Origanum vulgare L. essential oil (OEO-PbH) as an active ingredient in the management of FPs. The formulation has been shown to possess good qualities in terms of stability and sterility. Non-invasive measurements revealed changes in some physiological skin parameters. An increase in transepidermal water loss (TEWL) and erythema index was noted, while skin surface water content (SWC) decreased during eight weeks of treatment. The macroscopic evaluation revealed that the FPs dried and shrunk after topical treatment with OEO-PbH. Clinically, patients presented a lowering of the number of lesions on the treated area of 20–30% after one month of treatment and around 50% after the second month. Histopathological examination suggests that topical treatment with OEO-PbH may induce histological changes in the epidermis, dermis, and fibrovascular cores of FPs, including a loss of thickness, reduced size and number of blood vessels, and low cellularity. These changes may contribute to the observed reduction in size of FPs after treatment with OEO-PbH.Skin tags, also known as fibroepithelial polyps (FPs) or acrochordons, are soft, pigmented excrescences, with a prevalence of 50–60% in the population, occurring especially in the fourth decade of life. To date, FPs have been efficiently eliminated using minimum invasive methods such as surgical removal, cauterization, laser irradiation, and cryosurgery. Over-the-counter treatments are also of interest for patients due to their non-invasive character, but their clinical efficiency has not been clearly demonstrated. This study was designed in order to evaluate the efficacy of a modern-pharmaceutical-formulation-type poloxamer-based binary hydrogel, having  The skin, also known as the largest organ in the body, is a dynamic and complex anatomic structure. It is divided into three main layers: epidermis, dermis, and subcutaneous tissue . The epiWhile the skin is permanently in direct contact with exogenous factors which can imbalance its function, such as pollutants, irritants, allergens, and infectious agents, it is mandatory to maintain a solid skin barrier function for healthy functioning of the skin . In addiFPs, also known as acrochordons or commonly known as skin tags, have a high prevalence rate (approximatively 50–60%) within the adult population. These soft and pigmented excrescences tend to occur the most frequently during the fourth decade of life . MicroscOne of the possible mechanisms involved in the occurrence of FPs entails a hormonal disequilibrium, e.g., increased levels of estrogens and androgens, especially in overweight people. This imbalance can lead to a hyperproliferation of keratinocytes and fibroblasts due to the high affinity of α- and β-estradiol for these cells . FurtherCurrently, the effective removal of FPs involves minimally invasive techniques such as cryosurgery, cauterization, surgical excision, and laser therapy. While non-invasive treatments available over the counter have piqued the interest of patients, their clinical efficacy has not been plainly established yet . Nowadays, there is a growing demand for botanical and natural dermato-cosmetic products due to the current “back to nature” trend. Therefore, natural origin compounds are more likely to be used in cosmetics, cosmeceuticals, or pharmaceuticals to the detriment of synthetic substances ,19. WhilPlant-derived active phytochemicals have sparked curiosity ever since ancient times and are at present investigated in depth due to their various applications in medicine, veterinary medicine, the food industry, cosmetics, and natural dye production ,24. In tLamiaceae species , Melaleuca alternifolia (Maiden and Betche) Cheel, and Cinnamomum zeylanicum Blume, thus being choices for anti-microbial agents and preservatives [Citrus aurantium L., Lavandula officinalis  Mill., Origanum sp., Mentha x piperita L., Helichrysum italicum (Roth) G. Don) [Cymbopogon winterianus Jowitt, Piper aduncum L.) [Calendula officinalis L.) [Kunzea oil) [Curcuma aeruginosa L.) [Myrtus communis L., Origanum vulgare L.) [Origanum sp., Salvia triloba L.) [The actual tendency of researchers is to develop novel and modern plant-based cosmeceutical formulations by means of micro- and nanotechnology to overcome some physio-chemical limitations of natural compounds. EOs are characterized by an irritant and instable profile, hence the need for delivering efficient drug delivery systems. Both micro- and nanoencapsulation methods offer controlled release of active components, enhance the biological activity, protect the volatile compounds from degradation and evaporation, and improve the overall appearance of the finite product ,29. Furtrvatives ,32,33,34 G. Don) , mosquitncum L.) ,37, UV-Balis L.) , anti-pszea oil) , lightennosa L.) , anti-acgare L.) , and wouloba L.) .Origanum vulgare L.) is a versatile member of the Lamiaceae family owing to both its culinary and medicinal attributes. It is located and cultivated mostly in the Mediterranean region, but also in several areas of Europe, Asia, North Africa, and America with mild, temperate climates [Origanum vulgare L. essential oil (OEO), especially in the Greek one, accompanied by their precursors (p-cymene and γ-terpinene) [O. vulgare L. has been added to Europe’s List of Priority Species due to its unique biological characteristics and economic significance [O. vulgare L. has been intensively studied for novel therapeutic applications in the dermato-cosmetic field [Oregano (climates . In termrpinene) . O. vulgificance . The essic field . In thisic field . In addiic field . To improve the solubility and the bioactivity of OEO, we previously developed a modern pharmaceutical formulation, namely a poloxamer-based binary hydrogel (OEO-PbH) containing polymeric micelles as carriers for OEO. In comparison with other conventional semisolid topical vehicles, this polymeric hydrogel is superior due to its main components, namely the two poloxamers, which present the following advantageous characteristics: (i) they are biocompatible, non-toxic, and non-irritant, and therefore ranked as GRAS  by the FDA and included with individual monographs in the European and US Pharmacopoeia; (ii) in aqueous medium, at room temperature, they form gels with longer residence time on the skin, resulting in higher OEO local concentrations available for therapeutic effect; (iii) they are considered to be efficient penetration enhancers; (iv) they act as solubilizers for poorly hydrosoluble active ingredients (including OEO), encapsulating them in polymeric micelles, thus achieving their better safety profile when topically applied ,50. AlsoTherefore, in this study, several attributes of OEO-PbH, namely its susceptibility to microbial contamination and stability over time, were investigated to complete the quality control tests previously performed and reported. Moreover, a clinical study of this innovative hydrogel was carried out involving both non-invasive methods as well as histological evaluation.Considering the recommendations of the regulatory authorities presented in pharmacopoeias and national formularies for extemporaneous compounding formulations , the lonAs was previously reported , at 24 hAlong with the evaluation of organoleptic properties, the study of pH profile of a formulation over time provides information about the chemical stability of the preparation, with it being possible that the variations in this parameter correlate with decomposition processes of the vehicle components and/or the active ingredient.After 24 h, OEO-PbH showed a very slightly acidic pH (6.34 ± 0.05), attributed to the two poloxamers used as gelling agents . Figure w/v) throughout the 6 months of stability testing at room temperature and varied within a narrow range (from 98.60 ± 1.29% to 100.80 ± 0.80%)  . The penetration degree values of OEO-PbH that resulted from the penetrometry measurements during the long-term stability study are presented in It can be observed that these values increased insignificantly over time, ranging between 99.30 ± 1.36 mm at 24 h after preparation and 100.97 ± 1.48 mm after 6 months , thus inThe results of the spreadability test performed at room temperature 24 h after the preparation of the experimental OEO containing hydrogel were presented in our previously published study . It was When the spreadability test was repeated for the remaining time points , similar and almost overlapping extensiometric profiles were obtained with the one recorded on day 1 after preparation. For the clarity of the graphic representation, in The total count of colony-forming units (CFUs) was determined by counting the colonies that developed on agar plates, with values below 300 colonies/plate for bacteria and below 100 colonies/plate for fungi being considered to be microbiologically satisfactory.To detect bacterial growth and establish the total count of CFU/g sample, the change in color of the medium was followed, depending on the number of test tubes and the dilutions. All of the tested samples were negative. As described in the methodology, Salmonella spp., Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were absent. The preservative efficacy test showed that none of the microbial strains were isolated from the 48 h samples, with the inoculated culture media remaining sterile.The effect of OEO-PbH on FPs was evaluated along the eight weeks of topical application by means of mexametric, corneometric, and tewametric measurements, expressed in arbitrary units (A.Us.). Compared to the normal values registered at time 0, the erythema index showed a slow growing trend a. It canThe FPs that were excised at the initial dermatology consultation without any prior treatment were processed for use as a benchmark for histological changes induced via the application of the topical OEO-PbH. A microscopic examination of these FPs revealed a typical histological composition with a central core of loose fibrocollagenous tissue surrounded by a thin layer of epidermis.After one month of topical treatment with OEO-PbH, the microscopic examination of the FPs revealed a thinner epidermis in most cases, as well as a more edematous dermis and a marked increase in inflammatory cells at the same level. These histological changes suggest that OEO-PbH may have anti-inflammatory effects and may affect the thickness of the epidermis.After two months of topical treatment with OEO-PbH, the microscopic examination of the FPs revealed that the fibrovascular cores presented the most obvious change with a loss of thickness, reduced size and number of blood vessels, and low cellularity. These changes suggest that OEO-PbH may influence the fibrous core of FPs, leading to a reduction in size and changes in the blood supply to the FPs.Overall, the microscopic examination of FPs at different time points suggests that topical treatment with OEO-PbH may induce histological changes in FPs, including changes in the epidermis, dermis, and fibrovascular cores . These cO. vulgare var. vulgare. Forty-three