Salvia miltiorrhiza has numerous compounds with extensive clinical application. “Sweating”, a processing method of Traditional Chinese Medicine (TCM), results in great changes in pharmacology and pharmacodynamics. Previously, chromatogram of 10 characteristic metabolites in S. miltiorrhiza showed a significant difference after “Sweating”. Due to the complexity of TCM, changes in metabolites should be investigated metabolome-wide after “Sweating”. An untargeted UPLC/MS-based metabolomics was performed to discover metabolites profile variation of S. miltiorrhiza after “Sweating”. Multivariate analysis was conducted to screen differential metabolites. Analysis indicated distinct differences between sweated and non-sweated samples. 10,108 substance peaks had been detected altogether, and 4759 metabolites had been identified from negative and positive ion model. 287 differential metabolites were screened including 112 up-regulated and 175 down-regulated and they belong to lipids and lipid-like molecules, and phenylpropanoid and polyketides. KEGG analysis showed the pathway of linoleic acid metabolism, and glyoxylate and dicarboxylate metabolism were mainly enriched. 31 and 49 identified metabolites were exclusively detected in SSM and NSSM, respectively, which mainly belong to carboxylic acids and derivatives, polyketides and fatty acyls. By mapping tanshinones and salvianolic acids to 4759 identified metabolites library, 23 characteristic metabolites had been identified, among which 11 metabolites changed most. We conclude that “Sweating’’ has significant effect on metabolites content and composition of S. miltiorrhiza. Salvia miltiorrhiza Bunge, referred to as “Danshen” in China or “Tanshen” in Japan, which is an important species in Salvia genus with great medicinal values1, was first recorded in Shennong Bencao Jing, the earliest famous extant monograph of traditional Chinese pharmacology in China2. The dry roots and rhizomes of S. miltiorrhiza, called Danshen, have been extensively used in the clinical treatments of many different diseases, such as coronary heart disease3, hyperlipidemia4, cerebrovascular disease5, Alzheimer’s disease6, hypertension7, cancer8 and osteoporosis9.10. The bioactive components of Danshen are mainly divided into fat-soluble tanshinones belonging to diterpene and water-soluble caffeic acid-derived salvianolic acids12. Thus far, more than 40 tanshinones and over 50 salvianolic acids have been well isolated and identified from roots of S. miltiorrhiza. Tanshinones mainly include tanshinone I, tanshinone IIA (TsIIA), tanshinone IIB, cryptotanshinone and dihydrotanshinone I, and salvianolic acids mainly consist of salvianolic acid A , salvianolic acid B , lithospermic acid, danshensu, caffeic acid, and rosmarinic acid14.Because Danshen has a broad prospect of clinical application, lots of effort has been made to investigate the main constituents and bioactive ingredients of Danshen22. The initial processing of medicinal materials is from the harvest of medicinal parts to the formation of commercial medicinal materials, such as preliminary treatment and drying23. At present, initial processing methods mainly include slicing while the herbs are still fresh, drying and packing24. Medicinal materials are formed through the above-mentioned processing, during which physical and chemical changes take place, such as the removal of non-medicinal parts, the maximum retention of active substances, the reduction of toxic components, and the mutual transformation of chemical components23. The medicinal materials forming during initial processing are the raw materials of Chinese herbal slices. Their quality directly affects the quality of Chinese herbal slices, and then affects the clinical curative effect of TCM25.The initial processing of TCM is important and closely related to the quality of TCM’s productsSweating” is the main initial processing method for Danshen roughly as follows: the fresh roots of S. miltiorrhiza are piled up and covered to produce heat and emit moisture to the surface of the materials26. “Sweating” is beneficial not only to the drying of herbs, but also to regulate and promote the enzyme system and microbial community activity in herbs tissues, and accelerate the process of biotransformation and chemical transformation of primary and secondary metabolites, which directly affects the quality of medicinal materials27. Pharmacological activities of medicinal materials can be changed by “Sweating”. Some related pharmacodynamic studies have shown that the antioxidant activity28, brain protective effect, and myocardial protective effect of “Sweating” S. miltiorrhiza (SSM) are stronger than those of non-sweating S. miltiorrhiza (NSSM)29. Cocktail probed drug method has been used to study the effects of SSM and NSSM on CYP450 enzyme activity in rats. The result has shown that there are significant differences in effects of SSM and NSSM on CYP450 enzyme activity in rats28. The unique pharmacology and efficacy of SSM is a reflection of hundreds of bioactive compounds rather than a single or several ones. In addition, the various activities of components in Danshen are considered responsible for the therapeutic effects of Danshen30. Therefore, it is necessary to study the changes of the composition and content of chemical components in roots of S. miltiorrhiza after “Sweating”. However, due to the complexity of Traditional Chinese Medicine (TCM), it’s far from enough to be merely focused on several main bioactive components in TCM but ignore the whole changes of the metabolites31, and it’s difficult to comprehensively and accurately investigate the effect of “Sweating” on the chemical composition of Danshen by only determining some main components. Consequently, studies of the overall chemicals profile of “Sweating” on Danshen are necessary to better understand the mechanism of “Sweating”.“32. The LC–MS method is a useful tool to analyze the components of herbal medicine, which provides efficient visualization and identification of the metabolites33 It shows metabolite selectivity and sensitivity to targeted analysis, and detects low concentration compounds that may not be detected by other methods34. In this study, we used S. miltiorrhiza without “Sweating” (NSSM) and Danshen just after “Sweating” (SSM) as experimental materials. UPLC-MS-based metabolomics combined with multivariate statistical analysis was applied to reveal the metabolic profile differences between NSSM and SSM so as to investigate the effect of “Sweating” on the global metabolic profile of Danshen.Untargeted metabolomics is a valuable technique for comprehensive analysis and comparison of end-product metabolites in the process of cell regulatory processes, and their levels can be considered as the ultimate response of the biological system to changes in endogenous and/or exogenous factorsSweating” process without long-term storage leading to complex components loss, with which may result in non-unique difference. Additionally, we compared the changes of some main characteristic bioactive components in Danshen before and after “Sweating” to specifically reveal the effect of “Sweating”. The results gave a deeper insight into the mechanism behind “Sweating” of Danshen and a better explanation of the scientific connotation of “Sweating”.The difference of components between SSM and NSSM materials in this study merely comes from “Sweating” processing and identify critical metabolites responsible for metabolomics variation by “Sweating” processing.Methanol extracts from the roots of SSMs and NSSMs were measured by a UPLC system fitted with Q-Exactive quadrupole-Orbitrap mass spectrometer  to investigate the metabolic changes during the “+) and Fig. -), respectively. L-2-chlorophenylalanine (C9H10ClNO2) with RT (retention time) at 2.514 min and LPC (17: 0) (C25H52NO7P) with RT 6.430 min was added to the extracts as two internal standards . Metabolites in Danshen samples were identified according to internal standards and metabolomics databases of the Human Metabolome Database (HMDB), Lipidmaps (v 2.3) and METLIN by the software of Progenesis QI V2.3. A total of 10,108 substance peaks were detected by UPLC-MS in the samples of SSM and NSSM, among which 4759 metabolites were identified including 2241 metabolites from negative ion model (ESI-) and 2518 metabolites from positive ion mode (ESI+), respectively  of SSM and NSSM is shown in Fig. anine C9H0ClNO2 wiely Fig. .Figure 1Unsupervised principal components analysis (PCA) was carried out on the detected 10,108 substance peaks, QC samples and the two kinds of samples SSM and NSSM were classified into different metabolite profile with a significance of 0.05 by PCA, showing good stability and repeatability of the LC/MS detection Fig. a. In ord2 value of PCA and Q value of PLS-DA are shown in Fig. 2X and Q2  was also applied to investigate the metabolites difference between SSM and NSSM samples Fig. b. The caTo visualize the result of fold change analysis of 10,108 substance peaks, a volcano plot was drawn by transferring the fold change (FC) value of each substance peak to log2 (FC), and transferring the P value (P = 0.05) of Student’s t test to –log10. Among all of the differential metabolites, upregulated metabolites (SSM compared with NSSM) were symbolized with red dots gathered into a group and downregulated metabolites with blue dots gathered into a group Fig. a. As shoTo identify differential metabolites between samples, a fold change ≥ 1.6 (up-regulated) or ≤ 0.625 (down-regulated) in SSM compared to NSSM, and these metabolites were screened using a variable importance in projection (VIP) value (VIP ≥ 1.0) from the OPLS-DA model. A total of 287 differential metabolites had been identified from total 4759 identified metabolites between SSM and NSSM by VIP and FC value, containing 130 and 157 metabolites from the positive and negative model, respectively Fig. .Sweating” notably changed the metabolic profile of Danshen.Of these differential metabolites, 112 metabolites were up-regulated and 175 metabolites were down-regulated in SSM compared with NSSM. The majority of differential metabolites were lipids and lipid-like molecules, phenylpropanoid and polyketides, benzenoids, organic acids and derivatives, organic oxygen compounds, and there were 30 unclassified chemicals Fig. a. These 35. Research has shown that phenylpropanoid and polyketides possess anticarcinogenic, anti-inflammatory, neuroprotective, immunomodulatory, antihypertensive, and metabolic effects36. Benzenoids have the effect on anti-inflammatory37. All of these results indicate that changes in these differential metabolites are responsible for great change in Danshen’s pharmacological effects during the process of “Sweating”.Emerging literature has indicated that lipids and lipid-like molecules have less anti-inflammatory properties, higher antioxidant effects, immunomodulatory effect, and decrease the risk of cholestasis, and improve clinical outcomes of certain subgroups of patients38. Our result showed that 51 metabolites mapped to “metabolism” and they had KEGG ID. Subsequently, KEGG pathway enrichment analysis was conducted to identify differences in metabolic pathway between SSM and NSSM samples. 