pellets were kept frozen until subsequent use. For anaerobic bacteria, i.e., gapA and bfra genes was used as control for the presence of aerobic bacteria or B. fragilis, respectively.Real-time PCR amplification was performed on total bacterial DNA obtained from colon biopsies cultured in aerobiosis or anaerobiosis, as previously described, in order to detect the presence of genes for the virulence factors under study. The presence of E. coli strain EF4 for gapA, cdt, and cif; E. coli strain J96 for cnf1; E. coli Nissle 1917 for clbA and clbQ (pks+); and B. fragilis for bfra and bft.Amplification reactions were performed with Bio-Rad CFX96 platform, using a 96-well PCR multiplate from Bio-Rad. Samples were analyzed by means of SYBR Green Mastermix (Qiagen QuantiTect SYBR Green PCR Kit (1000) or Bio-Rad SsoAdvancedTM Universal SYBR Green Supermix). As positive controls, we used cif, cnf1, gapA, bfra, bft, clbA, clbQ, and cdt) to obtain a specific melting curve for each amplicon.Amplification conditions were optimized for each target used  using SsoAdvancedTM Universal SYBR Green Supermix (Bio-Rad) was 95 °C for 5 min, followed by 39 cycles at 95 °C for 30 s, 55 °C for 1 min, 72 °C for 1 min, 95 °C for 10 s, and 55 °C for 5 s. The fluorescence measurements were followed on-line by the software of the instrument. Two PCR replicates were carried out for each standard solution and control.The thermal profile using QuantiTect SYBR Green PCR Kit  for the amplification of Total DNA was extracted from biopsies by using the QIAamp Fast DNA tissue kit , according to the manufacturer’s instructions. This kit uses a combination of mechanical, chemical, and enzymatic lysis to homogenize samples. Briefly, biopsies were destroyed by mechanical agitation in tubes containing beads. Homogenization of the tissue and simultaneous stabilization of DNA released from the disrupted tissue was obtained by optimized chemistry. Digestion with a buffer containing proteinase K completed the lysis of samples, and genomic DNA was subsequently purified using the QIAamp Mini Spin Columns provided.DNA samples were genotyped using Illumina Global Screening Array on Illumina iScan high-throughput screening system in the Institute of Clinical Molecular Biology . The raw-intensity data were analyzed using the GenCall algorithm available in the software GenomeStudio to call the alleles.p-value < 1 × 10−5; removal of markers whose p-value of difference in missingness between adenoma and colorectal cancer cases and healthy controls was <1 × 10−5; and removal of samples that were outliers, identified using principal component analysis (deviation of more than 6 times interquartile range). A total of 279 samples were kept after the QC.Genotypes were QCed, removing samples and markers with the following criteria: exclusion of samples with ≥15% missing rates; exclusion of markers with non-called alleles; exclusion of markers with missing call rates > 0.05; exclusion of samples with ≥5% missing rates; exclusion of related samples (PI-HAT > 0.1875); exclusion of samples whose genotyped sex could not be determined; exclusion of samples with high heterozygosity rate (more than 3 times standard deviation from mean); removal of SNPs (Single Nucleotide Polymorphism) located in sexual chromosomes; removal of markers with Hardy–Weinberg equilibrium SNPs not genotyped were imputed by means of Sanger Imputation Server. The release 1.1 of Haplotype Reference Consortium was used as reference panel, and the imputation was done using the EAGLE2 + PBWT pipeline ,51,52,53To calculate the Polygenic Risk Score, the study GCST007856  depositeThe sample size was planned to assume a three-fold prevalence of bacterial virulence factors genes in pre-cancerous PA compared to controls (A.F.’s preliminary data). A sample size of 75 cases and 150 controls was estimated to be sufficient to assess the association with an 80% power.Subjects’ characteristics for the whole population and stratified by outcome  were summarized by means of the levels of the variables (with%) for categorical variables or by means of quantiles and mean with standard deviation for continuous variables.cif, cdt, cnf1, pks+, bft). The role of bacterial toxins genes on the outcome was also analyzed considering the sum of aerobic toxins and the sum of all toxins.Potential exposure factors were represented by the presence of bacterial toxins genes , physical activity, and other health status and lifestyle variables.Compliance with the study was evaluated in terms of percentage of missing values. Variables with high level of missing values were considered only for descriptive purposes; no imputation methods were used.p-value < 0.1 were considered in the multivariate models. Backward and stepwise methods were applied to identify the multivariate models with a step-by-step iterative construction that involved the selection of independent variables to be considered in the final model.In univariate analysis, non-parametric tests were performed for comparisons between groups . Logistic regression models were used in univariate and multivariate analyses to assess if the presence of bacterial toxins (one or more) in colorectal tissue was positively associated with PA or ADK compared to disease-free colorectal tissues. Odds ratios and 95% confidence interval were reported as parameter results of the logistic regression models. All covariates were evaluated in univariate models, and all factors with univariate association within p-value < 0.05 as indicating a statistically significant difference, and CI were calculated at 95% level. All analyses were performed using the R software [All tests were 2-sided, accepting software . Logisti