volatile compounds were identified and quantified, with carvacrol and thymol as the main components. Furthermore, OEO-loaded poloxamer binary hydrogel was prepared and evaluated in terms of physicochemical properties . The formulation exhibited good skin compatibility in terms of consistency and spreadability. A biological evaluation of OEO-PbH was also conducted. Both OEO and OEO-PbH presented significant anti-microbial activity. Furthermore, an in vitro assessment of HaCaT human keratinocytes on human dendritic cells was conducted. OEO-PbH demonstrated anti-proliferative, anti-migratory, and pro-apoptotic effects on HaCaT cells, while no injurious effect on dendritic cells was detected. In addition, the formulation determined a decrease in inflammatory cytokines  without affecting IL-10 [The present study expands on the prior investigations conducted by our research group. Using gas chromatography–mass spectrometry, we previously characterized the phytochemistry of OEO extracted via hydrodistillation from the aerial parts of Romanian ng IL-10 . The next step in our comprehensive investigation involved a morphological characterization of OEO-PbH by means of scanning electron microscopy analysis. A regular aspect was observed after OEO encapsulation. In vitro release and skin permeation studies showed a sustained release of OEO through synthetic and pig ear membranes. Moreover, we conducted an extensive in ovo assessment on CAM in order to establish the potential effects of OEO-PbH on angiogenesis and the biocompatibility of the tested formulation. OEO-PbH was well tolerated and did not present an irritative effect on CAM. Additionally, it showed an anti-angiogenic effect on CAM, especially on the blood vessels . Stability testing is an important assay in the quality control of a pharmaceutical product, since it produces evidence on how various environmental factors  can influence and, consequently, determine variations in the quality of a pharmaceutical preparation over time . In the The findings of pH determination, correlated with those of visual examination, suggest three beneficial attributes of the studied experimental formulation: (1) chemical stability, as there was no indication that the degradation of poloxamers and essential oil components occurred over the 6 month period; (2) thermodynamic stability of polymeric micelles and consequently the maintenance of their ability to solubilize the OEO, as there was no significant change in the formulation pH that could have destabilized the micelles ; and (3)The results of the OEO content assay showed that the OEO concentration in the formulation was kept above 90% at 25 °C for 6 months, complying with the specification of current pharmacopoeias and revealing the uniformity of active ingredient content and its chemical stability under the storage conditions, with this last-mentioned attribute also being confirmed by the pH study. Consequently, one can consider that no loss in potency of OEO occurred at the end of the stability test .For pharmaceutical semisolid preparations, consistency, or structural strength, assessed through its two components, hardness and spreadability, is an important rheological feature which can significantly affect several characteristics, including stability, homogeneity of incorporated active ingredient, bioadhesivity, and finally therapeutic effect . TherefoIn a previous paper, our research group demonstrated that OEO-PbH elicited anti-proliferative, cytotoxic, anti-migratory, and pro-apoptotic effects against HaCaT human keratinocytes . FurtherBecause of the strong irritant effect of EOs, the micellar encapsulation method was used in order to assure the safety of the product and to decrease the skin irritation caused by pure EOs ,62. For The use of non-invasive methods for the evaluation and quantification of a series of skin parameters offered new perspectives in the clinical research area and enriched the scientific field due to the easiness, safety, and precision of these measurements. Some of the most common non-invasive methods currently used are corneometry, pH-metry, mexametry, tewametry, cutometry, and sebumetry ,65. As mMexametric measurements contribute to the evaluation of cosmetic, cosmeceutical, and dermatological treatments by measuring the concentration of melanin and hemoglobin in the skin . MelaninSkin hydration is commonly characterized by two parameters, namely the water holding capacity of the stratum corneum, also known as skin surface water content (SWC), and the loss of transepidermal water (TEWL), with both features being beneficial in order to appreciate the effects of dermato-cosmetic treatments or to appraise several skin conditions. A dysfunctional skin process is highlighted by a decline in SWC and an increase in TEWL ,71. The The effects of OEO-PbH on skin tags were monitored both macroscopically and microscopically, while the changes in some physiological skin parameters were appreciated using non-invasive measurements. Within this evaluation, some of the skin parameters  that could give us relevant information regarding the effect of OEO-PbH on FPs were assessed. The non-invasive measurements confirmed an increase in the irritation degree and in TEWL, while SWC decreased during the eight weeks of evaluation compared with the normal values of these skin parameters measured in the first meeting with the subjects and also with those provided in the literature ,76.As a result of the erythema evaluation, OEO-PbH can be considered to be safe for human administration. Products with a strong irritative effect will be conducted to a significantly increased erythema index  . AccordiThe macroscopic evaluation of the FPs and the surrounding area after the application of OEO-PbH revealed that the FPs dried and shrunk, without visibly affecting the area around them. The feedback of the subjects was also essential for the evaluation of the hydrogel effect. From a clinical standpoint, a decrease in the percentage of lesions in the treated area could be observed, namely, after the first month of treatment it was approximately 20–30%, and after the second month of treatment it was approximately 50%, respectively. Lesions bigger than 5 mm were not influenced by the applied substance throughout the two months of treatment. The skin of the treated area did not present any changes after one month of treatment, but presented mild xerosis in 70% of the cases after two months, which rapidly subsided in 100% of cases after the application of emollient products. Erythema or descuamation were not clinically evident throughout the treatment. Other signs of adverse effects were not found upon clinical examination. Symptomatically, after the first month of treatment, 20% of patients reported mild recurrent pruritus (itching), especially after showering, which affirmatively remitted shortly after applying emollient topical products. This symptom usually accompanies xerosis . None ofXerosis, also known as dry skin, xeroderma, or xerosis cutis, is determined by structural and functional changes in keratinocytes due to an imbalance between hydrophilic and lypophilic content . GeneralHistopathological examination of FPs adds a significant layer of understanding to the underlying mechanisms through which OEO-PbH exerts its beneficial effects on skin tags. The initial observations from untreated FPs provided a baseline for comparison, revealing a standard composition with a central core of fibro-collagenous tissue enveloped by a thin layer of the epidermis. Following one month of topical treatment with OEO-PbH, microscopic examinations reported an intriguing finding: a thinner epidermis in most cases, coupled with a more edematous dermis and a substantial influx of inflammatory cells. These histological alterations suggest that OEO-PbH may elicit anti-inflammatory responses and also affect the thickness of the epidermis. This is in line with our in vitro studies which demonstrated the anti-proliferative and pro-apoptotic effects of OEO-PbH on keratinocytes . A reducO. vulgare var. vulgare and characterized [® F127), Pluronic L 31 purchased from Sigma-Aldrich-Chemie , and purified water were used. To obtain the poloxamer-based binary hydrogel loaded with OEO, oregano essential oil previously extracted from the species cterized , Poloxamw/w OEO was prepared via the “cold method” [w/w Pluronic F127 (P127) and 1% w/w Pluronic L 31 (P31) were slowly dispersed in cool (4 °C) purified water. The obtained dispersion was kept at 4 °C (refrigerator) for at least 24 h, stirring from time to time until the complete dissolution of poloxamers, indicated by the transparency of the formed solution. OEO in concentration of 5% w/w was added to the cold solution of poloxamers, while continuously stirring at room temperature, and a clear hydrogel was formed.The polymeric-micelle-based hydrogel loaded with 5%  method” , as prev method”  and in o method”  studies.w/w OEO was packed in an amber tightly closed glass jar, and stored at room temperature (25 ± 2 °C) for six months. Throughout the stability study over time, the OEO-PbH formulation was assayed for organoleptic characteristics, pH, OEO content, and consistency on days 7, 14, 28, 60, 90, and 180.After being physicochemically characterized at 24 h after preparation through visual examination, pH, and consistency determination , the polVisual examination was performed to observe the possible changes over time in organoleptic characteristics .m/v) and stirred for 15 min at room temperature. Then, the potentiometric analysis [For pH determination, the hydrogel was previously diluted in water to a concentration of 5%  to 10 mL. After adequate dilution, the absorbance of the ethanolic solution was measured in 1 cm quartz cells at room temperature. The determination of OEO content was repeated three times for each experiment. The OEO content of the samples was calculated using the following equation, describing the calibration curve of OEO: y = 0.72x + 0.0372 (= 0.9996), where y is the absorbance at 216 nm and x is the OEO concentration (mg %); the value of the correlation factor, R2, was 0.9996. The OEO content in the sample was determined throughout the stability study via ultraviolet–visible (UV-VIS) spectrophotometry at 216 nm , according to the methodology described below. A total of 0.200 g hydrogel sample  was accurately weighted and transferred to a 10 mL glass amber volumetric flask; then, it was diluted with ethanol 96%  following the procedure described by the European Pharmacopoeia . The sprSalmonella spp., Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus) were determined. The testing method and the interpretation of the results were performed according to the European Pharmacopoeia, 10th Edition [For microbial contamination control, the total count of the CFUs of aerobic microorganisms  and the absence of pathogenic or conditionally pathogenic microorganisms . The plates were incubated at 35 °C for 24 h.The working material was represented by 