19 metabolites had been found enriched in 30 KEGG pathways  of each pathway, linoleic acid metabolism was the most enriched pathway, glyoxylate and dicarboxylate metabolism and phenylpropanoid biosynthesis were also the main enriched pathways  database, looking first at information about pathwaysays Fig. b.39. Linoleic acid help to protect liver safety in obese and overweight women and fight against mortality41. Study also found biomarkers of S. miltiorrhiza that can improve hemorheological disorder and vascular endothelial function of microcirculation dysfunction rats are mainly related to linoleic acid metabolism, glutathione metabolism, and glyoxylate and dicarboxylate metabolism etc. All of the research indicated that changes in the core metabolites and their corresponding pathways culminate in improvement on pharmacological function of Danshen after “Sweating”.Previous study indicated that linoleic acid and related metabolic pathways are associated with bone mineral densitySweating” was at 7.54 folds, while the content of psoromic acid was only about 5% compared to NSSM , as a plant hormone, was found to have the function to promote anti-inflammatory responses and therapeutic benefits+) and 12 from negative ion models (ESI-). 99 substance peaks were exclusively determined in NSSM with 49 metabolites identified, which contained 27 and 22 differential metabolites from ESI+ and ESI-, respectively  or a proteomic study should be performed to investigate the change in genes or enzymes of Danshen during “Sweating” processing to explain the change of metabolites content of SSM.Tanshinones and Salvianolic acids are the two kinds of characteristic bioactive compounds possessing magnificent bioactivities in Danshen. In this untargeted metabolomic study, a total of 23 specific compounds of Tanshinones and Salvianolic acids had been detected . Lmurine-2-chlorophenylalanine was obtained from Shanghai Hengchuang Bio-technology Co., Ltd. . LysoPC17: 0 was purchased from Avanti Company of the United States, and all of the chemicals and solvents were analytical or HPLC grade.S. miltiorrhiza was with all kinds of size collected, washed thoroughly and put in an open, clean, dry and cool place, so as to avoid overstocking during the process of natural water loss, preventing heat production. Then, reed heads were removed from the roots and picked over ones with uniform thickness and randomly divided them into two groups. One group (SSM) was processed by “Sweating”, and the other group (NSSM) was unprocessed and directly stored at – 80 ℃. The steps of “Sweating” processing which are screened by modifying report from Liang et al. are as follows46: More than 100 kg fresh roots with different sizes were piled up for seven days and covered with a sack to produce heat so as to make the internal moisture of root tissues spread outward in an open place. In order to make the whole roots sweat evenly, the roots were spread out for a night and then covered with sack every two days. When the internal sections of the roots turned purplish red, end “Sweating” and gather them up. Six independent sample pairs were collected from two groups and each sample pair of the same size were ground into powder after being lyophilized in a speed vacuum and sieving with 50 order mesh were stored at − 80 ℃.The same batch of The powdery samples (80 mg) after being precisely weighed were transferred to 1.5 mL Eppendorf tubes, and two small steel balls were added to each tube. Two internal standards 17:0 Lyso PC (1-heptadecanoyl-sn-glycero-3-phosphocholine), 0.01 mg/mL; (20 μL each) , 0.3 mg/ mL, both of them were prepared with methanol and 1 mL mixture of methanol and water  were added to each sample, and the samples were pre-cooled at − 20 ℃ for 2 min. Then the samples were ground at 60 Hz for 2 min, and stood under ultrasonication at ambient temperature for 30 min, and placed at − 20 ℃ in refrigerator for 20 min. Samples were centrifuged at a speed of 13,000 rpm, 4 °C for 10 min. 300 μL of supernatant in a brown glass vial was dried in a freeze concentration centrifugal dryer, and then added 400 μL mixture of methanol and water  to each sample, vortex for 30 s, and ultrasonicated for 2 min. Samples were centrifuged again at 13,000 rpm, 4 ℃ for 10 min. The supernatants (about 150 μL) from each tube were collected using crystal syringes, filtered through microfilters at 0.22 μm and transferred to LC vials, and stored at − 80 °C for LC–MS analysis. QC samples were prepared by mixing aliquots of 6 SSM and 6 NSSM to be a pooled sample, and used to balance the chromatography-mass spectrometer system.47 source  was used to analyze the metabolic profiling in both ESI positive and ESI negative ion modes. An ACQUITY UPLC BEH C18 column  was employed in detecting both positive and negative modes. The binary gradient elution system consisted of (A) 0.1% (v/v) formic acid dissolved in water and (B) 0.1% formic acid (v/v) dissolved in acetonitrile and separation was achieved using the following gradient: 0 min, 5% B; 1.5 min, 5% B; 3 min, 30% B; 7 min, 60% B; 9 min, 90% B; 11 min, 100% B; 13 min, 100% B; 13.2 min, 5% B; 16 min, 5% B. The flow rate was at 0.35 mL/min and column temperature was at 40 ℃. All the samples were kept at 4 ℃ during the detection, the injection volume was at 5 μL each time, and the mass range was from m/z 100 to 1000. The resolution was set at 70,000 for the full MS scans and 17,500 for HCD MS/MS scans, and the Collision energy was set at 10, 20 and 40 eV. The mass spectrometer operated as follows: spray voltage, 3500 V at negative and positive mode; capillary temperature, 320 ℃; sheath gas flow rate, 40 arbitrary units; auxiliary gas flow rate, 10 arbitrary units.A Dionex Ultimate 3000 RS UHPLC system fitted with Q-Exactive quadrupole-Orbitrap mass spectrometer equipped with heated electro-spray ionizationThe QCs were injected at regular intervals (every 6 samples) throughout the analytical run to provide a set of data from which repeatability can be assessed, and were used to evaluate the stability of the mass spectrometer system during samples detection.48.The acquired LC–MS raw data were filtered by the software of Progenesis QI v2.3  through steps of baseline filtering, peak recognition, integration, retention time correction, peak alignment and normalization, and using the following parameters. Precursor tolerance was set at 5 ppm, product tolerance was set at 10 ppm, product ion threshold was set at 5%, and retention time (RT) tolerance was set at 0.02 min. Internal standard detection parameters were deselected for peak RT alignment, isotopic peaks were excluded for analysis, and noise elimination level was set at 10.00, minimum intensity was set to 15% of base peak intensity. The Excel file was obtained with three dimensions data sets including m/z (mass-to-charge ratio), peak RT (retention time) and peak intensities, and RT–m/z pairs were used as the identifier for each ion. The internal standard was used for data QC (reproducibility)https://www.nonlinear.com/progenesis/qi/)49 based on precise mass number, secondary fragments and isotope distribution, and qualitative by mapping on the Human Metabolome Database (HMDB), Lipidmaps (v 2.3) and METLIN database50.Metabolites were identified by the Progenesis QI Data Processing Software  ranks the overall contribution of each variable to the OPLS-DA model, and those variables with VIP ≥ 1.0 are considered relevant for group discrimination52.For the extracted data, delete the ion peaks whose missing values (ion intensity  = 0) is more than 50% in the group, and replace the 0 value with the half of the minimum value, and screen the qualitative compounds according to the score of the qualitative results of the compounds. The screening standard is 30 points (Full score 60 points), and if the score is lower than 30 points, the qualitative results are deemed to be inaccurate and deleted. Finally, the positive and negative data were combined to get a combined data matrix table which contained all the information extracted from the original data, and was imported into R ropls package. Principle component analysis (PCA) and  partial least-squares-discriminant analysis (O)PLS-DA were carried out to visualize the metabolic alterations between sweating and non-sweating groups, after mean centering (Ctr) and Pareto variance48.In this study, the default 7-round cross-validation and 200 times of response permutation testing (RPT) was applied with 1/seventh of the samples being excluded from the mathematical model in each round, in order to review the model quality and guard against over-fitting. The differential metabolites were selected on the basis of the combination of a statistically significant threshold of variable influence on projection (VIP) values obtained from the OPLS-DA model, P values from a two-tailed Student’s t test on the normalized peak areas and the adjusting P value for correcting was obtained by Benjamini & Hochberg method, and fold change (FC) value SSM verse NSSM, where metabolites with VIP values ≥ 1.0 and p values < 0.05 were considered as differential metabolites. While calculating FC values, if the datum displaying the content of metabolite is zero, it would be changed by the half of the minimal corresponding value in the table of data matrix (2.5E-06)53.The experimental data obtained in the present study is expressed as the mean ± standard deviation (SD) of six independent biological replicates, and the significance level of the metabolites between groups was calculated by one-way analysis of variancewith Student’s t test and fold change analysis using GraphPad Prism 7.00 softwareThe volcano plot was used to visualize the P value from student’s t test and the fold change (FC) value, which is conducive to screening differential metabolites, as shown in the figure with three different colors  to represent significantly up-regulated, down-regulated and non-significant differential metabolites in the experimental group.P-value) was used to measure whether the pathway enriched or not, and the pathway was considered as enriched pathway when P-value ≤ 0.0550.Mapping the differential metabolites to KEGG data to look for their KEGG ID and the pathway they belonged to, then counting the number of metabolites enriched in corresponding pathway. The -lg(Figure 1 Hierarchical Clustering of Expression Quantity of Identified Metabolites.Table 1 Data matrix of metabolomic analysis.Table 2 Differential metabolites between SSM and NSSM.Table 3 KEGG pathway enrichment data.Table 4 Top 50 differential metabolites from metabolomic analysis.Table 5 Extreme variant substance peaks and metabolites.Table 6 Specific Metabolites of tanshinones and salvianolic acids.