For the past 2 years, the coronavirus responsible for the COVID-19 infection has become a world pandemic, ruining the lives and economies of several nations in the world. This has scaled up research on the virus and the resulting infection with the goal of developing new vaccines and therapies. Natural products are known to be a rich source of lead compounds for drug discovery, including against infectious diseases caused by microbes . In this review article, we conducted a literature survey aimed at identifying natural products with inhibitory concentrations against the coronaviruses or their target proteins, which lie below 10 µM. This led to the identification of 42 compounds belonging to the alkaloid, flavonoid, terpenoid, phenolic, xanthone and saponin classes. The cut off concentration of 10 µM was to limit the study to the most potent chemical entities, which could be developed into therapies against the viral infection to make a contribution towards limiting the spread of the disease. The novel coronavirus disease 2019 (COVID-19) has led to a sudden change in the lifestyle of humans all over the whole world . The WorCoronaviruses (CoVs) belong to the family Coronaviridae, sub-family Coronaviridae and order Nidovirales. Viruses belonging to this family and sub-family are known to be large in terms of genome size (26−32 kb) , are envEven though research on the structure and mode of action of the SARS-CoV-2 virus is ongoing, several known antiviral drugs  have been used to manage cases of SARS-CoV infection, together with some anti-malarials like chloroquine and hydroxychloroquine, the antibiotic azithromycin, etc. –12. BesiSeveral excellent reviews have recently been published on NPs with the potential for treating SARS-CoV-2 infection –19. OtheAll pertinent information about the botanical description, conventional uses, phytochemicals and the pharmacological activities of the isolated compounds were collected from available recently published literature. The electronic databases employed for the assortment of relevant information include Scopus, NISCAIR, Scifinder, PubMed, Springer Link, Science Direct, Google Scholar, Web of Science, and an exhaustive library search. The chemical structures of the compounds were drawn using ChemDraw Ultra 8.0 software. PubChem and ChemSpider databases have been used to check the IUPAC names of the isolated phytoconstituents. The workflow for collecting the literature and writing the report has been provided in Fig. 1), Fig. Berberis from which the compound derives its name. Some medicinal properties of 1 reported include anti-inflammatory .FigXanthones are oxygen containing heterocyclic compounds commonly occurring in higher plants , fungi, lichen, and bacteria , 145, thCalophyllum blancoi against SARS-CoV. The genus Calophyllum is a rich source of phenolics and xanthones, possess great antibacterial [42), Table C. blancoi [50 value of 7.93 µM [Shen et al. studied the antiviral activity of acterial , 150, anacterial , antiplaacterial , anticanacterial  and antiacterial . BlancoxOut of 42 NPs belonging to the alkaloid, flavonoid, terpenoid, phenolic, xanthone and saponin classes obtained from our literature survey. Our results showed that majority of the NPs with inhibitory concentrations against the virus or its target proteins below 10 µM were terpenoids, alkaloids, flavonoids, and diarylheptanoids, respectively. The terpenoids (diterpenoids and triterpenoids) were the most abundant among the isolated NPs, constituting 26.19% of the isolated compounds. This was followed by alkaloids (19.05%), flavonoids (11.90%), diarylheptanoids (9.52%), phenolics (4.76%), saponin (2.38%) and xanthones (2.38%), which showed a similar trend to previous studies –19.pro, 3CLpro, SARS-CoV helicase, etc.) had earlier been published. We are presenting this review to encourage the testing of these compounds against the SARS-CoV-2 virus. In writing this manuscript, we must mention that the compounds were actually tested against the SARS-CoV-2 virus. Only ten of the 165 references date after 2020. It was the goal of the authors to summarize the results of compounds and plant species whose extracts have already been tested against earlier discovered coronaviruses so as to ease the task of discovery of NP antivirals that could be next generation drugs to treat infections caused by SARS-CoV-2. The results of the current study reveal that promising NPs as potential inhibitors of SARS-CoV-2 were terpenoids, alkaloids, flavonoids, and diarylheptanoids, respectively. The mechanism of action of some of the NP hits and the plant species from which they were isolated has been included in the study. It is worth mentioning that some of the medicinal plants and the NPs have shown good safety in vitro studies. This review could serve as a starting point for further development of these NPs hits as potential leads for COVID-19. Thus, it would be interesting in future to evaluate the toxicities and binding mode of some of these NPs using in silico approaches.Natural products (NPs) have provided privileged scaffolds in drug design . The nov


The aim of this study was to evaluate the potential of noug as a source for human nutrition. Diverse noug genotypes were evaluated for their content and/or composition of total lipids, fatty acids, proteins, and minerals using standard methods. The total lipid content (32.5–45.7%) and the proportion of an essential fatty acid, linoleic acid (72.2–77.8%), were high in noug, compared to other oilseed crops. The proportion of oleic acid, a monounsaturated fatty acid, was low in noug (5.2–9.2%). The breeding objective of increasing the oleic acid level in the highland, where noug is mainly cultivated, was limited, as the content of this acid was low in this environment. The seed protein concentration (25.4–27.5%) and mineral content were mainly affected by the cultivation environment, as the high temperature increased the amount of protein, whereas the soil condition was a major factor in the variation of the mineral content. Thus, noug is a unique crop with a high seed oil content, of which a high proportion is linoleic acid. With the exception of the seed oleic acid content, when grown in low-altitude areas, the genotypic variation contributes less than the cultivation environment to the nutritional attributes of noug. Hence, high-oleic-acid noug for lowland production can be targeted as a breeding goal. Oilseeds have the potential to contribute a vital source of nutrition to the human diet . VegetabGuizotia abyssinica Cass.) seeds are finely pounded using a mortar and pestle, until a thick fluid, locally known as litlit, is formed. Then, bread made from different cereals, sugar, salt, etc., are added to the litlit and further pounded. The mixture is then made into balls of different sizes and served as a food locally known as chifko [Currently, oilseeds are primarily used for oil extraction, and they are considered as the main sources of vegetable oil . The quas chifko . The curs chifko ,12. The s chifko . The oils chifko  have thes chifko . Minerals chifko ,17.Noug is one of the major sources of edible seed oil in Ethiopia ,19. The Hence, the objective of this paper was to investigate the quantity and quality of the nutrients of noug seeds, including lipids, fatty