10 g of the tested formulation, diluted in ethanol (EtOH) 10%. The lack of anti-microbial activity for EtOH 10% was tested using the disk-diffusion method. Blank paper disks , that 15 µL of EtOH was pipetted onto, were placed on the surface of a Mueller-Hinton agar, pre-inoculated with a 0.5 Mc Farland microbial suspension of reference strains  or black colonies, characteristic of Salmonella spp., were absent. For the identification of Escherichia coli, 10 mL of the 1/10 dilution of the tested sample was suspended in 100 mL of lactose broth, incubated at 45 °C for 24 h, and then re-inoculated on Mac Conkey solid media. After incubation, the lactose-positive (red) colonies characteristic of Escherichia coli were absent. For the identification of Pseudomonas aeruginosa, 10 mL of the 1/10 dilution of the tested sample was added to 100 mL of 2% glucose broth, incubated at 37 °C for 24 h, and then re-inoculated on the Istrati-Meitert solid media and on Pseudomonas agar. After incubation at 37 °C for 24 h, the characteristic colonies of Pseudomonas spp.  were absent. In 100 mL of Chapman broth, 10 mL of the 1/10 dilution of the tested sample was inoculated and incubated at 37 °C for 24 h, and then re-inoculated on Columbia agar +5% sheep blood (COS) and on Chapman agar. After incubation at 37 °C for 24 h, cream-yellow colonies on COS, often with hemolysis, or yellow colonies associated with a change in the color of the media in yellow, characteristic of Staphylococcus aureus, were absent.To determine the total count of CFU, 1 mL of the 1/10, 1/100, and 1/1000 dilutions was pipetted onto sterile Petri dishes; then, 15 mL of melted agar (simple agar for bacteria or Sabouraud for fungi) was added, at a temperature of approximately 45 °C. After homogenizing the contents, the plates were left at room temperature until the agar solidified, and then they were incubated accordingly (48 h at 35 °C for bacteria and 7 days at 25 °C for fungi). To determine the total count of CFU of coliforms, 1 mL of each dilution  was added to 10 mL of Mac Conkey broth and lactose broth, respectively (2 series with 3 tubes/dilution); then, the tubes were incubated at 37 °C and 45 °C for 48 h. To identify Staphylococcus aureus ATCC 6538P, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, Candida albicans ATCC 10231, and Aspergillus niger ATCC 16404 . For the PET, the microbial strains were selected according to the European Pharmacopoeia: 8 microorganisms/mL for all of the tested strains. A total of 0.1 mL from each suspension was inoculated in 20 g of the tested product, resulting in a final microbial concentration of 105–106 microorganisms/g. After incubation at 25 °C, 1 g of the inoculated product was dispersed on the surface of the culture media  immediately and at 6 h, 24 h, and 48 h after inoculation of the tested product. The microbial suspension was prepared at approximately 10A total of twenty volunteers who met the eligibility criteria were recruited by the dermatologist and enrolled in the study. This study was approved by the research ethics committee of the Victor Babes University of Medicine and Pharmacy Timisoara, Romania (Nr. 04 a/17 June 2022), and complied with the rules of good practice in biomedical research and with the principles established by the Declaration of Helsinki . Some inAt the initial dermatology consultation, one FP was removed from each participant and processed in the pathology laboratory (this was time frame 0). Afterward, the subjects applied the test product (OEO-PbH) twice a day for two months on the identified FPs. Patients were instructed to apply a small pea-sized amount of the tested product using a plastic applicator (received together with the OEO-PbH formulation) on the identified FPs. The study was conducted over eight weeks, and during which, the subjects came to the dermatologist’s office every two weeks for a non-invasive evaluation of the FP area. The following skin parameters were evaluated: erythema index, TEWL, and SWC. After one month of treatment with OEO-PbH, a second FP was removed from each participant and processed in the pathology laboratory (this was the time frame of 30 days). After two months of treatment with OEO-PbH, a third FP was removed from each participant and processed in the pathology laboratory (this was the time frame of 60 days). The purpose of these evaluations was to observe whether there were changes in FP composition and structure at the histological level.®MX18 probe, Tewameter®TM300 probe, and Corneometer®CM 825 probe, respectively, was used to evaluate erythema index, TEWL, and SWC, respectively. All of the measurements were performed using the same operator, in predefined temperature (20–23 °C) and humidity conditions (40–60%). The subjects were asked to wait for at least 15 min in the test room before starting the experimental part for the skin to adapt to the indoor environment. Measurements were taken in the same tested area and performed in triplicate, further using the average values.Three physiological parameters were evaluated in order to assess the skin response to OEO-PbH by means of non-invasive methods previously described by Simu et al. . A MultiAfter the FPs were removed from each of the twenty participants in the study, they were sent to the pathology lab for histological analysis. After grossing and histoprocessing, the paraffin-embedded FPs were then cut into thin sections, typically between 4 and 6 microns thick, using a microtome. The sections were placed on glass slides and then stained with H&E stain, which highlights the nuclei and cytoplasm of cells in the tissue. The H&E-stained slides were then examined under a microscope in order to evaluate the FPs for various histological features. This included assessing the composition of the fibrous core, the presence of hyperplasia or papillomatosis in the overlying epidermis, and the presence of other histological changes such as keratotic cysts or pigment in basal epidermal keratinocytes. This process was repeated for all twenty participants in the study, with three FPs per participant being analyzed histologically at different time points .p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).GraphPad Prism 8.01 software  was used in order to evaluate the measurements. Data were represented as mean ± SD and the results were analyzed by means of one-way ANOVA and Dunnett’s multiple comparison test  was highlighted. No change in organoleptic properties, pH, OEO content, or consistency of the Pluronic F 127/L 31 binary hydrogel loaded with 5% OEO was observed. The consistency tests (penetrometry and spreadability) confirmed that the two key rheological attributes promoting patient acceptability and therapeutic efficacy (prolonged residence time on the treated area and ease of application to the skin) were maintained after 6 months of storage at room temperature. Moreover, the hydrogel showed a lack of microbial contamination. The two-month-long clinical study  showed that OEO-PbH can be a therapeutic option for FPs smaller than 5 mm. This study, together with the previous ones quoted within this manuscript, offers a comprehensive scientific picture of a non-invasive modern pharmaceutical formulation for the management of FPs.


Diamond–Blackfan anemia (DBA) is a congenital bone marrow failure syndrome associated with malformations. DBA is related to defective ribosome biogenesis, which impairs erythropoiesis, causing hyporegenerative macrocytic anemia. The disease has an autosomal dominant inheritance and is commonly diagnosed in the first year of life, requiring continuous treatment. We present the case of a young woman who, at the age of 21, developed severe symptomatic anemia. Although, due to malformations, a congenital syndrome had been suspected since birth, a confirmation diagnosis was not made until the patient was referred to our center for an evaluation of her anemia. In her neonatal medical history, she presented with anemia that required red blood cell transfusions, but afterwards remained with a stable, mild, asymptomatic anemia throughout her childhood and adolescence. Her family history was otherwise unremarkable. To explain the symptomatic anemia, vitamin deficiencies, autoimmune diseases, bleeding causes, and myeloid and lymphoid neoplasms were investigated and ruled out. A molecular investigation showed the RPL5 gene variant c.392dup, p.(Asn131Lysfs*6), confirming the diagnosis of DBA. All family members have normal blood values and none harbored the mutation. Here, we will discuss the unusual evolution of this case and revisit the literature. GATA1 or TSR2, and there is only one case described with a recessive erythropoietin (EPO) mutation that presented with the typical histological findings of DBA. These entities are sometimes referred to as DBA-like [Diamond–Blackfan anemia (DBA) is a rare constitutional congenital bone marrow failure syndrome mostly caused by mutations in ribosomal genes resulting in ribosomopathy, which leads to progressive macrocytic normochromic anemia and erythroblastopenia . TypicalDBA-like . A few DDBA-like . The patDBA-like . AdditioDBA-like . It is rDBA-like .GATA1, which is an important erythroid transcription factor regulating processes in erythroid differentiation and maturation [HSP70 proteins, which act as chaperones for GATA1, also contributes to ineffective erythroid differentiation. The tumor suppressor p53, through its relationship with GATA1, also plays a role in the pathophysiology of DBA. The models of human primary cells have shown that mutations in RPS11 and RPS19 are associated with the stabilization and increased phosphorylation of p53, leading to the activation of its targets, and ultimately promoting cell cycle arrest and apoptosis [GATA1 acts as an inhibitor of p53 [RPL5, RPL11, and RPS19) could show a downregulation of its chaperone HSP70, leading to the increased phosphorylation of p53. The transduction of HSP70 into RPL11-haploinsufficient CD34+ cells leads to a significant increase in the proliferation of burst forming units and colony forming units [Another influencing factor is that mutations in some RP genes lead to a reduced expression of turation . Furtherpoptosis . It is kng units .The reduced globin synthesis is another mechanism considered in the physiopathology of DBA and consequently, the relation of globin to heme diminishes, raising the amount of free intracellular heme . Free inThe DBA phenotype and genotype is subject to ongoing research . We presA 21-year-old female patient of Tamil descent was presented in July 2021 to her family doctor due to orthostatic complaints. Persistent moderate macrocytic anemia was diagnosed. The hemoglobin was initially 82 g/L and the MCV 99.2 fl, and other values of the complete blood counts (CBC) were normal. A vitamin B12 deficiency was diagnosed and treated accordingly. After the substitution of cumulative 5 mg vitamin B12 over two months, the hemoglobin was 75 g/L with unchanged red blood cell (RBC) indices and the patient was referred