Cautionary pooled efficacy estimates from the studies that clearly reported efficacy data showed clinical resolution or improvement in 77.5% (n = 111) of burn wound infections, 86.1% (n = 310) of chronic wound/ulcer infections and 94.14% (n = 734) of dermatological infections. Over half of the reports that commented on safety (n = 8/15), all published in or after 2002, did not express safety concerns. Seven early reports (1929–1987), described adverse effects consistent with the administration of raw phage lysate and co-administered bacterial debris or broth. This review strongly suggests that the use of purified phage to treat superficial bacterial infections can be highly effective and, by various routes of administration, is safe and without adverse effects.Superficial bacterial infections, such as dermatological, burn wound and chronic wound/ulcer infections, place great human and financial burdens on health systems globally and are often complicated by antibiotic resistance. Bacteriophage (phage) therapy is a promising alternative antimicrobial strategy with a 100-year history of successful application. Here, we report a systematic review of the safety and efficacy of phage therapy for the treatment of superficial bacterial infections. Three electronic databases were systematically searched for articles that reported primary data about human phage therapy for dermatological, burn wound or chronic wound/ulcer infections secondary to commonly causative bacteria. Two authors independently assessed study eligibility and performed data extraction. Of the 27 eligible reports, eight contained data on burn wound infection ( The antibiotic resistance crisis threatens to return medicine to the pre-antibiotic era and is predicted to cost up to $100 trillion and lead to an annual 10 million premature deaths by 2050 [Bacteriophage (phage) are viruses of bacteria, independently discovered in 1915 and 1917 . The speAcinetobacter infection in the United States (US) using intravenous and intraperitoneal phage [Phage therapy is experiencing a renaissance in the geopolitical West, catalysed by the high-profile successful treatment of a patient in 2017 with a systemic multi-drug resistant al phage . Subsequal phage ,9. Alongal phage ,11,12. Ial phage ,12,13. Bal phage .Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus spp. and Escherichia coli as the most frequent pathogens, with S. aureus predominating in all geographical regions analysed [S. aureus (MRSA) and vancomycin-resistant Enterococcus [Pseudomonas spp., Acinetobacter spp. or S. aureus [S. aureus, Propionibacterium and Streptococcus spp. causing, among other pathologies, common skin infections such as acne, folliculitis, cellulitis and impetigo [Enterobacter, Proteus, Klebsiella, Aeromonas, Corynebacterium and Clostridium [The American Academy of Dermatology estimates that in 2013, 17.4 million Americans received treatment for burns, ulcers or wounds; while 40.8 million sought treatment for any form of skin infection. The cost of treating skin infections was estimated to be $8.1 billion, while the treatment of burns and wounds cost approximately $6.4 billion and ulcers approximately $5.4 billion ,16. Thesanalysed . Common rococcus ,20,21. S. aureus ,23. Impoimpetigo ,25,26,27stridium ,29.The safety and efficacy of intravenous phage therapy has been reviewed elsewhere , however® Epub Ahead of Print, In-Process & Other Non-Indexed Citations, Ovid MEDLINE® Daily, Ovid MEDLINE and Versions® (1946–2019) and Web of Science. The Web of Science Core Collection Citation Indexes searched were: Science Citation Index Expanded (1900–2019), Book Citation Index—Science (2005–2019) and the Emerging Sources Citation Index (2015–2019). The search was performed using the following terms: (“Bacteriophage*” OR “Phage*”) AND (“Acinetobact*” OR “Aeromonas” OR “Clostrid*” OR “Corynebact*” OR “Diptheroid*” OR “Enterobact*”OR “Enterococ*”OR “Escherichia*” OR “Klebsiella” OR “Propionibacterium” OR “Proteus” OR “Pseudomonas” OR “Staphylococ*” OR “Streptococ*”) AND (“Acne” OR “Boil” OR “Burn*” OR “Carbuncle*” OR “Cellulitis” OR “Cutaneous abscess” OR “Derm*” OR “Dermatitis” OR “Eczema” OR “Erysipelas” OR “Folliculitis” OR “Furuncle*” OR “Hidradenitis” OR “Impetigo” OR “Infect*” OR “Pyoderma” OR “Skin” “Ulcer” OR “Wound”) AND . In Ovid these terms were followed by the suffix ‘.mp.’ and they were searched as topics on the Web of Science platform. This systematic strategy was supplemented by hand searching of sources not widely available or indexed online [Three electronic databases were searched, without limits, for articles published up to October 2019: EMBASE (1980–2019), Ovid MEDLINEd online ,32,33, rd online ,35,36,37d online . A protoStaphylococcus, Pseudomonas, Streptococcus, Enterococcus, Escherichia, Aeromonas, Acinetobacter, Corynebacterium, Clostridium, Enterobacter, Proteus, Klebsiella or Propionibacterium; (B) they were published in the English language. Chronic wounds/ulcers complicated by osteomyelitis were included, but cases of osteomyelitis secondary to other causes  were excluded. Studies in which the clinical condition was stated but the infectious agent was not specified were assumed to be caused by one of the major pathogens examined in this study and therefore included. There were only three such studies, none of which were considered to report conditions unlikely to be caused by the bacterial genera in the search strategy [All studies underwent title and abstract screening. Eligible studies met the following criteria: (A) they contained primary data pertaining to human clinical phage therapy by any route of administration for one or more superficial bacterial infections  caused by bacteria of the genera strategy ,39,40. SDeduplication was performed using Endnote (version X8.0.1). Title and abstract screening was performed independently by two authors , with discrepancies resolved by a third author (JDJ). Full-text screening was performed independently by two authors , with discrepancies resolved by a third author (HJS). This review was conducted in accordance with the PRISMA  guidelines  and a PRn = 41) were added to the total patients with clinical resolution on the basis that recurrence, by definition, implies a period of clinical resolution. Slopek and colleagues reported three decubitus ulcer patients as having had ‘transient improvement’ [The following information was extracted from each study: author(s); date of publication; study location; study type; number of relevant reports; condition microbiology; clinical condition, and where possible patient age(s) and/or previous treatment(s); details of the phage treatment; treatment schedule and route(s), including details of other ongoing therapies  where reported; treatment efficacy; where possible, the numbers of patients ‘cured’, ‘improved’ or for which there was ‘no response’; comments or data regarding safety and adverse effects. The term ‘cured’ is used in ovement’ . These povement’ , with diSystematic searching yielded 9983 records published between 1929 and 2019. Eleven additional records were identified from other sources; three from a review article , seven fn = 8), Georgia (n = 7), Poland (n = 2), Russia (n = 3), India (n = 2), Belgium (n = 1), Egypt (n = 1), France/Belgium (n = 1), Belarus (n = 1) and the UK (n = 1). There were two clinical trials, four case reports and 21 case series. Two of the 21 case series were reported as ‘prospective’ studies; however, it was deemed that these better suited established definitions of case series [n = 24/27) studies reported microbiological data. This represented the application of phage therapy against nine of the 15 bacterial genera or species in the systematic search strategy , the US (n = 1), Belgium (n = 1), France and Belgium (n = 1), Georgia (n = 1), Russia (n = 1), Egypt (n = 1) and the United Kingdom (n = 1). Testing of the in vitro efficacy of phage against a patient’s bacterial isolate (phage sensitivity testing) was reported or implied for six of eight reports. Phage administration routes were oral (n = 1), topical (n = 6) or oral and local (n = 1). Topical administration included direct application (n = 3), the use of dressings soaked with phage solution (n = 2) or a biodegradable phage-containing bandage (n = 1). Prior neutralization of stomach acid was reported in one of two reports of oral phage administration. The phage treatments were unpurified phage lysates (n = 1), phage cocktails (n = 3), a phage-containing biodegradable bandage (n = 1) or unclear (n = 3).Eight records reported data regarding 156 patients with burn wound infections , of whicA precise efficacy estimate cannot be derived, as co-administered therapies, phages and reporting timepoints and methodology differed between all records. Notwithstanding these caveats, and given that most infections reported were refractory to antibiotics, a crude and cautionary estimate of efficacy can be derived, partially excluding one report in which outcome data were unclear for 45/54 patients . The remn = 5/8) studies did not comment on safety or adverse effects. Of the three studies that did, representing 20 patients, none reported adverse effects.Most (n = 5), Poland (n = 2), Russia (n = 2), India (n = 2) and Georgia (n = 1). The chronic wounds/ulcers treated were venous ulcers , diabetic infections , decubitus ulcers , non-specific chronic non-healing wounds  and community-acquired MRSA . Phage sensitivity testing was reported or implied for five of 12 studies. The routes of phage administration were topical (n = 5), oral and local (n = 2), local injection/instillation (n = 2), local injection or topical (n = 1) or unclear (n = 2). Topical administration represented the use of dressings soaked with phage solution (n = 3), direct application (n = 1) or a biodegradable phage-containing bandage (n = 1). Where used, oral administration was preceded by neutralization of stomach acid. One report documented wound rinsing with 4% sodium bicarbonate prior to the application of a phage-containing bandage to venous stasis ulcers. The phage treatments used were unpurified phage lysate (n = 2), monovalent phage suspension (n = 3), phage cocktails (n = 2), monovalent or cocktail based (n = 2), a phage-containing biodegradable bandage (n = 1) or unclear (n = 2).Twelve records reported data regarding 327 patients with chronic wound/ulcer infections . There wAs for burn wound infections, a precise efficacy estimate could not be derived from these studies. Nevertheless, notwithstanding the aforementioned caveats, and given that most infections reported were refractory to antibiotics, a cautionary crude estimate of efficacy can be derived, excluding one report in which outcome data for all 17 patients was unclear . The remn = 7/12) studies did not comment on safety or adverse effects. Of the five that did, representing 141 patients, four reported no safety issues or adverse effects. Slopek and colleagues 1987 was the last in a series of papers, in which an earlier manuscript commented on the ‘rather rare’ adverse effects [Most , with other reports from the US (n = 3), Poland (n = 1) and Belarus (n = 1). The infections treated were furunculosis , hidradenitis , acne , impetigo , carbunculosis , ‘strepto-staphylococcal epidermitis’ , abscesses , sycosis vulgaris , folliculitis , dermatitis , ecthyma , cellulitis , pyoderma , paronychia  and ‘various skin diseases’ . Phage sensitivity testing was reported or implied for 4 of 10 studies. The routes of administration were oral and local (n = 2), subcutaneous or local (n = 1) or subcutaneous injection alone (n = 2) or supplemented by intranasal administration (n = 1) or local application (n = 4). Oral administration was preceded by neutralization of stomach acid in all cases. The phage treatments used were unpurified phage lysate (n = 3), monovalent and/or cocktail based (n = 2) or unclear (n = 5).Ten records reported data regarding 1096 patients with various bacterial skin infections , of whicAs before, a cautionary crude estimate of efficacy was derived, excluding two reports where outcome data for all 308 patients were unclear ,46 and 1Staphylococcal phage lysate. Of the 149 patients for whom adverse effects were recorded, 28.8% had no adverse effects, 34.6% had mild localised erythema and soreness, 7.2% had generalized responses , 1% had an undefined ‘severe’ reaction and no data were available for the remaining 28.4% [Eight of 10 studies, representing 934 patients, commented on safety or adverse effects. Of these, one report from 2017 did not report any adverse effects or clinical or laboratory abnormalities. The remaining seven studies, dated 1929 to 1987, identified adverse effects. The earliest record, published 1929, reported that ‘extremely mild’ adverse effects were observed in the ‘majority’ of patients that received injected subcutaneous raw ng 28.4% . One yeang 28.4% . The thing 28.4% . Later Bng 28.4% . Similarng 28.4% . There wng 28.4% . ‘No sysng 28.4% .This systematic review presents the available English language clinical data about the use of phage therapy for the treatment of superficial bacterial infections, specifically burn, chronic wound/ulcer or dermatological infection. A total of 27 relevant sources representing 1688 patients were identified, stretching back over 90 years. It is important to appreciate that this greatly underestimates the global extent to which phage therapy has been used. While phage therapy was disregarded by Western medicine, Russia and other Eastern European countries, particularly Georgia, continued to employ phage therapy , and todn = 310) and dermatological infection , although efficacy was not as high among burn wound infections . It should be noted that many of the patients in these studies were also receiving antibiotic therapy, although in most cases patients’ infections were deemed antibiotic resistant. The comparatively lower level of clinical resolution among burn wound infection patients (49.6%), reflects data from three studies. One study only reported degrees of improvement and did not document whether any patients had achieved clinical resolution (n = 30). This was despite reporting that post-phage therapy 60% of patients’ wounds were culture negative, wound discharge had stopped for 40% of patients and phage therapy had had a ‘good’ or ‘excellent’ effect on the take of skin grafts for 60% of patients [6 plaque forming units (PFU)/mL to just 102 PFU/mL [6 and 108 PFU/mL. The other study was not, by the author’s admission, intended to evaluate efficacy and faced significant delays between patient identification and treatment [n = 65).The studies identified in this review demonstrate that when used appropriately phage therapy should generally be considered highly effective. Although precise efficacy estimates could not be derived because no reports were directly comparable, cautionary crude efficacy estimates revealed that clinical resolution or improvement was achieved in most cases of chronic wound/ulcer treatment , including a US Food and Drug Administration approved phase I safety trial. This is consistent with the absence of adverse effects from other recent applications of phage therapy for other indications, including intravenous use [Surprisingly, 12 of the 27 studies identified by this review did not comment on safety or adverse effects. This likely reflects the inherent tendency of scientific reports to only document positive findings, with the authors not deeming the absence of adverse effects remarkable. As phage therapy seeks to regain traction it will be vital that all future reports comment on adverse effects, even if absent. Of the 15 studies that commented on adverse effects, none of the eight studies published after 2002 reported adverse effects (nous use , and liknous use . This renous use ,47, one nous use ,43,45, anous use , likely nous use . The autnous use . This hanous use . Neverthnous use ,76,77, wThe reliance on English language sources available on the internet is an insurmountable limitation of this review. Inaccessibility was the most common reason that manuscripts were excluded during the full text screen, typically reflecting manuscripts published in volumes that were unavailable online or concealed by a paywall. Had all 26 such manuscripts been available in full, nine would have only been available in a foreign language. It was for this reason that the authors sought to supplement the data included in this review by including primary clinical data from secondary sources. This reflects the unique importance for any English language literature review of phage therapy to engage with less accessible sources. As with any systematic review, the quality of the data extracted is dependent upon the quality of primary reporting. Critical appraisal of the manuscripts included in this review revealed a variety of shortcomings, from not commenting on the presence or absence of adverse effects to inconsistencies with patient numbers reported or a lack of clarity about the condition or clinical microbiology of patients. Systematic reviews are also inherently limited by whether relevant studies contain key search terms in the fields searched and are available or appropriately indexed in the databases used. As renewed interest in phage therapy grows, it is important that all reports of phage therapy present their findings clearly and fully, especially regarding adverse effects and that, where possible, reports are published in indexed open-access journals.In summary, this review has collated English-language evidence to support the view that phage therapy for superficial bacterial infections should generally be considered both safe and effective. We hope this will encourage the development of complimentary reviews of phage data currently not available in English. Phage therapy for such infections has a long history and offers a potentially cheap, easy-to-use adjunct or alternative to antibiotics. Aside from reducing the morbidity and mortality of these infections, the use of phage also has the potential to reduce the amount of antibiotics prescribed for such infections, some of which often require multiple courses of antibiotics. Moreover, with the potential to generate bespoke formulations, phage therapy can offer, by definition, ‘personalised infection medicine’.This article does not contain any studies with human participants or animals performed by any of the authors.