acids, proteins, and minerals, sampled from a broad range of materials, including cultivars, landraces collected from wide geographic areas in Ethiopia, and “breeding populations”, selected based on lipid content and yield parameters. The nutritive value of noug and opportunities for production of high-nutritive-value noug in Ethiopia were evaluated, and comparisons of the lipid and fatty acid composition with other major oilseed crops were carried out.Basically, three different noug materials were used in the present study: (i) twenty-eight landrace populations, (ii) ten “breeding populations”, and (iii) seven released cultivars . The lanAn incomplete block design within each site was used. Each genotype was planted on a 3 m by 2.1 m (6.3 sqm) plot in seven rows, with a 30 cm distance between rows. A distance of 1 m between the plots was used. A pollination net of 3.2 m (l) × 2.3 m (w) × 2 m (h) was used to cover the plants on each plot, just before flowering, to prevent gene flow between genotypes from different plots. Hand pollination was carried out between plants within each plot by gently rubbing mature flower heads (capitula) in the morning every other day for two weeks. Each genotype and plot was separately harvested at maturity and threshed, and clean seeds were used for laboratory analyses. The lipid content and fatty acid composition were evaluated separately for the seeds from each genotype, plot, and site. Due to resource limitations, 21 of the 28 landrace populations, nine of the 10 “breeding populations”, and six of the seven cultivars were used for protein and mineral content analyses.® reagent grade) used for digesting the fine powder of noug samples for mineral content analysis was supplied by Avantor™ .The reagents used for oil extraction, fatty acid derivatization, and gas chromatography (GC) analyses were supplied by Merck KGaA  and were deemed suitable for these uses: acetic acid , methanol (anhydrous), chloroform (min 99.8%), sulphuric acid (95.0–98.0%), and hexane (≥95%). The methyl-heptadecanoate (≥99%) used as an internal standard for GC analysis of the oil was purchased from Sigma–Aldrich . The nitric acid , before adding 1.25 mL chloroform and 0.9 mL Millipore water, followed by vortexing. After centrifugation for 2 min at 3000 rpm, 50 mL of the chloroform phase of the extracted solution was transferred to a new glass tube, placed on a sand adjusted to 70 °C, and evaporated under a weak beam of liquid nitrogen. Following the complete evaporation of the chloroform solution, the fatty acids in the glass tube were methylated by adding 2 mL of 2% H2SO4 in methanol and incubating for 45 min at 90 °C. After the methylated solution was cooled down to room temperature, 200 nmol methyl-heptadecanoate (17:0-ME) was added to the solution as an internal standard, followed by the addition of 0.9 mL Millipore water and 2 mL hexane. The methylated solution was centrifuged for 2 min at 2000 rpm, and then 200 μL of the hexane phase containing the fatty acid methyl esters (FAMEs) was transferred to GC vials for analysis on Shimadzu GC model 17A , connected to a flame ionization detector [The content and composition of total seed oil and fatty acids were evaluated according to Bligh and Dyer , with thdetector . The prodetector . This indetector .The total nitrogen content of each seed sample was determined on two sample replicates by dry combustion using an automated nitrogen analyzer  . The pro® 8300 inductively coupled plasma optical emission spectrometer  [3 and 3 mL distilled water at a pressure and temperature of 375 psi and 185 °C, respectively. Blank solutions (without sample) were used as the control, and cross contaminations were checked. The mineral contents were recorded in micrograms of the mineral per gram of flour added to the solution (μg g−1) for each analyzed sample.A mineral content analysis of noug seeds was conducted on two sample replicates for each sample using a Perkin-Elmer OptimaMA, USA) ,36. EachAn evaluation of the data was carried out using the statistical package, SAS version 9.4 . Due to the unbalanced dataset, the mixed model (PROC MIXED), followed by the Tukey-Kramer test, was used to calculate the effect of the genotypes and location, as well as their interaction on the analyzed components, such as the total lipid content, content of various fatty acids, and mineral and protein content. Principal component analysis was carried out to understand the genotypic relationship for the combined variation in total lipid, fatty acids, mineral, and protein contents.NG-85) to 45.7% (NG-99) (Guizotia (21–33%) [The mean lipid content of the analyzed genotypes across the three environments ranged from 32.5% ( (NG-99) , with ab(21–33%)  and simi(21–33%) . Mixed mNG-113) to 77.8% (NG-93); the oleic acid concentration ranged from 5.2% (NG-87) to 9.2% (NG-113); and palmitic and stearic acids ranged from 7.8% (NG-113) to 9.6% (NG-87) and 7.2% (NG-111) to 10.0% (NG-113), respectively (Helianthus annuus L.), which is the closest crop to noug [The fatty acid composition of the noug genotypes evaluated in the present study corresponded well with previous investigations, which found linoleic, oleic, palmitic, and stearic acids to be the major fatty acids in noug seeds ,24,26,27ectively . In prevectively . Such re to noug ,40,41.NG-84, has been reported in previous studies as such a high oleic acid genotype when grown at low altitudes, with a decreased oleic acid content at high altitudes and high oleic content (52%) when grown at 21 °C–25 °C under green-house conditions [p < 0.01) Spearman rank correlation between high oleic acid values at low altitudes and the standard deviation of oleic acid content at various altitudes, independent of the altitude of origin of the samples , which Furthermore, a high content of oleic acid, when grown at high altitudes, was rarely found among the evaluated material. Therefore, our findings indicate limited opportunities to breed for and produce high levels of oleic acid in noug grown at high altitudes, while it would be worthwhile to focus on developing high-oleic-acid noug cultivars, specifically for production in lowland areas of Ethiopia and elsewhere. Opportunities to produce high-linoleic and oleic acid noug varieties would contribute an additional asset to the highly nutritious noug oil.p < 0.05) variation in the seed protein concentration among genotypes (p < 0.05) higher seed protein concentration was noted when noug was grown in Ginchi (27.5%), compared