to the hematology department of a tertiary hospital for further investigations. In June 2022, we saw the patient for the first time in the outpatient clinic; she was 21 years old and was in good health, but she complained of progressive fatigue. In the clinical examination, she was 145 cm tall and her weight was 40 kg (BMI 19.0). She has a series of congenital malformations  and no oThe review of her medical files showed that at birth, after an uncomplicated pregnancy, she had a ventricular septal defect, a double thumb on the right side, relative macrocephaly, and a lateral cleft lip. At the age of 10 weeks, she was hospitalized due to anemia-related heart failure. Her condition stabilized after transfusions. An elevated level of erythroid adenosine deaminase (eADA) activity suggested an underlying diagnosis of DBA, but at that time, genetic testing was not performed. As her hemoglobin levels were subsequently stable (above 80 g/L), therapies such as steroids were not administered. In childhood and adolescence, the hemoglobin values were always stable in the range of 85–100 g/L. There was a spontaneous closure of the ventricular septal defect around the age of 5 years with asymptomatic cardiac performance.Due to her short stature , she was subjected to somatropin supplementation from the age of 5 7/12 to 11 7/12. Under this regimen, she grew 28 cm (from 98 cm to 126 cm), after stopping this treatment, she reached a height of 145 cm . The cleft lip was surgically corrected with good results; the polysyndactyly does not limit the patient in her daily life.To better characterize the worsening of the anemia observed since 2021, bone marrow (BM) investigations were performed. The aspiration showed scarce spicules and provided little information. The BM biopsy revealed normal cellularity for her age, but a highly atypical CD4-predominant T-lymphocytosis , an impairment of the maturation of all three cell lines, and mild myelofibrosis (maximum MF-1). The molecular pathological examinations showed no evidence of a clonal rearrangement of TCR gamma. A PET/CT scan showed no evidence of lymphoma. The karyotype analysis was unsuccessful due to insufficient metaphases, and recurrent findings typical of a myelodysplastic syndrome were not detected in fluorescence in situ hybridization (FISH). In the array-CGH, a copy neutral loss of heterozygosity of the short arm of Chromosome 3 and 8 was shown. Due to the suspicion of evolution in a myeloid disease, an NGS using the fragment analysis of 65 genes and 300 known recurrent rearrangements was evaluated. No somatic mutation was identified.Finally, due to the consistent suspicion of a bone marrow failure syndrome, a heterozygous mutation in the RPL5 gene, variant c.392dup, p.(Asn131Lysfs*6), was detected with the TWIST Comprehensive Exome (Twist Bioscience) NGS panel. Combined with the clinical features of the patient, the diagnosis of DBA/Aase-Smith Syndrome II could be confirmed. In the family history, all relatives have a normal height according to their ethnic background; there was no history of malformations or anemia in her parents, sister, grandparents, uncles or aunts. The genetic testing of the parents and sister did not show the mutation and their CBCs were within the normal range .During follow-up, a worsening of the anemia was observed. At that time, the diagnosis of sarcoidosis was suspected and she received short-term steroids (prednisolone). Once the diagnosis of sarcoidosis was ruled out, the steroid treatment was quickly discontinued. This short-term administration of the steroid did not improve the anemia. The nadir values of the hemoglobin were 58 g/L and RBC transfusions were needed. The transfusion requirement was of 2 RBC units every 5 months. A 3-month trial of Eltrombopag using an aplastic anemia dose was initiated and was well-tolerated but failed to improve the anemia and was therefore withdrawn. In the last 12 months, a further worsening of the anemia (nadir Hb 40 g/L) with a greater transfusion requirement of 2 RBC units every 3 months was observed. A new attempt with steroids (0.5 mg/d) was made, and this time for longer time. A total of 28 days after the start of therapy, a reticulocyte response of 51 G/L (previously 10 G/L) with the stabilization of Hb values above 80 g/L was observed. Currently, steroid dose tapering is ongoing. The relevant aspects of this case are summarized in RPL5 gene, c.392dup, p.(Asn131Lysfs*6), was detected. This frameshift mutation leads to an early stop codon, and thus, nonsense-mediated mRNA decay. Pathogenic variants in the RPL5 gene are associated with DBA [We present here a DBA case in which anemia was diagnosed a few weeks after birth, but afterwards, the patient had an indolent course until the age of 20, when the patient began to show a clear worsening of the anemia, thus requiring transfusions and treatment. Although, due to her malformations, a congenital syndrome was suspected since birth, a confirmation diagnosis was not made until referral to our center for an evaluation of the anemia. After the exclusion of a myelodysplastic syndrome, lymphoma, and inflammatory diseases, a heterozygous gene variant in the with DBA . The cliup, p.Asn31Lysfs*6The majority of DBA cases will present with severe anemia during the first year of life and they will require continuous treatment . There aFurthermore, patients with inherited bone marrow failure syndromes (IBMFSs) are more likely to develop secondary malignant diseases compared to the general population. In DBA, this is not only true for MDS and AML, with an observed/expected ratio of 28.8 and 352.1, respectively , but alsRegarding treatment for DBA, 80% of patients initially respond to steroids. A trial of 2 mg/kg/d is frequently used; reticulocytes will typically appear after two weeks in responders. If there is no response after four weeks, the treatment should be discontinued. If there is an increase in reticulocytes and hemoglobin levels, steroids should be decreased to the minimal dosage to maintain a hemoglobin of 90 g/L. This dose should not exceed 0.3 mg/kg/d if erythrocyte transfusions are easily available (0.5 mg/kg/d if they are not) . Due to RPS19 mutation who was refractory to steroids and transfusion-dependent was included and became transfusion-independent even 10 months after the discontinuation of eltrombopag before relapsing. This patient regained a response after re-establishing the treatment for the whole follow up of 5 years [Eltrombopag is an orally available mimetic of thrombopoetin, which is a regulator of platelet production and the maintenance of human stem cells. Currently, eltrombopag is used for immune thrombocytopenic purpura , thrombo 5 years . These fMore awareness of unusual DBA presentations is needed. While testing for the causative mutation in DBA is not necessary to establish the diagnosis, it is important for the follow-up of these patients, due to the increased risk of transformation into myeloid diseases and other tumors, as well as for genetic counseling. Multidisciplinary clinical follow-up is also necessary for the management of non-hematological problems. Furthermore, more data about the underlying genetic profile will allow for a better understanding of the geno- and phenotype relationship in this heterogenic disease.


Carm1can direct MuSC renewal. In the absence of MPP7, TAZ can support regenerative progenitors and activateCarm1expression, but not to a level needed for self-renewal. Facilitated by the actin polymerization-responsive AMOT, TAZ recruits the L27 domain of MPP7 to up-regulateCarm1to the level necessary to drive MuSC renewal. The promoter of Carm1, and those of other common downstream genes, also contain binding site(s) for YY1. We further demonstrate that the L27 domain of MPP7 enhances the interaction between TAZ and YY1 to activateCarm1. Our results define a renewal transcriptional program embedded within the progenitor program, by selectively up-regulating key gene(s) within the latter, through the combination of protein interactions and in a manner dependent on the promoter context.Stem cells regenerate differentiated cells to maintain and repair tissues and organs. They also replenish themselves, i.e. self-renewal, for the regenerative process to last a lifetime. How stem cells renew is of critical biological and medical significance. Here we use the skeletal muscle stem cell (MuSC) to study this process. Using a combination of genetic, molecular, and biochemical approaches, we show that MPP7, AMOT, and TAZ/YAP form a complex that activates a common set of target genes. Among these targets,


Acute pain management after open abdominal surgeries is an essential goal in perioperative management.. Recently, serratus-intercostal plane block (SIPB) was suggested as an analgesic technique for upper abdominal surgeries.This prospective, randomized, controlled study included sixty adult patients scheduled for open upper abdominal surgeries. Patients were allocated into two equal groups: SIPB group  and control group . In the S group, SIPB was performed in the midaxillary line at the eighth rib level followed by continuous infusion of local anaesthetic for the first postoperative day. In the C group, no block was done. The primary objective of the study was to control postoperative pain on the first postoperative day as assessed by the numerical rating scale (NRS). Secondary outcomes included perioperative hemodynamics, total postoperative analgesic consumption, number of analgesic requests, and incidence of postoperative nausea and vomiting.P < 0.001). The postoperative morphine consumption was lower in the S group than in the C group . The incidence of PONV was significantly lower in the S group than in the C group .The mean postoperative NRS reported in group S was statistically lower than that in group C (2.4±0.7, 3.9±0.31, SIPB was associated with a better analgesic profile compared with the control group after upper abdominal surgeries. Further studies are recommended to determine block safety in special patient groups, including bariatric and laparoscopic surgeries. Diverse analgesic modalities have been used to control pain after abdominal surgeries; including neuraxial techniques, abdominal wall block, and systemic analgesics.3 As an alternative, abdominal wall blocks have been used for different open and laparoscopic abdominal surgeries; however, the ambiguity of the sonographic view in obese patients and the fear of iatrogenic injury to the viscera could complicate the procedure.4The epidural technique, which is the gold standard analgesia technique for abdominal surgeries, is limited by technical difficulties, hemodynamic effects, and contraindications in certain groups of patients.th to the 11th intercostal nerves, providing adequate analgesia for the control of pain after upper abdominal surgeries.5 The suggested advantages of SIPB include the ease of the technique, the solid landmarks, and the possible  administration in the supine position.6Recently, few studies