Small cell lung cancer (SCLC) is a fatal malignant tumor with a poor prognosis for patients who relapse after first-line treatment. There are few effective treatments for SCLC patients with relapse. Elucidation of the molecular network related to treatment resistance is an important issue for this disease. In this study, the molecular signature of SCLC specimens after treatment failure was generated. Several pathways, e.g., “cell cycle”, “homologous recombination”, “DNA replication”, and “p53 signaling” were identified as the enriched pathways in this signature. Aberrant expression of MCM2, MCM4, MCM6, and MCM7 were detected in SCLC clinical specimens after treatment failure. This signature contains molecules involved in treatment resistance and will contribute to the study of SCLC molecular pathogenesis.MCM) 2, MCM4, MCM6, and MCM7. The overexpression of these MCM genes was confirmed in SCLC clinical specimens. Knockdown assays using siRNAs targeting each of these four MCM genes showed significant attenuation of cancer cell proliferation. Moreover, siRNA-mediated knockdown of each MCM gene enhanced the cisplatin sensitivity of SCLC cells. Our SCLC molecular signature based on SCLC clinical specimens after treatment failure will provide useful information to identify novel molecular targets for this disease.Small cell lung cancer (SCLC) is a highly aggressive cancer, and patients who become refractory to first-line treatment have a poor prognosis. The development of effective treatment regimens is urgently needed. In this study, we identified a gene expression signature of SCLC after treatment failure using SCLC clinical specimens (GEO accession number: GSE162102). A total of 1,136 genes were significantly upregulated in SCLC tissues. These upregulated genes were subjected to KEGG pathway analysis, and “cell cycle”, “Fanconi anemia”, “alcoholism”, “systemic lupus erythematosus”, “oocyte meiosis”, “homologous recombination”, “DNA replication”, and “p53 signaling” were identified as the enriched pathways among the genes. We focused on the cell cycle pathway and investigated the clinical significance of four genes associated with this pathway: minichromosome maintenance ( Lung cancer is the one of the most malignant cancers, with high morbidity and mortality rates  .Platinum-based chemotherapy is used as the initial treatment for patients with ED-SCLC, and it has improved survival . A cliniS1PR1, MAD2L1, CDKN2A, STIL, NDC80, NCAPG, PAD51AP1, TTK, PRM2, EZH2, PRC1, UBE2C, RFC4, CENPF, TCP2A, HMGB3, TYMS, SOX4, MCM2, and SMC4) [SCLC is rarely treated surgically and obtaining tumor samples suitable for genome-based studies is especially difficult for ED-SCLC. Regardless, several genome-based studies have been conducted so far to elucidate the malignant phenotype of SCLC cells ,9. In rend SMC4) .TP53 and RB1 are the most frequently mutated genes in SCLC [RB1 plays a pivotal role in tumor progression and the subsequent mutation burden of SCLC [SOX2, FGFR1, LRP1B, KIAA1211, and PTEN) [PI3K/AKT/mTOR pathway  in SCLC [PI3K/AKT/mTOR pathway are strongly associated with poor survival of patients with ED-SCLC [PI3K/AKT/mTOR-mediated signaling enhances SCLC tumorigenesis, and that the involved genes are promising therapeutic targets for SCLC.Several previous studies have performed whole genome analyses using SCLC clinical specimens ,17,18,19 in SCLC ,15,16,18nd PTEN) ,14,16,17In this study, we identified a SCLC expression signature using clinical specimens obtained from SCLC patients after treatment failure. Normal tissues, primary lesions, and metastatic lesions were collected from three patients (autopsy specimens), and comprehensive gene expression analyses were performed. Our mRNA expression signature data was deposited in the GEO (Gene Expression Omnibus: GSE 162102) database and is accessible.MCM) genes , which are involved in the cell cycle and cell replication. The replisome is a complex molecular machinery that are essential for DNA replication and are therapeutic target for human cancers. The MCM-family are major component proteins of DNA replisome [MCM genes contributed to an aggressive nature in SCLC cells. Furthermore, siRNA-mediated knockdown of MCM genes enhanced the cisplatin sensitivity of SCLC cells. There have been few reports of gene expression profile in treatment failure of SCLC clinical specimens. This signature contains molecules involved in treatment resistance and will contribute to the study of SCLC molecular pathogenesis.To discover genes involved in the aggressive nature and drug resistance of SCLC cells, we focused on genes that are highly expressed in cancer cells. The highly expressed genes were found to be enriched in “cell cycle”, “Fanconi anemia”, “alcoholism”, “systemic lupus erythematosus”, “oocyte meiosis”, “homologous recombination”, “DNA replication”, and “p53 signaling” pathways. Next, we performed functional analyses focusing on four minichromosome maintenance  and the Japanese Cancer Research Resources Bank , respectively.® Universal Elite ABC Kit  using SCLC tissues obtained by autopsy. The expression of synaptophysin, chromogranin A and MCM family members  in normal lung tissue was obtained from the Human Protein Atlas ). In western blotting, cell lysates were prepared using a RIPA buffer . A total of 20 µg protein was separated on SuperSep TMAce   and transferred onto polyvinylidene fluoride membranes . The procedures have been described previously [Immunostaining was performed with a VECTASTAINeviously ,23,24,25In this study, we extracted total RNA from SCLC autopsy specimens and evaluated gene expression using Agilent SurePrint G3 Human GE v3 8x60K microarrays. The raw microarray data were registered in GEO (GSE 162102). Our selection strategy is presented in TM Plus DNA Reagent Kit  according to the manufacturer’s protocol. Apoptotic cells were detected using a PE Active Caspase-3 Apoptosis Kit (BD Biosciences). These SCLC cells were analyzed using a flow cytometer . BD FACSDiva Software  was used to examine the flow cytometry data. The procedures used for transfecting siRNAs and for the functional assays  were described in our previous studies [The cell proliferation assay was determined by XTT assays using a Cell Proliferation Kit . In the cell cycle assay, SCLC cells were treated with BD Cycletest studies ,23,24,25TM RT Master Mix . Subsequently, we evaluated the expression of the gene by TaqMan Real-Time PCR Assays. qRT-PCR reactions were run using the StepOnePlus Real-Time PCR System . TaqMan probes and primers are shown in The methods used for RNA extraction from clinical specimens and cell lines and for qRT-PCR have been described previously ,23,24,25MCM2, MCM4, MCM6, or MCM7 knockdown on cisplatin sensitivity, SCLC cell lines were seeded at 4000/well in a 96-well plate. The following day, cisplatin  was administered for 72 h with or without transfection of the MCM-targeting siRNAs. Cell viability was assessed by XTT assay, and the IC50 of cisplatin was determined.To determine the effect of cisplatin combined with Statistical analyses were performed using GraphPad Prism 7  and JMP Pro 14 . One-way analysis of variance (ANOVA) and Tukey’s post-hoc test were used for multiple group comparisons.In this study, we obtained autopsy specimens from three patients who died of drug-resistant metastatic SCLC. The characteristics of the patients and their clinical specimens are shown in To confirm the histological type, the autopsy specimens were stained with hematoxylin and eosin and subjected to immunostaining of synaptophysin, chromogranin A, and CD56, which are specific markers of neuroendocrine tumors including SCLC .https://genecodis.genyo.es/ (accessed on 4 December 2020)), revealing that these genes are associated with the following pathways, “cell cycle”, “Fanconi anemia”, “alcoholism”, “systemic lupus erythematosus”, “oocyte meiosis”, “homologous recombination”, “DNA replication”, and “p53 signaling”  were extracted. Subsequently, KEGG pathway analysis was performed using the GeneCodis program , which are involved in the cell cycle pathway  .MCM2, MCM4, MCM6, and MCM7, we performed siRNA-mediated knockdown assays in SBC-3 and H82 cells. RT-PCR and Western blotting showed that expression levels of both mRNA and protein were markedly reduced by both siRNAs , which were identified as having roles in the “cell cycle” and “DNA replication” pathways. Cancer cells exhibit dysregulation of the cell cycle, leading to uncontrollable cell proliferation. Therefore, aberrantly expressed genes involved in the cell cycle and DNA replication are potential therapeutic targets. The replisome is a complex molecular machinery that closely contributes to DNA replication. Notably, some of the genes that make up replisome were upregulated in treatment failure patients with SCLC, e.g., cell division cycle 7 (CDC7), cell division cycle 45 (CDC45), MCM2, MCM4, MCM6, and MCM7. Control of replisome is an important concept in cancer treatment. The MCM complex is a eukaryotic replicative helicase that functions as a molecular motor unwinding duplex DNA to generate single strands of DNA templates for replication [Our present study based on KEGG pathway analysis showed that the genes upregulated in SCLC tissues were associated with the “cell cycle”, “Fanconi anemia”, “alcoholism”, “systemic lupus erythematosus”, “oocyte meiosis”, “homologous recombination”, “DNA replication”, and “p53 signaling” pathways. Notably, a previous study of differentially gene expression analysis of SCLC by using GEO datasets (GSE6044 and GSE11969) showed that DNA replication pathway including patients . These alication . The MCMlication . Severallication . It has lication ,28,29,30MCM4 attenuated the aggressiveness of lung adenocarcinoma cells [MCM2, MCM4, MCM6, and MCM7 enhanced the cisplatin sensitivity of SCLC cells. Proteins of the replisome complex, including MCM proteins, are target molecules for SCLC treatment.In cancer cells, the aberrant expression of MCM family members has been reported in a wide range of cancers, and cancer cell malignant phenotypes can be suppressed by knockdown of these genes ,32,33,34ma cells . In thisMCM2 [CDC7) was overexpressed in SCLC tissues after treatment failure. CDC7 is a crucial kinase required for initiating the replication machinery [Ciprofloxacin is a broad-spectrum antibiotic that binds to DNA gyrases and exhibits antibacterial activity by inhibiting DNA replication. A previous study showed that ciprofloxacin at low concentrations blocked the DNA helicase activity of the MCM complex . Based oMCM2 . MoreoveMCM2 . Our expachinery , and itsachinery ,38,39. Iachinery ,41. In mIn this study, we created a molecular signature of patients with SCLC who became treatment failure. This signature contains a number of molecules that may control treatment resistance. Based on this signature, the understanding of molecular pathogenesis for treatment-resistant SCLC will be accelerated.MCM2, MCM4, MCM6, MCM7, CDC45, and CDC7, were identified as upregulated in the SCLC tissues and may be potential therapeutic targets for treatment-resistant SCLC cells. The treatment-failure signature for SCLC derived from this study will contribute to elucidating the molecular pathogenesis of cancer cells that have acquired treatment resistance.In conclusion, we successfully identified an expression signature of SCLC after treatment failure using clinical specimens. Among the genes upregulated in SCLC tissues compared with normal tissues, “cell cycle” and “DNA replication” were identified as enriched pathways. DNA replisome-related genes,


Qishen Granules on Heart Failure Rats via Hippo Pathway” [In the article titled “Antiapoptosis and Antifibrosis Effects of Pathway” , there wMeasurement of Serum Biochemical Markers:At the end of the cardiac function examination, rats were anesthetized with 1% pentobarbital sodium (50 mg/kg) by intraperitoneal injection, and blood samples were collected from the abdominal aorta and centrifuged at 1000 ×g for 20 min to obtain serum.Serum levels of ALD and PIIINP were measured by radioimmunoassay. BNP, ANP, MMP-2, and MMP-9 were detected by enzyme-linked immunosorbent assay following the instructions of kits .