with Holeta  a, clearlBasically, all genotypes grown at Ginchi showed negative values for the first principal component, while the same genotypes grown at Holeta showed positive values b, corresThe nutritional quality of noug can be divided into two parts: (i) the quality of noug oil production, which is principally determined by the total lipid content and composition of the fatty acids; and (ii) the quality of the whole seed/cake for human nutrition, which is basically determined by the fatty acid, protein, and mineral composition. The total lipid content (of importance for vegetable oil production) in noug had a close parity with that found in a range of other representative oilseed crops, all of which have recently been described in a monograph of important oil crops ,51,52,53Considering the total lipid content, noug has the potential to develop into an oil crop of great importance. However, the nutritive value and quality of oilseeds is not only dependent on the total lipid content, but even more so on the fatty acid composition of the seed oil . The fatThe protein concentration and composition are of importance if the oil crops should be used for human consumption as whole seed or seed cake. Additionally, when it comes to the seed protein concentration, noug is similar to the other oil crops. As can be seen in −1), as compared to many of the other seeds of the oilseed crops , Noug is unique among oil crops in having a high total seed lipid content, with a high proportion of linoleic acid, contributing to the health-promoting properties of its vegetable oil. For the use of its seed cake, whole-seed, or seed meal, the fatty acid composition, with a high content of linoleic acid content, is beneficial, and noug also has a high seed protein concentration and a high Fe content, contributing to its nutritional potential. Unfortunately, the Zn content and oleic acid content are on the lower end of that found in other oilseed crops, partly hampering the quality of both the vegetable oil and whole seed/cake of noug when used for human consumption. Noug genotypes with a high oleic acid content are available when grown at low altitudes in Ethiopia, making them suitable to breed for high-oleic-acid-content noug varieties for these growing environments. However, when these genotypes are grown at high altitudes, the oleic acid content is reduced. Thus, a lack of opportunities to grow high oleic acid genotypes at high altitudes seems to prevail. The environmental conditions are also of higher importance than the genotypes in determining the grain protein concentration and mineral content in the seeds, and both factors are of importance if the oil crops are to be used as whole seed/cake for human consumption. Thus, for a high seed protein concentration, dry conditions, reducing the yield, are needed, which is not a suitable solution, while the soil mineral content is a major factor determining the mineral content of noug seeds. Despite the minor influence of the genotypes on the quality attributes, novel genomic tools, such as the recently described transcriptome-based SNP markers and the developed KASP markers, e.g., in , might b


The 31 main components of Lianhua Qingwen (LHQW) were obtained through a literature and database search; the components included glycyrrhizic acid, emodin, chlorogenic acid, isophoroside A, forsythia, menthol, luteolin, quercetin, and rutin. Sixty-eight common targets for the treatment of novel coronavirus pneumonia (NCP) and influenza virus pneumonia (IVP) were also obtained. A “component-target-disease” network was constructed with Cytoscape 3.2.1 software, and 20 key targets, such as cyclooxygenase2 (COX2), interleukin-6 (IL-6), mitogen-activated protein kinase14 (Mapk14), and tumor necrosis factor (TNF), were screened from the network. The David database was used to perform a Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway enrichment analysis and gene ontology (GO) biological process enrichment. Results showed that the key targets of LHQW in the treatment of NCP and IVP mainly involved biological processes, such as immune system process intervention, cell proliferation, apoptosis and invasion, toxic metabolism, cytokine activity, and regulation of the synthesis process. KEGG enrichment analysis revealed that 115 signalling pathways were related to the treatment of LHQW. Amongst them, IL-17, T cell receptor, Th17 cell differentiation, TNF, toll-like receptor, MAPK, apoptosis, and seven other signalling pathways were closely related to the occurrence and development of NCP and IVP. Molecular docking showed that each component had different degrees of binding with six targets, namely, 3C-like protease (3CL), angiotensin-converting enzyme 2 (ACE2), COX2, hemagglutinin (HA), IL-6, and neuraminidase (NA). Rutin, isoforsythiaside A, hesperidin and isochlorogenic acid B were the best components for docking with the six core targets. The first five components with the best docking results were isoforsythiaside, hesperidin, isochlorogenic acid B, forsythin E, and quercetin. In conclusion, LHQW has many components, targets, and pathways. The findings of this work can provide an important theoretical basis for determining the mechanism of LHQW in treating NCP and IVP. Lianhua Qingwen (LHQW) is composed of 13 traditional Chinese medicines (TCMs), namely, Forsythiae Fructus , Lonicerae Japonicae Flos , Ephedare Herba , Armeniacae Semen Amarum , Gypsum Fibrosum , Isatdis Radix , Dryopteridis Crassirhizomatis Rhizoma , Houttuyniae Herba , Pogostemonis Herba , Rhei Radix et Rhizoma , Rhodiolae Crenulatae Radix et Rhizoma , l-Menthol , and Glycyrrhizae Radix et Rhizoma . According to the pathological theory of TCM, LHQW is good medicine for dredging collaterals, dispersing the lung, opening with pungent and bitter drugs, and clearing away heat and detoxification . LHQW haGiven the complexity of the composition and mechanism of traditional Chinese medicine (TCM), systematically explaining the mechanism of TCM in the treatment of diseases is difficult. Network pharmacology is a new technology that integrates system biology, multidirectional pharmacology, and computer analysis. It can systematically link drugs with diseases and observe the intervention and influence of drugs on disease networks –9. The sIn recent years, network pharmacology has been widely used to systematically explain the mechanism of action of TCM and TCM compound prescription in the treatment of diseases –12, and