have reported serratus-intercostal plane block (SIPB) as an effective analgesic technique for upper abdominal surgeries. The diffusion of local anaesthetic (LA) in this fascial plane blocks the cutaneous branches of the 7In this study, continuous SIPB; using catheter technique was investigated as an adequate analgesic option for patients undergoing elective open abdominal surgeries. The primary objective of the study was to control postoperative pain assessed by the numerical rating scale (NRS) on the first postoperative day. Secondary outcomes included perioperative hemodynamics, postoperative analgesic consumption, number of postoperative analgesic requests, and postoperative nausea and vomiting (PONV).This prospective, randomized, controlled study was conducted after Medical Research Ethics Committee, Institutional Review Board of Mansoura University approval  and clinical trial registry (PACTR202105543447214). Informed consent was obtained from eligible candidates, including all adult patients of American Society of Anaesthesiologists physical status I and II, who were scheduled for open abdominal surgeries through a unilateral incision. Patients with chest wall deformities, neuromuscular disease, and a known allergy to the study medications were excluded from the study.7; SIPB Group  and control group . All patients were subjected to routine preoperative assessment, including medical history, physical examination, and laboratory investigations, and additional assessment was performed as per attending anaesthesiologist’s recommendations. All included patients were educated about the use of a 10-mm NRS for pain assessment .A randomization list, in blocks of 10, was used to allocate patients into two equal groups -1 of fentanyl, 1-2 mg kg-1 of propofol, and 0.5 mg kg-1 of atracurium. A properly sized endotracheal tube was inserted and fixed in place after confirmation of correct positioning. Anaesthesia was maintained using sevoflurane in 40% oxygen-air mixture.Upon arrival to the operative room, patients were monitored using basic monitors . General anaesthesia was induced by intravenous (IV) administration of 2 µ kgth rib in the midaxillary line. An 80-mm Tuohy needle was introduced via an in-plane approach until the needle tip was adjacent to the 8th rib between the serratus and external intercostal muscles. A test for correct placement of the needle was performed using 2-3 mL of normal saline, and then 20 mL of 0.25% bupivacaine was injected between the external intercostal and the serratus anterior muscle after negative blood aspiration. After bolus injection, a 20-gauge catheter  was inserted into the interfacial plane and fixed in place using adhesive tape  was placed sagittally at the 8ive tape .After catheter insertion and fixation, a continuous infusion of 0.125% bupivacaine was initiated, at a rate of 4 mL h, through the catheter using an elastomeric infusion pump, with no injection was performed in C group.-1) was administered. Patients were admitted to a high dependency unit for 24 h monitoring after surgery. Thereafter, the catheter was removed and its site was inspected for any sign of edema, redness, hematoma, or discharge.For both groups, IV 50 µg fentanyl bolus was administered when intraoperative rescue analgesia was deemed indicated by the attending anaesthetist or if the hemodynamic measurements increased by >20% of the basal recording (in the absence of other causes). Additionally, 1 g of acetaminophen was given 30-minutes before skin closure in both groups. After skin closure, the patient was extubated upon fulfilling the extubation criteria and adequate reversal of the muscle relaxant. The postoperative analgesia order included regular IV paracetamol (1 g/6 h) administered to all patients. If postoperative NRS ≥4, a rescue dose of IV morphine  measured at induction, 15 min, 30 min, and hourly until end of surgery, analgesic bolus requirements, NRS , analgesic bolus requirements, incidence of PONV, and any catheter site complications. Data were collected and recorded by an independent nurse trained in the study protocol.8 the mean postoperative pain score after upper abdominal surgeries was (NRS 4.8±1.6). Adopting a 25% reduction of the mean pain score after SIPB as an accepted effect size to attain a study power of 0.80 with an alpha error of 0.05; 27 patients per group were required. Adapting for 10% dropout, 30 patients were sufficient. G*power software version 3.1.9.7 was used for the sample size calculation.Depending on the results of a previous study,P value was considered significant if less than 0.05.IBM SPSS (USA) version 22 was used for statistical analysis of the collected data. Data were explored for normality of distribution and presented as mean ± standard deviation, median (minimum-maximum), or number (percentage). Statistical differences between the two studied groups were analyzed using the independent samples t-test, Mann-Whitney test, or chi-square test as appropriate. In this study, 63 patients scheduled for open upper abdominal surgery through a unilateral incision were recruited from the study from May 2021 to July 2022. As shown in Patient basal data and perioperative characteristics, duration, incision type, and type of surgery were comparable between the two groups . PeriopeP < 0.001]. Postoperatively, the reported NRS was statistically lower in group S than in group C at 0, 2, 4, 8 hours after surgery. Likewise, a statistically significant reduction in both the mean postoperative NRS value and the total postoperative morphine consumption (mg) was noticed lower in group S than in group C . In addition, P 0.045 respectively).None of the cases in group S showed any edema, redness, hematoma, or discharge at the site of catheter insertion.In this study, continuous SIPB was used for perioperative analgesia open abdominal surgeries through a unilateral incision. Compared with IV opioid, SIPB showed a better analgesia profile, less postoperative morphine consumption and decreased incidence of PONV. To the best of our knowledge, this is the first study to apply continuous SIPB for analgesia after unilateral abdominal incisions.10 Inadequate analgesia is associated with higher rates of postoperative complications, delayed ambulation, and prolonged hospital stay.11Acute pain management after open abdominal surgeries is an essential goal in perioperative management, especially in the era of enhanced recovery programs.15Analgesic practices for abdominal incisions include a wide range of techniques. IV analgesics either bolus or continuous infusion were associated with respiratory depression, delayed intestinal motility, and PONV. Opioid crisis worldwide have triggered the use of opioid-saving strategies, including neuraxial, regional, and facial plane blocks.17Neuraxial analgesic techniques, including intrathecal, epidural and paravertebral blocks, have proven to have great analgesic efficacy. Nevertheless, neuraxial procedures are considered technically difficult, and they also carry some risk in special groups of patients, e.g., stenotic valve diseaser receiving anticoagulants.20Facial plane blocks, such as transversus abdominal plane (TAP) and rectus sheath blocks, have been proven to decrease pain scores after abdominal surgeries, especially with the evolution of ultrasound technology. However, difficulties are usually encountered in obese patients and in patients with previous incisions that obscure the proper visualization of the interfacial planes or are even disturbing. In addition, preemptive use of TAP block can cause escape of LA outside the targeted space after surgical incision. In addition, serious complications such as vascular injury, abdominal visceral injury, liver trauma, and nerve injuries have been reported.21 using the acronym BRILMA block . Advancement of the technique follows, with subsequent modification to target the lower intercostal nerves at the level of the 8th rib; modified BRILMA block.  The block was effective for somatic analgesia after gastrectomy and cholecystectomy in a small series of cases.22 Afterwards, more than one study reported the effectiveness of SIPB for open upper abdominal surgeries.23 SIPB lacks an analgesic effect on visceral pain, yet control of the somatic component of postoperative pain can result in satisfactory pain scores and minimize the required rescue analgesics.The first description of SIPB was in 201324 Compared with the oblique subcostal TAB block; SIPB compared with the rectus sheath block significantly improved the quality of analgesia and lowered the analgesic requirement in patients undergoing laparoscopic cholecystectomy.5 Interestingly, bilateral SIPB was effectively used as a rescue analgesia after bariatric surgery, achieving an adequate NRS pain score after 10 min and lasting for 46 hours.24Several studies have used the SIPB bolus technique for analgesia after unilateral abdominal surgeries.23In a prospective randomized study, 102 patients were divided into a control group receiving IV morphine and an interventional group receiving SIPB. The SIPB group showed lower pain scores, lower postoperative opioid consumption and improved quality of recovery (QoR-15 scores) 24 h postoperatively.25 Lower PONV can increase patient satisfaction and shorten the in-hospital length of stay.26PONV is an important patient outcome that was recorded in our study and was found to be significantly lower in patients who received SIPB. This can be explained by the lower postoperative opioid consumption, which is included in the Apfel score as one of the risk factors for PONV.Limitations of the study included the heterogeneous group of patients with different surgical approaches and incisions and variable pain profiles. However, this can support the wide applicability and effectiveness of the SIPB. Secondly, the use of an elastomeric pump, which has a fixed preset infusion rate, should be used in all patients, rather than individualized. Nevertheless, simplicity, non-electricity, and better patient mobilization motivated the authors to use an elastomeric pump instead of electricity-driven syringe pumps. Assessment of dermatomal distribution of analgesia was limited by the surgical wound and dressing. The serum level of LA was not measured in our study, the LA concentration reached after bolus and continuous infusion in SIPB can assess the degree of systemic absorption, verify block safety, and help determine the mechanism of action of the block. A double-blind protocol could avoid data collection bias and the placebo effect; nevertheless, this could not be achieved in this study because of the nature of the intervention.Continuous SIPB can be added to the arsenal of analgesic techniques used for analgesia after abdominal surgeries with a unilateral incision. In our study, SIPB was associated with a better analgesic profile and lower analgesic consumption. Further studies are recommended to determine block safety in special patient groups, including bariatric and laparoscopic surgeries.