Scientific Reports 10.1038/s41598-020-76806-8, published online 16 November 2020Correction to: The original version of this Article contained typographical errors.In the Introduction section,23.”“In humans, the dominant face-specific brain response peaks approximately 170 ms after the stimulus onset for review,now reads:23.”“In humans, the dominant face-specific brain response peaks approximately 170 ms after the stimulus onsetIn the Discussion section, under the heading ‘Classification of faces and non-faces’,23.”“This time window both precedes and coincides with the face-specific response in humans around 170 ms for review, seenow reads:23.”“This time window both precedes and coincides with the face-specific response in humans around 170 msAlso in the Discussion section, under the heading ‘Threat and emotional information of facial expressions’,37.”“In rodent studies, the early responsiveness of the amygdala has been characterized in the classical studies of conditioned fear for review, seenow reads:37.”“In rodent studies, the early responsiveness of the amygdala has been characterized in the classical studies of conditioned fearAnd,38,39.”“This phenomenon is well known in both human and non-human animals for reviews, seenow reads:38,39.”“This phenomenon is well known in both human and non-human animalsAnd,38.”“In mammals, the lateral amygdala-nucleus is associated with fear conditioning, and the basolateral and basomedial nuclei are associated with anxiety and fear-related freezing for review, seenow reads:38.”“In mammals, the lateral amygdala-nucleus is associated with fear conditioning, and the basolateral and basomedial nuclei are associated with anxiety and fear-related freezingThese errors have now been corrected in the PDF and HTML versions of the Article.


The tumour suppressor p53 is essential for maintaining DNA integrity, and plays a major role in cellular senescence and aging. Understanding the mechanisms that contribute to p53 dysfunction can uncover novel possibilities for improving cancer therapies and diagnosis, as well as cognitive decline associated with aging. In recent years, the complexity of p53 signalling has become increasingly apparent owing to the discovery of the p53 isoforms. These isoforms play important roles in regulating cell growth and turnover in response to different stressors, depending on the cellular context. In this review, we focus on Δ40p53, an N-terminally truncated p53 isoform. Δ40p53 can alter p53 target gene expression in both a positive and negative manner, modulating the biological outcome of p53 activation; it also functions independently of p53. Therefore, proper control of the Δ40p53: p53 ratio is essential for normal cell growth, aging, and responses to cancer therapy. Defining the contexts and the mechanisms by which Δ40p53 behaves as a “good cop or bad cop” is critical if we are to target this isoform therapeutically. DNA is the repository of all genetic information within a cell and it is under constant threat of damage from a variety of environmental and endogenous sources . Defectsp53 has been widely described as the “guardian of the genome” due to its ability to coordinate multiple and diverse signalling pathways involved in cellular homeostasis and the DNA damage response ,5,6,7,8.A seminal discovery in 2005 revealed the existence of several p53 isoforms that have distinct functions . One of TP53 mutations present in about 50% of all human cancers ,5115,51]More recently, post-translational degradation of the FLp53 protein via the 20S proteasome was shown to generate Δ40p53 , adding Most of our knowledge on Δ40p53 function comes from studies performed in cancer cell lines ,64,65,66WAF1p21 and PUMA expression when expressed in excess to FLp53, both at the basal level or following treatment with DNA damaging agents. However, Δ40p53 had no transcriptional activity in the absence of FLp53 on these genes [BAX and pro-apoptotic activities, contradicting previous findings that Δ40p53 exhibited only dominant-negative effects on FLp53 [WAF1p21, IL-8, HDM2 and FAS in response to ectopic Δ40p53 in TP53 null hepatocellular carcinoma (HCC) cells [PIDD [In melanoma cells, Δ40p53 has been shown to repress se genes ,16. The se genes . This waon FLp53 ,17. In cC) cells . In addiC) cells . Furtherls [PIDD .WAF1p21, to a greater degree than FLp53 alone [WAF1 protein was reduced by expression of Δ40p53 in H1299 cells, whilst it was maintained in Saos-2 cells. Together, these studies indicate that the effects of Δ40p53 on FLp53 activity differ between these cells and that the function of Δ40p53 is highly dependent on its expression relative to FLp53 [A study by Hafsi et al. showed that Δ40p53 had no transcriptional activity and that its effects on FLp53 were cell line dependent. Δ40p53 activity was shown to increase under genotoxic stress and mediate p53-inducible target genes, including upregulation of 53 alone . This re53 alone ,20. AccoWAF1 expression, indicating a transient inactivation of FLp53 functions [Control over the cell cycle is tightly regulated by FLp53 and Δ40p53. Courtois et al. showed that Δ40p53 is highly expressed at the onset of S phase in serum-starved cells when compared to FLp53 and the isoform becomes predominant up to 30 h after serum stimulation, where its expression was associated with a drop in p21unctions ; and Rayunctions . These runctions . This suPIDD and suppression of WAF1p21 in melanoma cells [When Δ40p53 was stably overexpressed the number of dead cells was significantly higher in Δ40p53-lentivirus infected melanoma cells compared with empty vector controls. This was accompanied by a reduction in viability and induction of apoptosis , but there were no alterations in cell cycle distribution. These results were only observed in cells which expressed FLp53, suggesting that Δ40p53 cannot induce apoptosis independently of the full-length protein. Activation of p53 by Δ40p53, which is reflected by the phosphorylation of serine 15, was a response to proteotoxic stress caused by the high level of Δ40p53 relative to FLp53. This resulted in increased expression of p53 as well as ma cells . Hence, ma cells .WAF1p21 expression. Moreover, Δ40p53 suppressed clonogenicity and inhibited cell proliferation and survival in the presence or absence of FLp53. Despite an increase in WAF1p21, there was no increase in BAX and PUMA expression, caspase-3 and -7 activity, or the percentage of apoptotic cells, suggesting that the suppression of cancer cell growth was not a result of increased apoptosis. However, Δ40p53-induced tumour suppressor activity was attenuated in hot-spot mutant Δ40p53-R175H cells and was reversed following knockdown of Δ40p53. These studies showed that Δ40p53 may have independent functions from FLp53, exerting tumour suppressor activity, rather than simply functioning as an antagonist of FLp53 [It has been demonstrated that Δ40p53 promotes cellular senescence in HCC cells, as shown by an increase in the percentage of senescence-associated β-galactosidase -positive cells and of FLp53 .A model of one p53 dimer binding to a half-site on the consensus DNA sequence and then further stabilized through binding of a second dimer has been proposed , indicatDue to an absence of the HDM2-binding domain, Δ40p53 may increase the stability of mixed oligomers containing both Δ40p53 and FLp53. Co-transfection of FLp53 with HDM2 in p53-null Saos-2 and H1299 cells, resulted in an almost complete disappearance of FLp53. Interestingly, co-transfection with HDM2 had no effect on Δ40p53 expression levels. Co-transfection of Δ40p53, FLp53 and HDM2 resulted in the maintenance of a detectable level of FLp53 with increased phosphorylation on serine 15 also in the presence of HDM2, which strongly suggests that Δ40p53 is able to protect from HDM2-dependent degradation . A studyΔ40p53 is localised predominantly in the cytoplasm, while FLp53 is mostly nuclear. In melanoma cells, Δ40p53 cytosolic localisation was associated with a higher proportion of FLp53 within the cytoplasm, indicating that this isoform likely contributes to p53 mis-localisation within the cytoplasm in melanoma and this is likely to play an important role in defining the function of Δ40p53 . TherefoΔ40p53 is expressed in normal tissues and cell lines at varying concentrations ,75. The TP53 [TP53 mutation status [In ovarian cancer, Δ40p53 mRNA expression was analysed in 166 patients subdivided into 91 serous, 30 endometrioid and 45 mucinous ovarian cancer patients by RT-qPCR . This isTP53 , using tn status .TP53 occur in only 3% of primary and 10 to 20% of relapsed B-cell precursor ALL cases. Therefore, it seems that p53 variants could impair the canonical p53 pathway. Recently, Oh et al. [TP53 by using RT-PCR and western blot. The authors showed that Δ40p53 expression was significantly increased in relapsed patients when compared to control bone marrow cells. Moreover, the authors used a mathematical model to predict the formation of hetero-complexes containing Δ40p53 and FLp53 using their relative expression levels. There was a higher probability of Δ40p53-FLp53 hetero-oligomers in relapse samples than in primary samples, suggesting that Δ40p53 may impair p53 transcriptional activity and that it is associated with the progression of the disease. Even though the small number of healthy controls (n = 4) and the heterogeneity of the groups are limitations of this study, their findings present some important insights into the expression of the p53 isoforms in leukaemia [Mutations in h et al.  investigeukaemia .TP53 (<25%) and this is dependent on their subtype. TP53 is mutated in 72% of HER2-enriched and 80% of basal cases, whereas less than 30% of luminal cases harbour TP53 mutations [TP53 [Similar results (to ALL) have been found in breast cancer. Breast cancers exhibit a low frequency of mutations in ns [TP53 ,82, indins [TP53 . We alsons [TP53 . Furtherns [TP53 .TP53 mutations when compared to FLp53. However, no differences were detected between control and tumour samples, demonstrating that in RCC the TP53 mutation status could influence Δ40p53 levels independently of the cellular context. Furthermore, no associations were found between high/low Δ40p53 levels and patient survival [Δ40p53 mRNA expression was also evaluated  in renalsurvival .Takahashi et al.  characteAlthough mRNA evaluation of p53 isoforms can help to answer quantitative questions, there is a necessity for the development of novel tools to facilitate studies in this area and to predict with accuracy the role that Δ40p53 plays in cancer. The relative mRNA expression evaluation methods require only small amounts of an expressed target, thus making it suitable for studying the low expressed p53 isoforms, but are limited as they only