https://pubchem.ncbi.nlm.nih.gov/), the target protein of the compound was obtained from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database (http://tcmspw.com/tcmsp.php), National Center for Biotechnology Information (NCBI) database (https://pubchem.ncbi.nlm.nih.gov/), and https://dx.doi.org/10.1093/nar/gkz382 (SWISS) database (http://www.swisstargetprediction.ch/). At the same time, the protein database UniProt (http://www.uniprot.org/uploadlists/) was used to convert the targets to a unified gene name.The main components of LHQW can be identified and quantified through literature retrieval. Through verification and conversion of the PubChem database (https://www.genecards.org/) is a platform that provides all known human genes in genome, proteome, transcription, heredity, and function [GeneCards database (function . NCP- anhttps://string-db.org/) to construct a protein-protein interaction  network map. The database parameter was set to “Homo sapiens,” and the protein type was set to the rating criterion (confidence level >0.9). Then, the PPI network file was downloaded. The active components, their corresponding LHQW targets, and PPI were input into Cytoscape 3.2.1 to construct the “native component-target” network of LHQW. The topology parameters of the network were analysed using the network analyser function of Cytoscape and derived. The related topological data, such as degree (DG) and betweenness centrality (BC), were examined. DG and BC are important topological parameters for evaluating the specific gravity of a node in the network. DG can reflect the number of links between one node and other nodes in the network, and BC reflects the ratio of the number of paths passing through the node to the total number of shortest paths in all the shortest paths in the network. The larger the topology parameters of the node are, the more critical the target in the network is [To determine the interaction between viral pneumonia potential therapeutic targets and targets of LHQW, this study introduced the selected targets into the STRING network platform . GO enrichment can be used to understand the main action process of the target. The KEGG pathway can be adopted to understand the role of this target in metabolism, signal transduction and other pathways by observing the distribution of targets in the pathway. The main signalling pathways and biological processes involved in the efficacy of LHQW were obtained by screening the target genes with P < 0.05. The P value is the statistical significance of target enrichment/annotation, and P < 0.05 indicates that the pathway has analytical significance. The smaller the P value is, the more significant the enrichment result is.The core targets of LHQW in the treatment of viral pneumonia were subjected to gene ontology (GO) biological process and Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway enrichment analyses by using the David database (https://www.rcsb.org/), and the target protein and component were pretreated using AutoDock 1.5.6 and Discovery Studio 2.5 software. Then, the target and component were docked using AutoDock Vina. Generally, negative values of binding energy are associated with the stable conformation of ligand binding to the receptor. If the binding energy is less than −5 kcal/mol, then the ligand and receptor can bind spontaneously; binding energy ≤−7 kcal/mol denotes good binding [The protein structure file was downloaded from the Protein Data Bank (PDB) website . Main ingredients of LHQW (red triangle) and medicinal composition of LHQW (green diamond).To further explore the mechanism of LHQW, 68 target proteins of LHQW against viral pneumonia were input into the STRING database, and the PPI network file was obtained . The medP < 0.05 were screened out. In general, the GO terms were classified into three parts: biological process (BP), molecular function (MF), and cellular component (CC). The visualisation results of BP, CC, and MF enrichment analyses were obtained using OmicShare, as shown in Twenty key targets were annotated with GO terms, and 91 terms with P < 0.05 were selected. The visualisation results of the top 20 KEGG pathways with the most significant P value were obtained using OmicShare, as shown in The enrichment analysis of KEGG signalling pathways was carried out on 20 key targets, and 115 signal pathways with The occurrence and development of a new coronavirus and influenza virus exhibit similarities and differences after infection. The former mainly involves two targets (3CL and ACE2) –30, wherThe top five components for combination with each target are shown in Dimax = (Yi − Ymin)/(Ymax − Ymin), and OD = (D1D2... DK)k1/ (k is the index number) [To further analyse the results of each component combined with the targets, this study performed a multi-index comprehensive analysis and multi-index data processing by referring to the general evaluation normalisation method reported in literature , 37. In  number) , 39. Theα, IL1, etc.), and chemokines , and upregulates the expression of COX2 and NOS2, which induce the synthesis of inflammatory mediators, such as PGE2 and NO [κB, as regulatory factors of the inflammatory response, bind to the IL-6 response element in the promoter of the acute phase protein gene [α is an important member of inflammatory cytokines [α can regulate immune cells, and as an endogenous heat source, it can cause fever and interfere with apoptosis. As an early cytokine of viral pneumonia, the expression of TNF-α can regulate other proinflammatory factors downstream. An increase in other cytokines stimulates the production of TNF-α and triggers the cytokines that are dependent on TNF-α. IL-6 and TNF-α are the key cytokines [α, as an early cytokine, can regulate other proinflammatory factors downstream. An increase in other cytokines stimulates the production of both and initiates the TNF-α-dependent cytokine cascade reaction, thus forming a vicious spiral that can cause a cytokine storm and aggravate the patient's condition. The activation and inhibition of these targets not only aggravate the disease but also promote each other, resulting in a vicious cycle that is closely related to the inflammatory injury of the lung; it is also the key target of this study. Therefore, an in-depth study of these targets can help reveal the scientific nature and connotation of the treatment of IVP and NCP.In this study, 31 main components of LHQW were qualitatively and quantitatively obtained by compiling literature, and 