Sal), the main component of a famous herb Rhodiola rosea L, enhances memory performance and reduces fatigue. Therefore, this study assessed the effect of Sal on memory impairment induced by a long-term intake of ethanol (EtOH) in rats and investigated its relevant mechanisms using gut microbiota metagenomic analysis and hippocampal transcriptomic analysis.Salidroside . The rats in the Model and Sal groups intragastrically (i.g.) received 2 g/kg EtOH for 30 consecutive days, whereas the CON group was given an equal volume of distilled water. Meanwhile, the rats in the Sal group were administered i.g. 30 mg/kg Sal 60 min after EtOH intake. All rats were tested in the eight-arm maze for their memory function every 3 days. On the 30th day, metagenomic analyses of gut microbiota and transcriptomic analyses of the hippocampus were performed.Eighteen male SD rats were divided into the normal control group (CON group), EtOH model group (Model group), and Sal treatment reduced the total time to complete the eight-arm maze task, decreased the number of arm entries, and abated the working memory error that was significant from the 9th day. Additionally, Sal intervention improved the gut microbiota composition, such as the increased abundance of Actinobacteria and Bifidobacterium, which was related to the metabolism of amino acids and terpenoid carbohydrate, endocrine function, and signal transduction by neurotransmitters. In the hippocampus, the EtOH intake differentially expressed 68 genes , compared with the CON group, whereas Sal intervention affected these changes: 15 genes increased whereas 11 genes decreased. And, enrichment analyses revealed these genes were related to the structural components of the ribosome, mRNA splicing process, protein translation, mitochondria function, and immunological reaction. Finally, a correlation analysis found the memory impairment was positively correlated with the abnormal upregulation of Tomm7 but negatively correlated with decreased abundance of gut Alistipes_indistinctus, Lactobacillus_taiwanensis, Lactobacillus_paragasseri, and Lactobacillus johnsonii.Compared with the Model group, Sal improved memory impairment caused by long-term EtOH intake in rats, which may be related to its regulation of gut dysbiosis and hippocampal dysfunction. In recent times, long-term alcohol use has imposed a tremendous disease burden on human health worldwide. Long-term alcohol use is associated with more than 200 diseases . The lonLong-term alcohol use leads to neurological dysfunction in different brain regions, such as prefrontal cortex, hippocampus, and amygdala, which are involved in memory processes . The hipLong-term alcohol intake leads to disturbed gastrointestinal function, which can cause diverse symptoms in the body, including high cognitive dysfunction . The gutRhodiola rosea L is a perennial herb, which has therapeutic effects on mental disorders, such as anxiety and depression  is the main component of Rhodiola rosea L, and like its parent herb, it can enhance mental strength and reduce fatigue , dopamine (DA), serotonin (5-HT), and acetylcholine (Ach), to stimulate CNS activity, especially enhance learning and memory  test. After the confirmation of Sal therapeutic effects, the relevant mechanisms were explored by metagenomic analyses of gut microbiota and the hippocampus. Additionally, a triple correlation analysis was performed between behavior and gut microbiota changes and hippocampal gene expression changes. This work not only provides a basic research evidence for the development of Sal as a promising candidate and potential strategy to treat memory deficit in alcoholic conditions, but also address the correlation between the gut microbiota and the hippocampus functionally interact to mediate memory impairment caused by a long-term ethanol consumption.Taken together, the above evidence points to a possible therapeutic role of Sal group. Briefly, in the subsequent 30 days, the Model and Sal groups were continuously administered with EtOH  by the intragastric (i.g.) route. The Sal group was administered with Sal  60 min after alcohol intake, whereas the CON and Model groups were given the same volume of distilled water. Memory was assessed by the EAM behavior experiment on days 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 of the experiment. Sal (purity ≥98%) was purchased from Shaanxi Wanyuan Biotechnology Co .Eighteen SD rats  were purchased from the Animal Laboratory Center of Qiqihar Medical University. All rats were individually housed with controlled diet and free access to water on a 12-h dark/light cycle at a constant humidity of 55 ± 5% and temperature of 22 ± 2°C. All experimental protocols were approved by the Animal Ethics Committee of Qiqihar Medical University (QMU-AECC-2023-87). The alcoholism modeling method was slightly modified based on the experiment of Cognitive Status Measurement was slightly modified based on the experiment of Rats were trained once a day for 7 days prior to testing, while maintaining 80% of their normal feeding throughout the experiment. At the beginning of training, rats were placed on the central platform of the maze for 15 s, followed by removal of the eight radiating arm baffles. Rats freely chose to enter the end of the radiated arm for foraging for 10 min or completed foraging for all arms prematurely, indicating the end of training. Following each training session, the maze was wiped with acetic acid to eliminate the influence of odors on subsequent testing. Food was placed only at the end of arms 1, 2, 5, and 7 (working arm), and no food was placed in arms 3, 4, 6, and 8 (reference arm) throughout the test. The order of placement of the entire food arm remained unchanged in this experiment. Repeated entries into the working arm were recorded as working memory errors (WMEs).Test indicators were as follows:(1) Total time for rats to complete all working arms of the eight-arm maze: time to pass arms 1, 2, 5, and 7. If not completed within 10 min, time was recorded as 10 min. (2) Total number of working memory errors: the total number of errors within 10 min; error was defined as repeated entry into the same working arm. (3) Total number of arm entries: total number of arm entries of rats in a test.Changes were detected and analyzed on experimental days 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30. The specific experimental flow is shown in At the end of the behavioral testing on day 30, the rats were decapitated immediately to collect blood samples. Rat skull was dissected, and the hippocampus was rapidly collected. Additionally, 2 g of intestinal contents were collected from all rats and rapidly frozen in liquid nitrogen and transferred to a −80°C freezer. All operations were performed on a sterile ice bath.The metagenomic sequencing of intestinal contents was performed according to this order: sample DNA extraction, library sequencing, data quality control, species annotation and analysis, gene set construction and analysis, functional annotation analysis, and other processes. In this project, metagenomic analysis was performed using the Wekemo Bioincloud .① Sample extraction testing and sequencing of intestinal flora® Ultra ™ DNA Library Prep Kit for Illumina . Qualified DNA samples were randomly interrupted into fragments of approximately 350 bp in length using a Covaris ultrasonic disruptor , and the entire library preparation was completed by end repair, deployment A tail, sequencing adaptor, purification, and PCR amplification of the DNA fragments. Finally, the AMPure XP system purified the PCR products, detected insert size of the library using Agilent2100 , and quantified library concentration using real-time PCR . Indexed coding samples were clustered on the cBot Cluster Generation System using the Illumina PE Cluster Kit . Following cluster generation, DNA libraries were sequenced on the Illumina Novaseq 6000  platform, and 150 bp double-ended reads were generated.Genomic DNA was extracted from intestinal content samples using the CTAB method. Libraries were constructed using the NEB Next ② Data analysisData quality control and de-host sequence: Metagenomic sequencing was performed using the Illumina NovaSeq high-throughput sequencing platform to obtain metagenomic raw data for bacteria, fungi, and archaea in intestinal content samples. Raw sequencing data were pre-processed using the Knead data software.③ Species and functional annotationsKraken2 and self-built microbial nucleic acid databases  were aligned to calculate the number of sequences containing species in each group of samples, and then Bracken was used to predict the actual relative abundance of species in the samples. Sequences after quality control and de-hosting were aligned (DIAMOND based) to the Protein Data Bank (UniRef90) using the HUMAnN2 software. PCoA analysis was based on specie abundance tables and functional abundance tables. Lefse analysis and mining were performed to detect differences in species composition and functional composition between samples .④ Resistance gene annotatione-value <1e-3) -i80 (identity percent >80%)]. The relative abundance of antibiotic resistance genes in each group of samples was obtained from the alignment  .① Sample collection and preparation® UltraTM RNA Library Prep Kit (Illumina), and the resulting mRNA was subsequently randomly interrupted with divalent cations in the NEB Fragmentation Buffer. The fragmented mRNA was used as a template and random oligonucleotides as primers to synthesize the first strand of cDNA in the M-MuLV reverse transcriptase system, followed by degradation of the RNA strand with RNaseH and synthesis of the second strand of cDNA with dNTPs under the DNA polymerase I system. The purified double-stranded cDNA was end-repaired, A-tailed, and connected to the sequencing junction. The cDNA of 250–300 bp was screened by AMPure XP beads and amplified by PCR, and the PCR products were purified by AMPure XP beads again to obtain the library. After library construction, initial quantification was performed using a Qubit 2.0 Fluorometer , and the insert size of the library was subsequently checked using an Agilent 2100 bioanalyzer. After the libraries passed the test, Illumina sequencing was performed to generate 150 bp paired-end reads.RNA was extracted using standard extraction methods, and RNA samples were tested for RNA integrity using the Agilent 2100 bioanalyzer  for quality control. The mRNA with polyA tails was enriched by Oligo(dT) magnetic beads using the NEBNext② Data quality control and differential expression analysisTo ensure the quality and reliability of data analysis, filtration of raw data is necessary. It mainly includes removal of reads with adapter, removal of reads containing N (N indicates that base information cannot be determined), and removal of low-quality reads (reads with Qphred ≤20 bases accounting for more than 50% of the entire read length). Additionally, clean data were subjected to Q20, Q30, and GC content calculation. All subsequent analyses were based on clean data for high quality analysis. Indexing of the reference genome was constructed using HISAT2v2.0.5, and paired-end clean reads were aligned to the reference genome using HISAT2 v2.0.5. StringTie (1.3.3b) for novel gene prediction. Differential expression analysis between the two compared combinations was performed using the DESeq2 software (1.16.1).