provide a qualitative assessment of their expression levels as either present or absent. Our own studies have measured the relative mRNA expression of the p53 isoforms semi-quantitatively using real-time PCR in breast cancer samples with isoform-specific primers . HoweverTaken together, these studies support both beneficial and detrimental roles of Δ40p53 in cancer in a tissue-specific manner. These outcomes should be interpreted with prudence. Δ40p53 may act in a p53-independent manner  or it maDisruption of normal levels of p53 affects its physiological functions, playing a key role in aging and in neurodegenerative diseases ,87. Agin+/+) and observed a progeroid phenotype similar to accelerated aging characterised by memory loss, neurodegeneration, and premature cognitive decline [+/+) were crossed with mice lacking endogenous p53 (p53−/−), the phenotype was reversed, suggesting that to exert its pro-aging action, p44 requires FLp53, supporting the conclusion that p53 has longevity-assurance activity [Δ40p53 or p44 in mice (owing to its molecular mass of 44-kDa) has been shown to play critical roles in the mouse at all developmental stages. In the developing embryo, p44 is detected well before FLp53 and in embryonic stem cells p44 is required for the maintenance of a highly proliferative pluripotent state . p44 has decline . A high  decline , as well decline ,94. Inteactivity ,96,97 an+/+ mice also exhibit hyperphosphorylation of tau, synaptic impairment and premature cognitive decline [+/+ phenotype could be reversed by Mapt haploinsufficiency, the gene encoding tau [TP53, suggesting that p44 and p53 are not involved in this regulation. Nevertheless, overexpression of p44 altered the phosphorylation status of tau and the profile of these tau kinases [Tau is a microtubule-binding protein that contributes to microtubule stabilization. Analysis of post-mortem human brain specimens and mouse models has shown that aging and memory deficits associated with neurodegenerative diseases are related to the accumulation of hyperphosphorylated tau ,99,100.  decline . Pehar eding tau , mutatioding tau . Interes kinases . Additio kinases .Mapt downregulation in APP AD models was able to rescue memory loss, implying a promising role of tau linked with APP [Given that aging is the main risk factor for late-onset AD, similar changes, albeit more severe, can be detected in AD patients . A key pwith APP ,108. Thewith APP . This stIt is clear to question whether the role of Δ40p53/p44 in aging and longevity could be also associated with stem cell regulation, given that p53 has been implicated in pluripotent and embryonic stem cell reprogramming ,111. p53The role of Δ40p53 in cancer stem cell regulation is poorly understood. Takahashi et al.  found a TP53 being a prototype tumour suppressor gene [As indicated throughout this review, ∆40p53 can interact with FLp53 to fine-tune its activity in both a stimulatory and inhibitory fashion. However, this interaction is highly complex and dependent on the ratio between the isoforms, as well as the cellular context ,20. Withsor gene , modulatSeveral small molecules that are already used clinically have the potential to alter cellular levels of ∆40p53 and thereby affect p53 pathway activity. While the focus of their current clinical use is not related to ∆40p53, their application in this context is outlined throughout this section.HDM2 antagonists, such as Nutlin-3 , may incTP53 is not mutated . An example of this scenario can be found in breast cancer, where TP53 mutations are present in only a quarter of cases [This approach may be applicable in instances where ∆40p53:FLp53 is elevated, which is associated with worse prognosis, and of cases , an incrof cases , and thiof cases ,69.TP53 (n = 13). None of the patients with mutant TP53 (n = 3) responded to AMG 232 [With HDM2 antagonists  already being investigated as potential orally administered antineoplastic agents , targeti AMG 232 . Additio∆40p53 expression may be induced through ER stress. Tunicamycin and thapsigargin both cause ER stress through blocking protein folding and ER transit, and non-competitive inhibition of the ER calcium-ATPase respectively. Treatment with these drugs has been found to induce ∆40p53 expression in a cell-specific context  as discuThe 20S proteasome has been found to cleave FLp53, generating ∆40p53 . Small mSeveral research groups have successfully altered ∆40p53 in vitro using an array of transfection and transduction, as well as ionizing radiation. While the practicality of using some of these approaches therapeutically varies due to questions around specificity and safety, others might be developed and fine-tuned into novel treatments modify p53 pathway activity.TP53 loss-of-function mutations to provide alternative sources of the protein and its isoforms. Transfection or transduction of ∆40p53 has been performed in a number of cell lines ,21,51,52Site-directed mutagenesis of constructs may replace either the first or the second in-frame start codon with alanine, allowing translation of ∆40p53 or FLp53 respectively . Viral vBAX and CDKN1A) indicated dose-dependent effects (both inhibitory and stimulatory). However, this dose-response was not observed when looking at apoptosis, a function thought to be primarily mediated through TAD II [Constructs may also contain hairpin structures near the first start codon, enhancing ∆40p53 expression through the blockage of cap-dependent RNA translation . Maskingh TAD II  and may h TAD II ,64,66.Transduction may also be exploited to eliminate FLp53 expression from one allele. Using adenovirus vectors, Ota et al. deleted exon 2, which harbours the majority of TAD I, and thereby induced ∆40p53 expression. The group was not successful in deleting exon 2 from both alleles . SimilarTP53, as ∆40p53 was found to suppress proliferation in HCC cells regardless of TP53 mutation status [Increasing ∆40p53 or the ∆40p53:FLp53 ratio may be of therapeutic benefit in cancers where ∆40p53 expression has been associated with improved prognosis , or whern status . Howevern status ,92. Thusn status . Similarn status ,121.Antisense oligonucleotides are short DNA sequences that can bind to RNA transcripts making them susceptible to degradation by RNase H. Antisense oligonucleotides that target the 5’ terminal region of the p53 transcript were found to limit both FLp53 and ∆40p53 expression. An antisense oligonucleotide (TCCCAGCCCGAACGCAAAG) targeting a sequence upstream of AUG1 at the base of hairpin G56-C169 was found to cause a decrease in both FLp53 and ∆40p53 expression. A different antisense oligonucleotide (AACGTTGTTTTCAGGAAGTAG) targeting a sequence between AUG1 and AUG2 on the atypical loop at the 3’ end of hairpin U180-A218, reduced the expression of FLp53, but raised levels of ∆40p53 in transfected MCF-7 cells. This effect may be the result of mRNA truncation between the two start codons, increasing accessibility to AUG2 .∆40p53 knockdown through RNA interference presents an opportunity to target the p53 isoform produced through alternative splicing of intron 2. Small-interfering RNAs (siRNAs) against intron 2 may partially inhibit ∆40p53, but would not eliminate alternative translation of this isoform through the IRES. However, thus far only FLp53 and isoforms other than ∆40p53 have been targeted by RNA interference ,64,122 (Silencing of FLp53 and the ∆40p53 isoform was achieved through CRISPR/Cas9 technology. A single guide RNA (sgRNA) designed to excise a proportion of exon 4 was employed due to a CCT protospacer adjacent motif (PAM) within the exon . Thus faTP53 towards ∆40p53. Site-directed mutagenesis of the guanines in intron 3 decreased the excision of intron 2 by around 30%, indicating a potential increase in ∆40p53 [+ supplementation may also enhance G4 formation and limit retention of intron 2. Sodium ions (Na+) may have the opposite effect, being known to destabilise G4 structures [Formation of G4 structures within GC-rich regions of intron 3 promotes splicing of intron 2, guiding RNA processing towards fully-spliced p53 transcripts as opposed to ∆40p53 mRNA . This men ∆40p53 . Addition ∆40p53 ,53. As Gn ∆40p53 , K+ suppructures .Modulation of G4 formation by the aforementioned mechanisms only had limited effects on ∆40p53 protein levels , likely TP53 in cancer, heterogeneity in the response to altered Δ40p53 expression, limited understanding of the physiological function of ∆40p53, and potential side effects may limit the application of these modulators in clinical settings.Additionally, some modulation strategies may increase the sensitivity to existing chemotherapeutic agents and radiation therapy . Beyond TP53 to determine mutation status and detection of endogenous levels of both FLp53 and ∆40p53, is necessary prior to altering ∆40p53 levels, if it was to be used as a targeted therapy in cancer or aging related diseases. Such profiling is currently hindered by the lack of commercially available antibodies specific to p53 isoforms, including ∆40p53, and difficulties in distinguishing between isoform and FLp53 transcripts through RNA sequencing approaches. Additionally, p44 has been linked to premature aging and consequently an AD-like phenotype in mice [In conclusion, although research on this p53 isoform is continuing, there is still a need to understand the cellular contexts in which ∆40p53 acts in a positive or negative manner. While elevated levels of ∆40p53 have been associated with worse prognosis in some cancers, such as breast cancer ,69 and A in mice ,23,92. W