712 component targets were obtained from three databases. A total of 68 common targets for the treatment of viral pneumonia were obtained. The expression of COX2 is abnormal in IVP and NCP –43 and m2 and NO , 45. The2 and NO , 47, suc2 and NO , 49. NOSein gene , 51. Wheytokines , 53 thatytokines , 55 for α, IL-2, IL-4, IL-15, and IL-17, can be inhibited relatively. IL-17 signalling pathways regulate IL-17 family expression (first discovered by Th17 cells), and by binding to different receptors, they activate downstream signalling pathways JAK-STAT and NFKB to regulate the secretion of other inflammatory factors; IL-17 signalling pathways are also involved in the occurrence and development of lung injury [β and IL-6, which can activate signal transduction and the transcription protein pathway by inducing the expression of transcription factors. Th17 mediates inflammatory responses mainly by secreting IL-17, IL-21, IL-2, IL-4, TNF-α, and other cytokines [κB is located at the hub of the downstream signalling pathway in TLR [α, IL-1, IL-6, IL-8, and other inflammatory factors; they can also upregulate the expression of COX2 and NOS2, which can induce the synthesis of inflammatory mediators (PGE2 and NO). The accumulation of these cytokines can directly reflect the degree of inflammation. A moderate inflammatory response is a kind of defence reaction of the body, and it is conducive to the clearance of pathogens and the recovery of the body. However, an excessive reaction can cause cascading inflammatory reactions that are difficult to control. TLR and NF-κB may form a complex cycle network with the participation of certain cytokines, which can regulate each other and form a vicious cycle. Therefore, the 20 intervening key targets and their pathways are key to the treatment of viral pneumonia with LHQW.According to the KEGG pathway enrichment analysis in this study, the IL-17 pathway is the most important signal pathway. IL-17 signal pathways are important to the study of multiple coronaviruses. For example, SARS viruses induce the activation of pattern recognition receptors and IL-17 pathways . The Midg injury , 58. In g injury . Severalg injury , 61, andytokines , 63, butytokines . NF-κB iy in TLR . It can κB, TLR, MAPK, and other signalling pathways and downregulation of the gene expression of COX2 and iNOS. As a result, the production of proinflammatory factors, such as IL-6, TNF-α, and chemokines , is reduced to intervene in acute lung injury. Luteolin [κB signalling pathway and the expression of apoptosis-related factors  to reduce the immune damage on the host cells caused by the virus. It also regulates cell apoptosis and plays an antiviral role. Patchouli alcohol [α and IL-6. Forsythia A [κB, and Th17 signalling pathways. Phillyrin [α, IL-6, IL-1β, CXCL10, etc.) induced by SARS-CoV-2 by regulating the activity of the NF-κB signalling pathway. Therefore, forsythin can be used as a new strategy for coronavirus disease. Research has confirmed that glycyrrhizic acid [In addition, the results of molecular docking showed that the 31 main components contained in LHQW key targets had different degrees of combination. In particular, flavonoids and their glycosides and phenylpropanoids had the best binding result. The varied components contained in LHQW could affect multiple pathways by acting on multiple targets to achieve overall intervention in the development of viral pneumonia and overall treatment. Previous studies have found that chlorogenic acid , cryptocLuteolin , 76 has  alcohol  has anti alcohol , 79, cansythia A  can inhihillyrin  can signzic acid  is similκB and MAPK signalling pathways in activated T cells and inhibiting the continuous burst of cytokines. Most of the components reported in these studies are the main components of honeysuckle and forsythia suspense, which belong to the monarch medicine of the LHQW recipe and are the most important components in the treatment of diseases. They clear away heat, detoxify the body, and disperse wind heat. The results of molecular pairing in this study also showed that the main components of honeysuckle and forsythia account for the largest number and are the key to the efficacy of LHQW. The ingredients reported in the above-mentioned studies are basically consistent with the results of the current study, suggesting that the material basis analysis results of this study conform to the law of the monarch, the subject, and TCM to a certain extent. Presently, most studies focus on nonvolatile components, and the few reports on antiviral, anti-inflammatory, and immune regulation of the volatile components of LHQW focus on cooling, expectorant, and antibacterial effects and nasal congestion reduction [Several studies  have foueduction . The resAn abnormal immune system is the main common point in the occurrence and development of IVP and NCP. The pathway enrichment analysis and biological function annotation in this study showed that the mechanism of LHQW in the treatment of the two diseases is closely related to immune, inflammatory, and viral signalling pathways. Therefore, the mechanism of LHQW treating IVP and NCP could focus on restoring the physiological function of the immune system, reducing the persistent injury of inflammation to the body, inhibiting the invasion and proliferation of the virus, and promoting the apoptosis of the virus.LHQW contains 31 main components. Isochlorogenic acid B, isoforsythiaside, forsythoside E, rutin, quercitrin, hesperidin, and other components are the key to the simultaneous treatment of NCP and IVP by LHQW. These components can interfere with signalling pathways, such as IL-17, T cell receptor, Th17 cell differentiation, TNF, toll-like receptor, MAPK, and apoptosis by acting on targets, including 3CL, ACE2, COX2, HA, IL-6, and NA, to realise the anti-inflammatory, antivirus, and immune regulation comprehensive efficacy of LHQW. Although the mechanism of treating IVP and NCP differs and the different contents are mainly reflected in the manner of virus infection and the material basis of antivirus, the main links are closely related to regulating immunity, reducing inflammation, and combating the virus. The process embodies the characteristics of seeking common ground whilst reserving differences in the treatment process of IVP and NCP. It also shows the multicomponent, multitarget, and multichannel advantages of LHQW and provides a research method for the mechanism and substance foundation of the treatment of IVP and NCP.