③ Enrichment and pathway analysisGO enrichment analysis of differentially expressed genes  was performed using the clusterProfiler (3.4.4) software. Statistical enrichment of differentially expressed genes in KEGG pathways was analyzed using the clusterProfiler (3.4.4) software.All experiments were repeated three times. Data are presented as the mean ± standard error of the mean (SEM). One- or two-way ANOVA followed by Tukey’s post-hoc test was used for multiple comparisons. Data were analyzed using GraphPad Prism 8.0 software . Correlation analyses were performed using R software (V4.2.2).Sal group were more effective during the EAM task , whereas the Sal group had a decreased number of WMEs , whereas Sal intervention reduced the number of arm entries ; ② the scanned sequence (4-bp sliding window size). The removal condition of subsequent sequence was set as average mass score < 20 ; and ③ the sequence with a final length of <50 bp was removed (parameter MINLEN: 50) . Compared to the relatively scattered distribution in the Model group, the distributions in the CON and Sal groups were more concentrated, indicating that Sal intervention had an effect on the composition of the intestinal flora of rats in the model of alcohol-induced memory damage and brought the composition of the intestinal flora close to that of the control group. All valid sequences of the samples were annotated and classified by Kraken2 . If the samples were similar in species composition, the closer they were in the PCoA plot. The results showed  that Axi Kraken2  , and the relationships between different microbial groups, from phylum to species level (LDA = 4), are shown in the branching diagram ,B. The rSal group, three highly expressed metabolic pathways were involved: Endocrine system, Environmental adaptation, and Infectious disease_parasitic.Based on the search of the KEGG (Kyoto Encyclopedia of Genes and Genomes) database for comparative annotations, a KEGG secondary pathway map was obtained . No signSal groups mapped to the rat genome, respectively. Differentially expressed genes were screened by screening out detection rates (proportion of count not 0) <0.25, |log 2 (Fold Change)| > 1 & padj<0.05. The results revealed 68 differentially expressed genes in the Model group, compared with the CON group. Among them, 54 genes had elevated relative expression and 14 genes had decreased relative expression.Quality control processing was performed on the raw data of each sample using the fastp software  to obtaiThe top five relatively highly expressed genes were Cnmd , S100a9 , Tomm7 , Klhl40 , and Lilrb3a . The first five relatively low-expressing genes were AY172581.16 , AY172581.17 , Npsr1 , AY172581.5 , and Rxfp1 . Twenty-six differentially expressed genes were identified in the Sal group, compared to the Model group. Among these genes, 15 genes were elevated and 11 genes were decreased. The top five relatively highly expressed genes were Olr1694 , Ca3 , Synpo2 , AY172581.17 , and Spp1 . The top five relatively lowly expressed genes were Ch25h , Tubb2b , Tomm7 , Naa38 , and Mei1 . The results are shown in Sal and Model groups; they included biological processes (416 subclasses), cell components (122 subclasses), and molecular functions (114 subclasses). Among them, the top five terms were mitochondrial inner member, translation, immediate response, structural constitution of ribosomes, and mRNA splicing, via spliceosome  of the function of genes among the three groups revealed 314 GO terms with significant differences between the Model and CON groups. They included biological processes (197 subclasses), cell components (57 subclasses), and molecular functions (60 subclasses). Among them, the top five terms included mitochondrial inner membrane, translation, structural constituent of ribosome, immune response and cellular response to tumor necrosis factor . The coriceosome . The corTomm7 and Klhl40 mRNA in rat hippocampus. The upregulation of Tomm7 and Klhl40 mRNA was positively correlated with the abundance of Eubacteriumlimosum and negatively correlated with the abundance of Alistipesindistinctus, Lactobacustaiwanensis, Lactobacusparagi, and Lactobacusjohnsonii in the intestine of the Model group. Compared with the Model group, the Sal group had a significantly decreased mRNA expression of Tomm7 in the hippocampus upon administration of Sal. This decreased expression was positively correlated with the abundance of Alistipesindistinctus, Lactobacustaiwanensis, Lactobacusparagi, and Lactobacusparagillusjohnsonii in the intestine of the Sal group.Correlation analyses among data of behavioral, microbial genome, and hippocampal transcriptome were performed using the R package(R 4.2.2) . CompareSal on memory impairment caused by long-term administration of EtOH in rats, which was manifested as the reduction in total time to complete the EAM task, decreased number of arm entries, and abated working memory errors in the EAM tests. Additionally, the metagenomic analysis revealed that the composition and diversity of intestinal flora were significantly altered in rats with memory improvement after Sal intervention, which are mainly associated with metabolism of amino acids, terpenoids, and polyketides. For instance, EtOH caused a decrease in the relative abundance of Actinobacteria, Bifidobacteriaceae, Akkermansiaceae, and Lactobacillus and an increase in the relative abundance of Romboutsia ilealis in the intestinal tract of rats. The relative abundance of microorganisms in their intestinal contents improved with Sal intervention, especially the relative abundance of Bifidobacterium, Ligilactobacillus, Adlercreutzia, and Lactobacillus was increased. These findings confirmed the effect of Sal on intestinal microbes.In this study, we observed the ameliorative effect of Sal acts on the gut-brain axis, a transcriptomic analysis of hippocampal tissue was then performed. The overall results showed that 26 genes were differentially expressed after Sal intervention, compared with the Model group. Furthermore, enrichment analyses revealed that these differential genes were mainly involved in the mitochondrial inner membrane, translation, immune response, structural constituent of ribosome, and mRNA splicing. Finally, the multiple correlation analyses uncovered the correlation among the behaviors, gut microbiota, and changed expression of hippocampal genes. Altogether, these findings provide evidence that Sal can attenuate alcoholic memory impairment, which may be associated with an improvement in gut-brain axis function.To verify whether Sal treatment in rats administered with long-term EtOH. Additionally, according to the most popular theory in recent times, memory improvement by Sal treatment is likely linked to alteration in gut-brain function.Memory is the physiological process of the CNS by which information or data about intra- or extra-environments are encoded, stored, and retrieved when needed. Memory is usually evaluated using the EAM test. In this study, either spatial memory or working memory performance was greatly improved by Sal plays a beneficial immunomodulatory role by regulating immune cell differentiation, inflammatory signaling pathway activation, and inflammatory factor secretion to reduce inflammatory damage in various diseases  [8] or macrophage polarization  and 11 genes with decreased expression . One study found that by rapidly increasing carbonic anhydrase (CA) concentrations in the cortex and hippocampus (two brain regions involved in memory processing), the extracellular signal-regulated kinase (ERK) pathway in the cortex and hippocampus—a key step in memory formation—could be rapidly increased  have been suggested to be probably associated with the pathophysiology of schizophrenia and have a unique role in neuronal differentiation and cell viability. In patients with schizophrenia, Tubb2b protein expression is reduced in the anterior cingulate cortex and increased in the dorsolateral prefrontal cortex; however, it remains unchanged in the superior temporal gyrus or hippocampus. In contrast, alcohol consumption can lead to cytoskeletal changes  is a key regulator of cognition, especially in recognition and aversive memory. A study has found that injecting inhibitors of carbonic anhydrase in the hippocampal CA1 region or mPFC impairs short-term social recognition memory . The afo changes . In the Sal and Model groups revealed 652 significant GO terms between the two groups. The top five terms included mitochondrial inner membrane, translation, immune response, structural constituents of ribosome, and mRNA splicing, via spliceosome. The immune system is an important system that performs immune response and immune function, and alcohol abuse can lead to impairment of the immune system, which can be inherited by offspring through the maternal generation, resulting in severe immune deficiency  also play a role in this process . AnotherAlistipesindistinctus, Lactobacustaiwanensis, Lactobacusparagi, and Lactobacusparagillusjohnsonii in the intestine of the Sal group. Some studies suggest that Alistipes indistinctus can interact with other gut microbiota to influence gut barrier, immune system, and metabolism ; NMDC0000192.The animal study was reviewed and approved by the Ethics Committee of Qiqihar Medical University.YJ, RZ, PL, and XL designed the experiments. YJ, ZZ, TW, LL, TL, XL, and DX performed the experiments. YJ, SW, PL, and XL performed the statistical analysis. RZ, PL, DX, ZZ, and YJ wrote the manuscript. All authors contributed to this article and approved the submitted version.This study was funded by the National Natural Science Foundation of China (82104173), and Qiqihar Academy of Medical Sciences, Grant No. QMSI2019M-09; Heilongjiang Provincial College Students Innovation and Entrepreneurship Project, Grant No. 201911230056; The Science Research Foundation of Qiqihar Medical Institute No. 2021-ZDPY-014; The Science Research Foundation of Qiqihar city, China No. LHYD-2021015; The Science Research Foundation of Qiqihar city, Qiqihar city, China No. LHYD-202011.