The complexity of the human immune system is exacerbated by age-related changes to immune cell functionality. Many of these age-related effects remain undescribed or driven by mechanisms that are poorly understood. γδ T cells, while considered an adaptive subset based on immunological ontogeny, retain both innate-like and adaptive-like characteristics. This T cell population is small but mighty, and has been implicated in both homeostatic and disease-induced immunity within tissues and throughout the periphery. In this review, we outline what is known about the effect of age on human peripheral γδ T cells, and call attention to areas of the field where further research is needed. Age and functionality of the immune system are intricately connected, as has been well established throughout the literature in the fields of both innate and adaptive immunology ,6,7,8,9.When considering the effects of aging on the immune system, there are many other factors that should be included—such as environmental conditions and prior antigen exposure ,12. LifeEven as our understanding of the effects of age on the immune system is expanding, there are still many gaps in the field. A notable example of this can be observed in γδ T cells. This cell subset of the adaptive immune system uniquely exhibits characteristics of both innate and adaptive immunity, and is often thought to be “bridging the gap” between the two arms of the immune system ,17. Age The immune system broadly consists of two arms: innate and adaptive immunity. The innate immune system is considered the first line of defense against pathogens and damage, rapidly responding in a nonspecific manner. The adaptive immune system is slower to respond, and elicits its functions in a pathogen-specific manner. T cells and B cells make up the adaptive compartment and achieve their specificity via their T cell receptor (TCR) and B cell receptor (BCR), respectively. The TCR and BCR are both created by V(D)J Recombination, a semi-random process by which lymphocytes construct their antigen-specific receptors . The TCRTRGV)-encoding genes and eight functional T-cell receptor delta variable region (TRDV)-encoding genes [TRDV usage: Vδ2+ and Vδ2− T cells [Human γδ T cells have seven functional T-cell receptor gamma variable region  is a latent herpesvirus with immunomodulatory capabilities across the lifespan ,101,102.The effect of age on γδ T cell immunity to HCMV is arguably the most well-characterized ,114, andThe HCMV-driven mobilization of γδ T cells occurs in both immunocompetent and immunocompromised individuals . To dateIt has been well-established that age has a dramatic effect on the immune response to influenza ,121,122.In CD8+ T cell biology, the presence of CMV can positively affect the immune response to influenza in both mice and humans . This efMycobacterium tuberculosis (Mtb) infection and Bacillus Calmette–Guérin (BCG) vaccination on γδ T cells have been studied since the late 1900s [Mtb [The effects of both te 1900s ,133,134,te 1900s ,135. TB te 1900s . Additiote 1900s . A publite 1900s , and potte 1900s ,137. BCGte 1900s ,139, andte 1900s , althoug00s [Mtb ,141. Gen00s [Mtb ,143, esp00s [Mtb ; howeverThe γδ T cell response to bacteria is innate-like in nature, and also involves Vγ9+Vδ2+ T cells ,99. ThisChronic infections uniquely shape the immune system, and the interplay between age, coinfection, and disease is dynamic in nature. Not all chronic viral infections are created equally, and can be loosely sorted into three categories: latent, smoldering, and persistent . γδ T cePlasmodium falciparum [γδ T cells have been implicated in a multitude of other disease states, including the viruses West Nile and SARS-CoV-2 ,152,153,lciparum , and nonlciparum ,156,157.lciparum . A more lciparum . Until t


Yimeng scorpion is a specific geographical indication breed of Yimeng Mountain area in China. The complete mitochondrial genome sequence of Yimeng scorpion was determined for the first time (Accession number MN597087). It is mitochondrial genome  contains 13 protein-coding genes, 21tRNA genes, 2 ribosomal RNA genes and one large non-coding region (a possible control region). Moreover, tRNA-ASP-loss was observed from the Yimeng scorpion mitochondrial genome. The mitochondrial genome sequence of the Yimeng scorpion enriches data resource for further research on genetic mechanism and classification. Buthus martensii, also known as Mesobuthus martensii  using the invertebrate mitochondrial code. The tRNA genes were identified by ARWEN  begin with the common start codon “ATT”, 4  with “ATG”, 3  with “TTG”, 1 (ND5) with “ATC” and 2  with “ATA”. For termination codons, the seven protein-coding genes  terminated with “TAA”, then the remaining genes ended with incomplete stop codon T–– or AT. Except for ND6 and eight tRNA genes , all the mitochondrial genome genes were encoded on the H strand. The 12S rRNA (784 bp) gene and 16S rRNA (1367 bp) gene were located between the tRNA-Gln and tRNA-Leu genes, and separated by the tRNA-Val gene, with the same result as other arthropods. In addition, the small non-coding region, a putative control region, was located between 12S rRNA and tRNA-Gln genes with the length of 781 bp. Due to lack of tRNA-Asp, the Yimeng scorpion mitochondrial genome encodes only 21 tRNA genes, which has not been observed in any other non-scorpion mitochondrial genome published to date  was 14,840 in length, of which 14,023 nucleotides are coding DNA, and 817 nucleotides are non-coding DNA. It contains the 13 protein-coding genes, two rRNA genes, 21 tRNA genes and a possible control region. The arrangement and composition of the mitochondrial genome is also comparable to the case of other Arthropoda (Lavrov et al.


Investigations of β-alanine supplementation shows effects on metabolic (aerobic and anaerobic) participation and performance on swimming by a possible blood acidosis buffering. Considering this background, the objective of the present study was to analyze the effects of β-alanine supplementation on metabolic contribution and performance during 400-m swim.− 1 of β-alanine or placebo. Before and after the supplementation period, the total anaerobic contribution (TAn) and 30-s all-out tethered swimming effort (30TS) were assessed. Anaerobic alactic (AnAl) and lactic energy (AnLa) was assumed as the fast component of excess post-exercise oxygen consumption and net blood lactate accumulation during exercise (∆[La−]), respectively. Aerobic contribution (Aer) was determined by the difference between total energy demand and TAn. In addition to conventional statistical analysis , a Bayesian repeated measures ANOVA was used to evidence the effect probability (BFincl).Thirteen competitive swimmers underwent a 6-week, double-blind placebo-controlled study, ingesting 4.8 g.dayincl: 25.02); absolute AnLa , fatigue index  and total anaerobic participation .No differences and effects were found between groups, indicating no supplementation effects. Repeated measures ANOVA, with confirmation of effect, was indicate reduce in ∆Lactate (p: 0.001; BFThus, the results demonstrated that all changes presented were evidenced as a result of exposure to the training period and β-alanine supplementation doesn’t affect metabolic contribution and performance during 400-m freestyle. Training period alters energy provision, neuromuscular and metabolic parametersBeta-alanine supplementation demonstrated no effects on energy provision to 400m freestyle swimming performance.Beta-alanine supplementation doesn’t alter force parameters on tethered swimming testBeta-alanine supplementation doesn’t alter performance on 400m freestyle swimming−])  accumulation and low intramuscular pH are reported PEAK). The amount of blood lactate accumulation was the difference between [La−] at peak and baseline values (∆[La−]). [La−] was determined using a blood analyzer YSI-2300 .Immediately before, 3, 5 and 7 min after the 400-m performance, blood samples (25 μl) were collected from the earlobe to determine [La−], considering a metabolic equivalent of 3 mL∙O2∙− 1 kg− 1 for each unit of lactate accumulated during the maximal effort  was found .The method used to analyze energy contribution is useful to change paradigms related to metabolic contribution, identify better pacing strategies and estimate individual capacity to develop anaerobic performance . Thus, aThe absence of significant changes in swimming performance , best evidenced in the percentage of individual differences Table , can be − 1) and body mass (0.3 g∙kg− 1) [β-alanine supplementation has been used in various sports, such as swimming , 7, 17, g∙kg− 1) , 33, 34.− 1 for 1 week and 6.4 g·day− 1 for 4 weeks and combining with SB (0.3 g∙kg− 1) on the last day of tests. Their main findings were that β-alanine supplementation, β-alanine supplementation with SB addition to the effort and only SB addition to effort effectively similarly improved 200-m freestyle performance, and tended to improve 100-m performance [De Salles Painelli et al. (2013) investigated the effects of two supplementation strategies  on 100-m and 200-m swimming performance. In β-alanine alone, sixteen swimmers received 3.2 g·dayformance .The β-alanine supplementation dose and time of exposure on the study performed by Salles Painelli et al. 2013) were similar to our study; however, the authors found no significant differences in buffer capacity (fatigue index) or ergogenic aid  were found . This ca3 were si− 1) or PLA and combined co-administration of SB or PLA before two 100-m maximum freestyle bouts. Although pH increase was observed in both groups with SB, performance improvement was only observed when compared with PLA alone, and no differences were found for blood lactate concentration, evidencing that β-alanine supplementation did not provide significant alterations and effects were recurrent from SB administration. Under similar conditions of supplementation (time and quantity), the authors reported no alterations in performance, corroborating the present study. However, it should be noted that no changes in blood lactate concentration can be linked to the effort’s duration [Mero et al. 2013) investigated the effects of a six-week supplementation using only β-alanine  administrated 4.8 g·dayFinally, although several studies have demonstrated that β-alanine supplementation can improve high intensity exercise performance , 4, 17, The present study has some limitations, only three studies had their focus on swimming performance analysis, and none has quantified the metabolic contribution on β-alanine supplementation to make comparisons with the same method of quantification and modality. Therefore, this is the first study focused on shortening this gap in the literature. Even from the evidence of Stegen et al. (2013) the lack of intramuscular carnosine content measurement can be considered a limitation. Despite not measuring intramuscular carnosine, Harris et al. (2006) evidenced that the dose used in the present study seems sufficient to increase intramuscular content of carnosine even in a short period of supplementation. The chosen dose of β-alanine caused paresthesia on three athletes, showing which group was PLA and which was β-alanine. Finally, there were no nutritional control during supplementation, as feeding may influence the amount of carnosine originated by β-alanine supplementation.Despite the methodologic constraints inherent in studies involving competitive athletes , they are important to substantiate the efficiency of nutritional aids over performance and metabolic contribution in sports.Although metabolic, neuromuscular and energy provision parameters changes, no effect from supplementation was found, evidencing that all the alterations found were the result of exposure to training. Thus, it can be concluded that β-alanine supplementation doesn’t changed metabolic contribution and it was ineffective in improving performance during 400-m freestyle.