Streptomyces sp. strains of the CS collection isolated from the surface of leaf-cutting ants of the Attini tribe and compared them to four Streptomyces model species and Pseudonocardia sp. Ae150A_Ps1, which shares the ecological niche with those of the CS collection. We used a combination of phylogenetics, bioinformatics and dereplication analysis to study the biosynthetic potential of our strains. 51.5% of the biosynthetic gene clusters (BGCs) predicted by antiSMASH were unknown and over half of them were strain-specific, making this strain collection an interesting source of putative novel compounds.The rapid emergence of bacterial resistance to antibiotics has urged the need to find novel bioactive compounds against resistant microorganisms. For that purpose, different strategies are being followed, one of them being exploring secondary metabolite production in microorganisms from uncommon sources. In this work, we have analyzed the genome of 12  In the current scenario of emergence of new pathogens and the rapidly increasing rate of resistant bacteria to clinically used antibiotics, the need for compounds with novel bioactivities has arisen as an urgent worldwide concern . AccordiIn recent years, bioinformatics has become an essential tool in the field of new drug discovery mainly due to three facts: (i) the improvement in sequencing techniques as well as the reduction of their costs have contributed to the public availability of an enormous pool of genome sequences; (ii) the rapid development of new, powerful and ‘user-friendly’ bioinformatic tools; and (iii) the great amount of information that could be obtained without resorting to traditional expensive and time-consuming laboratory screening programs.Attini tribe cultivate basidiomycete fungi in their nests to provide food to their larvae. A well-known symbiotic relationship has been stablished between these ants and bacteria of the genus Pseudonocardia which defends the fungus cultivar from the attack of the fungal parasite Escovopsis sp. by secreting antifungal compounds. In this highly competitive environment, a third partner has been identified: bacteria of the genus Streptomyces have been usually isolated from the cuticle of leaf-cutting ants. These Streptomycete strains form a protective microbiome as they produce many bioactive compounds  against other invasive microorganisms [Insect microbiomes have attracted the attention of scientists in recent years as they comprise a valuable source of bioactive compounds ,6. Ants rganisms ,8.Streptomyces spp. have widely been recognized for their secondary metabolite production. They are, in fact, the source of 2/3 of the antibiotics currently available for human use [uman use . When thuman use ,11. ThusStreptomyces strains isolated from leaf-cutting ants  [Streptomyces coelicolor, Streptomyces albidoflavus, Streptomyces clavuligerus and Streptomyces arvemitillis. We also analysed the genome of Pseudonocardia sp. Ae150A_Ps1, which was isolated from the same ecological niche as the Streptomyces CS strain collection. We generated a similarity network using BiG-SCAPE tool to detect those BGCs shared by or specific for each strain, comparing them with the MIBiG database to identify the already known gene clusters. Furthermore, we manually analysed the antiSMASH predictions to refine the results obtained from the network, and we complemented the study with data from dereplication analysis of ethyl acetate extracts from cultures of these strains grown in R5A medium. 51.5% of the gene clusters detected were unknown, and half of them were strain-specific, therefore showing the great potential that this collection of strains has for novel drug discovery and how this combination of methods can help to select gene clusters to work with.With the aim of studying the diversity of compounds produced by an in-house group of ed them) , we perfStreptomyces sp. CS014, CS057, CS065a, CS081a, CS090a, CS113, CS131, CS147, CS149, CS159, CS207 and CS227 strains were isolated from leaf-cutting ants , as previously described [600nm = 0.2.escribed . To obtahttps://submit.ncbi.nlm.nih.gov/subs/genome/; accessed on 25 October 2021). To verify the quality of the sequence assemblies, the genome completeness was checked with BUSCO v.5.1.3 pipeline using the lineage dataset “bacteria_odb10” (OrthoDB v.10.1) and the assessment mode “genome” [Streptomyces coelicolor A3(2) , Streptomyces albidoflavus J1074 (NC_020990.1), Streptomyces avermitilis MA-4680 = NBRC14893 , Streptomyces clavuligerus ATCC27064  and Pseudonocardia sp. Ae150A_Ps1  by the Department of Biochemistry at the University of Cambridge . ,14,15. T“genome” . Commonl00000.1; ).A maximum-likelihood tree was generated by using the online tool autoMLST . GenbankPrediction of BGCs was carried out with the bioinformatic tool antiSMASH v5.2 , configuThe program BiG-SCAPE  was usedDictionary of Natural Products version 26:2 [Seven-day-old whole culture samples were extracted with an equal volume of ethyl acetate and analysed by HRMS-based dereplication against MEDINA Foundation in-house library  and the ion 26:2  to identAll known BGCs predicted by homology or detected by dereplication of the strain’s cultures were represented on a heatmap. The heatmap was generated using the Python module Seaborn. A complete method was used to calculate the hierarchical clustering and the Euclidean method to calculate pairwise distances. Strains were ordered according to the phylogenetic tree.High quality sequences are required to perform accurate and non-sesgated comparative genomic analysis. In this work, high quality genome assemblies from CS strains were used with an approximate coverage of 92% and low number of scaffolds, ranging from 2 to 9. Genome size and G + C content (%) averages were 7.8 Kb and 72%, respectively. Data regarding genome assemblies are summarized in To locate specific niche selection of bacterial communities, we carried out a phylogenetic analysis. 