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Flax is a flowering plant cultivated for its oil and contains various unsaturated fatty acids. Linseed oil is known as the “deep-sea fish oil” of plants, and is beneficial to brain and blood lipids, among other positive effects. Long non-coding RNAs (lncRNAs) play an important role in plant growth and development. There are not many studies assessing how lncRNAs are related to the fatty acid synthesis of flax. The relative oil contents of the seeds of the variety Heiya NO.14 (for fiber) and the variety Macbeth (for oil) were determined at 5 day, 10 day, 20 day, and 30 day after flowering. We found that 10–20 day is an important period for ALA accumulation in the Macbeth variety. The strand-specific transcriptome data were analyzed at these four time points, and a series of lncRNAs related to flax seed development were screened. A competing endogenous RNA (ceRNA) network was constructed and the accuracy of the network was verified using qRT-PCR. MSTRG.20631.1 could act with miR156 on the same target, squamosa promoter-binding-like protein (SPL), to influence fatty acid biosynthesis through a gluconeogenesis-related pathway during flax seed development. This study provides a theoretical basis for future studies assessing the potential functions of lncRNAs during seed development. Flax is an important oil-producing and textile crop; its seeds are extracted for their oil and the stalks are stripped for fiber. Flax is typically classified as oil type, fiber type, or oil and fiber type based on its economic properties and uses . The oilLncRNAs have a length of more than 200 bp and are generally transcripts that cannot encode proteins . They haFatty acids play an essential role in plant growth and development, primarily as a major component of cell membranes and a provider of energy and signaling molecules . Fatty aIn this study, two flax varieties, Macbeth and Heiya NO.14, were used to analyze the relative oil content of the seeds at different developmental time points. Combined with strand-specific transcriptome data at different developmental periods, lncRNAs related to fatty acid synthesis during seed development were screened out. According to the ceRNA theory and a previous small RNA transcriptome data set, the ceRNA network was constructed. Through lncRNA, miRNA, and mRNA correlation analysis, the mechanism of lncRNA related to fatty acid synthesis was further analyzed. The goal is to reveal the relationship between fatty acids and lncRNAs in flax seed development, analyze the mechanism behind how lncRNAs regulate flax fatty acids, and provide a theoretical basis for improving flax fatty acids.In order to clarify the regulation mechanism of flax fatty acid synthesis, oil-flax variety Macbeth (M) and fiber-flax variety Heiya NO.14 (H) were used as study materials. The two varieties were selected because Macbeth has a high oil content (467 g/kg), high linolenic acid content (56.8%), large seed size (6.0 g/1000 seed weight), lodging resistance, disease resistance, and high yield, and it is suitable for a variety of soils; it has been widely cultivated since its development in Canada in 2002 . Heiya 1TM Directed RNA Library Prep Kit for IlluminaR , according to the manufacturer’s recommendations. A total of 16 libraries were constructed for the two varieties at four time points, and index codes were added to each sample in the attribute sequence. The samples at each time point were taken from a fruit mixture of three plants of the same species, with two fruits taken from each plant. To preferentially select inserts of 150–200 bp in length, library fragments were purified with AMPure XP Beads . PCR was then performed with 3 μL of USER enzyme (NEB) incubated with the size-selected, adapter-ligated cDNA for 15 min at 37 °C. PCR was then performed with Phusion high-fidelity DNA polymerase, universal PCR primers, and index (X) primers. The PCR products were finally purified using the AMPure XP system, and the library quality was assessed using an Agilent Bioanalyzer 2100 and qPCR. Clustering of the index-encoded samples was performed in the acBot Cluster Generation System using the TruSeq PE Cluster Kitv3-cBot-HS , according to the manufacturer’s instructions. After cluster generation, library preparations were sequenced on the Illumina Hiseq platform  in order to generate paired-end reads.The total RNA was extracted using a plant tissue RNA extraction kit . RNA degradation and contamination were detected on 1.5% agarose gel. The RNA concentration and purity were measured using a NanoDrop 2000 Spectrophotometer , and the RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 System . After passing a quality control screening, 1.5 μg of RNA was used for each sample for library construction. rRNA removal was performed using a Ribo-Zero rRNA removal kit . Sequencing libraries were generated using the nebnexr UltraLinum usitatissimum ) was used as the reference genome, and TopHat2 [The raw reads of the strand-specific transcriptome were removed from the raw reads containing adapters and those of a low quality , after which the clean read was obtained.  TopHat2  was used TopHat2  was used TopHat2 ,31,32,33During the detection of differentially expressed lncRNAs, DESeq was used for differential analysis , and we p-value < 0.01, and the corrected FDR value < 0.01. The ceRNA network was visualized using Cytoscape 3.8.2 software.From the above analysis results, we found that the amount of differential lncRNA data was most abundant at M5 and M30. To further analyze how lncRNAs are related to fatty acid accumulation during seed development in the Macbeth variety, we selected the strand-specific transcriptome and small RNA transcriptome data of Macbeth with a high oil content at 5 day and 30 day for lncRNA-miRNA-mRNA association analysis and constructed ceRNA networks, in which small RNA and mRNA datasets were obtained from the previous studies in our lab ,41. The −ΔΔCT method [The relative expression of lncRNAs, miRNAs, and mRNAs during seed development was assessed by quantitative real-time polymerase chain reaction (qRT-PCR). The total RNA of lncRNAs and mRNAs was extracted using the RNA Easy Fast Plant Tissue Kit  and the total RNA of the miRNAs was extracted using the UNlQ-10 column-based Trizol Total RNA Extraction Kit . The stem-loop reverse transcription primers for miRNAs were designed using Primer Premier 5.0. SnapGene software was used to design the primers for the lncRNAs and mRNAs. Quantitative analysis was performed using ABI Quant Studio 6 fluorescent quantitative PCR . The relative RNA expression levels were calculated using the 2T method . Three rThe Macbeth (M) and Heiya NO.14 (H) varieties were used in our study a. As theThe raw data of the 16 strand-specific libraries were quality-controlled to obtain 188.35 GB clean reads, with more than 81,000,000 clean reads for each sample, all with a GC content greater than 47% and Q30 above 88% , which iTo more clearly compare the differences between the Macbeth and Heiya NO.14 varieties at each time point, data from 5 day, 10 day, 20 day, and 30 day after flowering were compared, using Macbeth as a control. Two lncRNAs (TCONS_00107244 and TCONS_00245620) were found to be significantly down-regulated at all four stages. We also found the highest number of specifically expressed lncRNAs at 10 day with 24, and the least at 5 day with 6, and 21 at both 20 day and 30 day c.To investigate the differences between Macbeth and Heiya NO.14 during seed development, we used the 5 day data from both varieties as a control and compared them with the data from 10 day, 20 day, and 30 day d,e. We fTo explore the functional differences of lncRNAs between the two cultivars at four time points, we performed functional annotation of the target genes of differential lncRNAs based on the GO database with Macbeth as the control, and the results showed that the cis-target genes of the two cultivars were annotated in 44 terms and the number of terms annotated at each time point was approximately the same. We showed that the GO results were mainly enriched in terms of metabolic process, cellular process, single-organism process, cell, cell part, organelle, membrane, catalytic activity, and binding, etc. a. The KEWe compared the 5 day data of Macbeth and Heiya NO.14 with the 10 day, 20 day, and 30 day data. The results of GO showed that there was no significant difference in terms of the genes enriched at different developmental stages between the two cultivars. The metabolic process, cellular process, and single-organism process, as well as the cell part, cell, and organelle had the most genes, and the number of genes involved in catalytic activity, binding and transporter activity was the highest in terms of molecular function. c. KEGG fInteractions between lncRNAs and miRNAs play an important role in regulating plant growth and development and in responding to stress . To consFlax seed oil is rich in various unsaturated fatty acids, especially ALA . To inveWe analyzed the basic characteristics of lncRNAs in flax, in which lincRNAs were found to be the most abundant, which is consistent with previous findings in soybean , peanut Acetyl COA produced by glucose metabolism is an important substrate in fatty acid synthesis . α-glucaWe found that the relative oil content of both varieties increased from 5–10 day, and we screened two lncRNAs (TCONS_00021781 and TCONS_00134305) that they shared during this process. Functional analysis found that the target genes of TCONS_00021781 were annotated to entries related to chlorophyllase (EC: 3.1.1.14). Chlorophyllase catalyzes the degradation of chlorophyll to form chlorophyll esters, during which a large amount of phytol is released to participate in fatty acid phytate biosynthesis . TherefoLncRNAs have been reported to function as ceRNAs in the post-transcriptional regulation of miRNA-mediated gene expression. Here, for the first time, we constructed a ceRNA network based on strand-specific transcriptome data and small RNA data. The miR156-SPL regulatory module is involved in multiple processes of plant growth and development ,60. The We analyzed the relative oil content of the seeds of the oil flax variety Macbeth and the fiber variety Heiya NO.14 at 5 day, 10 day, 20 day, and 30 day. It was found that the high expression of fatty acid accumulation-related lncRNAs in the Macbeth variety primarily occurred from 5–20 day, while Heiya NO.14 primarily occurred from 5–10 day. In Macbeth, ALA synthesis-related genes were highly expressed at 10–20 day. We performed strand-specific transcriptome analysis of two flax varieties based on the relative oil content trend. Based on the functional annotation information of GO, KEGG, and NR, a group of lncRNAs that may be related to fatty acid accumulation were screened. To further understand lncRNA function, we constructed a ceRNA network and found that MSTRG.20631.1 and miR156 co-regulated SPL6, SPL16, and SPL2, which play an important role in fatty acid accumulation by regulating the glucose metabolism-related pathways during flax seed development. We also validated our network by qRT-PCR. Our study provides a theoretical basis for studying the role of lncRNAs in fatty acid biosynthesis.