Despite the poor photochemical stability of capsules walls, polyacrylate is one of the most successful polymers for microencapsulation. To improve polyacrylate performance, the combined use of different acrylate-based polymers could be exploited. Herein butyl methacrylate (BUMA)-based lattices were obtained via free radical polymerization in water by adding (i) methacrylic acid (MA)/methyl methacrylate (MMA) and (ii) methacrylamide (MAC) respectively, as an aqueous phase in Pickering emulsions, thanks to both the excellent polymer shells’ stability and the high encapsulation efficiency. A series of BUMA_MA_MMA terpolymers with complex macromolecular structures and BUMA_MAC linear copolymers were synthesized and used as dispersing media of an active material. Rate and yield of encapsulation, active substance adsorption onto the polymer wall, capsule morphology, shelf-life and controlled release were investigated. The effectiveness of the prepared BUMA-based microcapsules was demonstrated: BUMA-based terpolymers together with the modified ones (BUMA_MAC) led to slow (within ca. 60 h) and fast (in around 10 h) releasing microcapsules, respectively. In the last decade, microencapsulation technique of effective liquid and solid agents has attracted wide interest from several industrial sectors, such as the food chemistry, building, health and beauty industries ,2,3,4. TIn this scenario, the combination of physical and chemical methods is an emerging approach, thanks to the hopeful benefits deriving from each of these techniques and the minimization of the already cited drawbacks. The physicochemical methods are mainly based on the formation of walls from preformed polymers, and among the most used methods, ionic gelation , complexNowadays, in the field of microcapsules preparation processes, one of the main issues to overcome is to obtain a stable product during storage with consistent encapsulation and release efficiency. The latter demand is quite difficult to reach because the common materials used for lattices formulation, e.g., alginates , polyacr1H nuclear magnetic resonance (NMR), Fourier-transform infrared and thermal analyses, and of microcapsules through dynamic light scattering and scanning electron microscopy studies, was conducted and is discussed here as well as the corresponding kinetics of active material release.To the best of the authors’ knowledge, few studies in the literature about microcapsules formation from Pickering emulsion procedure, tailoring different polyacrylate-based polymers, have been reported so far. Hence, in this work, we prepared novel polyacrylic lattices  linear copolymers) as protective materials for the microencapsulation of an active agent, by water-based free radical polymerization procedure during Pickering emulsion. These microcapsules were designed to: (i) be formulated exploiting environmentally friendly materials to achieve more biodegradable polymeric matter; (ii) preserve the active material from the surrounding environment; (iii) tune the effective agent release; and (iv) be re-dispersed in water to form a free-flowing non-viscous aqueous formulation. Therefore, a deep characterization of the prepared polymeric latex, via 2S2O8, ≥99.9%), sodium dodecyl sulfate , trifluoroacetic acid-d , sunflower oil, sodium chloride , iron  oxide nanopowder , ethanol (96%), methyl orange (MO), KNO3 10−2 M and distilled water Chromasolv® (≥99.9%) were supplied by Sigma Aldrich  and used without further purification.N-butyl methacrylate , methacrylic acid , methyl methacrylate , pentaerythritol triacrylate , methacrylamide , sodium persulfate (NaBUMA_MA copolymer (used as reference), two terpolymers, i.e., BUMA_MA_MMA and BUMA_MA_MMA_T3, and BUMA_MAC copolymers were synthesized via free radical polymerization. 3 three-necked round bottom flask was equipped with a reflux condenser having a nitrogen inlet adapter, an internal thermometer adapter and a mechanical stirrer. The flask was flushed with nitrogen, charged with 100 cm3 of distilled water, 0.5 g of sodium dodecyl sulfate (SDS) used as latex stabilizer [2S2O8 (mol Na2S2O8 = 1% mol mol−1∑acrylate monomers) and acrylate monomers, according to the desired polymer. The polymerization mixture was put in an oil bath, mechanically stirred at around 280–300 rpm, heated for 24 h at 69 °C and then gradually cooled down to room temperature. The reaction yields, determined gravimetrically, were around 100% for all the investigated systems.In a typical polymerization procedure, a 250 cmabilizer , Na2S2O82 at 50 °C for 24 h.For spectroscopic and thermal characterizations, an aliquot of the lattices prepared was previously dried under N−1. A single-bounce diamond crystal was used with an incidence angle of 45°. 1H NMR spectra were collected at 25 °C with a BRUKER 400 MHz spectrometer. All samples were prepared dissolving 8–10 mg of polymer in 1 cm3 of C2DF3O2.Fourier transform-infrared (FT-IR) spectra were obtained on a Spectrum 100 spectrophotometer (Perkin Elmer) in attenuated total reflection (ATR) mode using a resolution of 4.0 and 256 scans, in a range of wavenumbers between 4000 and 450 cm3 10−2 M to adjust the ionic strength  [Dynamic light scattering (DLS) analyses were carried out to study the lattices particles size distribution. Samples were analysed after a 1:100 dilution in KNOerature) ,52, usin−1;heating from 0 °C to 100 °C at 20 °C min2 min isotherm at 100 °C;−1;cooling from 100 °C to 0 °C at 20 °C min2 min isotherm at 0 °C;−1.heating from 0 °C to 150 °C at 20 °C min using an empty 40 µL aluminium pan as reference.Differential scanning calorimetry (DSC) analyses were conducted using a Mettler Toledo DSC1; the analyses were conducted weighting 5–10 mg of each sample in a standard 40 µL aluminium pan, using the following temperature cycles:3 glass jacket reactor 88 cm3 of sunflower oil, 224 mg of Fe3O4 and 721 mg of NaCl were added. The mixture was processed with a mechanical stirrer (around 380–400 rpm) for 15 min at room temperature. In a separate tube, 1 cm3 of a methyl orange (MO) solution with a concentration of 600 ppm was added to 16 cm3 of the as-synthesized latex  for 20–30 min at room temperature. The mixture was decanted for 2 h in order to separate the yellowish microcapsules from the supernatant oil phase. The oil phase was removed, and the resulting microcapsules were re-dispersed in water. The process was repeated until oil was no longer observable. In a 300 cmatex see . Then, 6An aliquot of the microcapsules obtained was dried for 2 h at room temperature on a blotting paper in order to perform scanning electron microscopy (SEM) images. This analysis was carried out using a SEM Hitachi TM-1000 .3 of distilled water (pH 5.5) under magnetic stirring (ω = 50–80 rpm). The absorbance spectra ) of each sampling (1.5 cm3) were acquired at regular time intervals for a total of 120 h, in order to monitor the release of MO over time. Moreover, to compute the exact MO amount, a calibration curve for MO in water was obtained at pH 5.5 (see 3 mol−1 cm−1. The encapsulation efficiency percentage (EE%) was computed according to [To deeply investigate the encapsulation and release capacities, methyl orange was chosen to make easier the correct evaluation through ultraviolet/visible (UV/Vis) spectroscopy. Specifically, once the MO-encapsulated microcapsules were obtained, 1 g of them were dispersed into 100 cm 5.5 see , followirding to .2 or NaOH, or by adding emulsification agents [3 side groups instead of hydroxyl functionalities, was attained. Specifically, two new kinds of BUMA-based polymers were obtained via free radical polymerization in water by adding (i) MA/MMA as comonomers plus T3, as branching agent (adopting MA/BUMA molar ratio of 0.25% mol/mol for both BUMA_MA_MMA samples) and (ii) MAC, improving both the polymer shells’ stability and the encapsulation efficiency. As a reference, BUMA_MA latex (having MA/BUMA molar ratio of 0.83 mol mol−1) was prepared as already described in literature [Several approaches have been already reported in literature to improve microcapsules formation either using hydroxyl functionalized monomers, such as MA neutralized by CaCln agents . Howevern agents . Herein,terature . −1 (δ-CH), the stretching of carbonyl ester groups -C=O in the range between ~1750 cm−1 and ~1600 cm−1 (v-C=O), and the characteristic absorption band for the symmetric stretching vibration of -C–O conjugated to carbonyl ester groups (1350–1100 cm−1) (s–C–Ov) [3 groups conjugated to carbonyl functionalities is also detectable in the range ~3100–2800 cm−1 (v–CH3) [−1 and a double C=O peak due to the simultaneous presence of ester and acid moieties along the macromolecular chain.(vs–C–O) . In the  (v–CH3) . As expe−1, is mandatory to avoid the flocculation of parts of polymer from water media, as also confirmed by literature data [1H NMR spectrum, where the presence of the branching agent cannot be highlighted due to its exiguous amount. For clarity, the reference and BUMA_MA_MMA 1H NMR spectra have been compared to the one relative to BUMA_MA_MMA_T3 sample  encapsulation and its release, other kinds of polyacrylate-based material were investigated. As reported by Stranimaier et al. , amide fTo unravel the stability of the as-prepared lattices, DLS measurements and DSC analyses were performed. At first, It is worth noting that in the latter case, the addition of only an apolar monomer, i.e., MMA, is not enough to ensure the formation of homogenous and stable latex, since polymeric aggregates of ca. 250 nm can be formed. Conversely, concerning the BUMA_MAC family, BUMA_MAC_75 latex seems to be the most stable one and DLS measurements reveal the presence of particles with a very small hydrodynamic diameter of around 12 ± 3 nm.gT, of a polymer is a key value for the occurrence of the locking phenomena, since it corresponds to the temperature at which a polymer switches from hard and glassy form to soft and viscous one. In particular, gT values higher than room temperature favour the fusion of the latex polymer particles in a continuous polymeric shell and, therefore, promote high microcapsules yield [Lastly, during Pickering emulsions a latex can be transformed into microcapsules when the interfacial nanoparticles are either linked or fused together to form a continuous wall. This step, usually called “locking”, can be achieved via several approaches, such as chemical cross-linking, in situ polymerization, thermal softening and solvent-instability of the particles . Howeveres yield . gT corresponds to the theoretical glass transition temperature value of the resultant copolymer or terpolymer; w1 and nw are the weight fractions of each component; gT,1 and Tg,n are the glass transition temperatures of BUMA, MA, MMA and MAC homopolymers, 293–501–378–486 K, respectively. Glass transitions were determined during the first heating scan ), they show a rough surface with an average diameter of 220–270 μm, as clearly visible in On the other hand, the reference BUMA_MA latex gave rise to microcapsules with both an average diameter comparable with the one of MC(BUMA_MA_MMA_T3) and, even, smaller . Furthermore, a series of BUMA_methacrylamide (MAC) copolymers were prepared, varying the molar percentage of MAC monomer (25–50–75% mol mol−1). At first, the macromolecular structures, thermal features and hydrodynamic volumes were investigated before and after the Pickering emulsion, i.e., the microencapsulation process used here; moreover, the morphology of the microcapsules obtained was assessed via scanning electron microscopy, demonstrating the formation of polymer shells with average diameters of 270 and 55 μm in the case of BUMA_MA_MMA_T3 and BUMA_MAC_75, respectively.Herein, butyl methacrylate (BUMA), methacrylic acid (MA) and methyl methacrylate (MMA) latex was synthesized via free radical polymerization in a water media using just 0.5% mol molThe microcapsules obtained were studied by kinetic release, focusing on rate of release and physical resistance of the microcapsules dispersed in water. Both polymeric lattices showed very promising features as protective envelopes. Notably, BUMA_MA_MMA_T3 latex seems to be the most suitable one in the case of slow release, due to its ability to form bigger microcapsules and thus protect for longer times the active agents entrapped from the surrounding environment. Conversely, for BUMA_MAC_75-based microcapsules fast kinetic release was observed. Hence, to the best of the authors’ knowledge the synthesis of such kinds of lattice and their use in Pickering emulsion for microencapsulation process of effective agents has not been reported so far. Specifically, the present research has led to an in depth comprehension of microcapsules formation and kinetic release of BUMA-based lattices that can be successfully adopted as protective polymer shells for the controlled delivery of active materials.