16S RNA locus has been traditionally used to group bacteria into operational taxonomic units (OTUs), but lately some inaccuracies have been reported using this method due to horizontal transfer events and variable rates of evolution. Nowadays, a broader spectrum analysis based on several housekeeping genes, not only on 16S RNA, has been stablished as the method of choice ,31.Streptomyces strains generally used as reference organisms, one Pseudonocardia strain that shared the ecological niche with the CS strains and some closely related soil bacteria selected by the online tool autoMLST  based on 82 housekeeping genes, including the twelve CS strains under study, four autoMLST . SurprisS. clavuligerus, and C2 that comprised CS113, CS159, CS207 and CS227 strains together with the well-known reference strains S. coelicolor, S. avermitilis and S. albidoflavus. According to this data, strains from the C1 clade are potentially more attractive candidates for new drug discovery screenings since they were less related to Streptomyces strains commonly used for secondary metabolite analysis  and another part with 100% homology with the MIBiG entry to antimycin (BGC0000958), clearly showing that they are two completely different BGCs. Thus, a manual curing of the results was needed to improve gene cluster delimitation. A total of 36 BGCs were manually curated .S. coelicolor A3(2)  identified a total of 541 BGCs in the 17 strains under study , distrib 38.71%; B. The lor genome C, a commS. clavuligerus.To identify a possible relationship between the ecological niche and secondary metabolite production profile, we studied the Streptomycete strains clustered into two different groups: one comprising the twelve CS strains and the other with the rest of the reference strains D. In theFrom the total of 390 BGCs detected in the CS strains, 155 were predicted as already known gene clusters (homology ≥ 85%), 33 presented a moderate homology  and 201 were putatively novel BGCs without significative homology to any BGC in the MIBiG repository . These dFrom the 155 known BGCs detected by antiSMASH in the 17 genomes used in this study, the production of 60 different already described compounds  could be inferred. Among them, 16 BGCs were shared by the CSs and the reference strains, 18 were exclusive of the reference strains and 26 were unique in the CS collection. Notably, most of the shared BGCs were classified as siderophores, terpenes, butyrolactones, ectoine and melanin, and the great majority of the exclusive BGCs fell into the PKS, NRPS and RiPP classes. This scenario is similar to that described by Murphy and co-workers , suggestIn order to complement the results obtained by antiSMASH prediction, we created a heatmap containing all BGCs that have a predicted homology over 85% to a known BGC and metabolites that had been detected by dereplication .Streptomyces strain, those being ectoine, hopene, desferrioxamine and geosmin, possibly due to their vital function in highly competitive soil environments and preventing osmotic and nutrient stresses  and isorenieratene (in CS131 and CS147 strains), both presumably involved in photoprotection. Surprisingly, Pseudonocardia sp. only shared the ectoine BGC with the rest of the strains, although it is phylogenetically related and shares the ecological niche with the CS isolates. Moreover, this ectoine BGC does not cluster together with the Streptomyces ones in the similarity network The fact that, based on bioinformatic analysis, only one of the 16 BGCs present across the With the aim of obtaining a more visual representation of shared and specie-specific BGCs, we generated a similarity network using the program BiG-SCAPE, which bound every BGC (represented by nodes) to others that have high similarity and to its corresponding MIBiG BGC entry . DiffereSurprisingly, six different node groups only included the same seven CS strains, CS014, CS057, CS065a, CS090a, CS131, CS147 and CS149. These groups of nodes corresponded to the BGCs of AmfS, griseobactin, RiPP-1, terpene-2, RiPP-2 and RiPP-3. These seven strains are close related according to the phylogenetic tree  which coStreptomyces strains. Ectoine and geosmin BGCs were also detected as present in all Streptomyces strains in the antiSMASH analysis previously reported in this work , which represented a total of 106 BGCs  should be used instead [Among the 42 known different metabolites potentially produced by the CS strains (according to antiSMASH), 21 were detected and identified by dereplication. The remaining 21 compounds that were not detected by dereplication were mainly classified as siderophores, terpenes, lanthipeptides, lasso peptides, lipopeptides, ectoine, butyrolactone and melanin. Our inability to detect these compounds could be due to two different reasons: (i) they were not being produced under our laboratory conditions or (ii) the extraction method using ethyl acetate is not suitable for this type of metabolites, and other solvents . NonactYP-A7193 , the nonYP-A7193  and bothYP-A7193 , we coulYP-A7193 ) and to YP-A7193 ). In-depYP-A7193 ,60. HomoS. clavuligerus)   and harbSometimes it is difficult to prioritize the best strains or BGCs candidates in new drug discovery. Here, we highlighted the importance of a careful bioinformatic study at the first stages of secondary metabolite studies based on genome mining. We proposed the combination of genome analysis by antiSMASH, the generation of a similarity network, phylogenetic analysis and sample dereplication as a useful approach to find the secondary metabolite wealth of our CS strain collection. We were able to predict known and unknown BGCs as well as the frequency of appearance in the genomes, and a way to identify which gene clusters could be the better candidates for new drug discovery. We were also able to identify two strains (CS081a and CS090a) that showed the best potential for novel bioactive compound discovery within the CS collection.