Living donor transplantation provides the best possible recipient outcomes in solid organ transplantation. Yet, identifying potential living donors can be a laborious and resource intensive task that heavily relies on the recipient’s means and social network. Social media has evolved to become a key tool in helping to bring recipients and potential living donors together given its ease of utilization, widespread access, and improved recipient’s comfort with public solicitation. However, in the USA, formal guidelines to direct the use of social media in this context are lacking.N = 125/174, 72%) indicated that their program provided education to use social media to identify potential living donors and most programs tracking referral source confirmed an increase utilization over time. The use of social media was compounded with program and recipient’s challenges including concerns about privacy, inadequate technology access, and knowledge gaps. In this review, we discuss the results of this national survey and recent literature, and provide suggestions to inform program practices and guidance provided to patients wishing to use social media to identify potential living donors.To better inform the landscape and opportunities utilizing social media in living donation, the OPTN Living Donor Committee surveyed US transplant programs to explore programs’ experiences and challenges when helping patients use social media to identify potential living donors (September 2019). A large majority of survey participants (Transplant programs should become competent in the use of social media for potential living donor identification to empower patients interested in using this tool. Social media education should be provided to all patients regardless of voiced interest and, when appropriate, revisited at multiple time points. Programs should consider developing a “team of experts” that can provide focused education and support to patients embarking in social media living donor campaigns. Care should be taken to avoid exacerbating disparities in access to living donor transplantation. Effective and timely guidance to patients in the use of social media could enhance the identification of potential living donors.The online version contains supplementary material available at 10.1007/s40472-022-00382-1. Patients in need of a life-saving organ transplant face an uncertain future. In the USA, organ wait times are usually long and variable depending on geography, organ type, and various patient factors –3. PatieData suggest that patients with robust social networks are more likely to succeed at identifying potential living donors , 16. TraThere are several concerns when using social media for living organ donor campaigns which often stem from the lack of supervision, absence of guidelines, and uneven agency among potential users –22. DataIn September 2019, the OPTN Living Donor Committee invited US living donor programs to participate in a survey exploring their (1) social media education and resources; (2) program perceived or reported patient challenges; (3) programmatic concerns and alleged challenges; (4) practice changes in response to experienced challenges; and (5) program preparedness to handle a sudden influx of potential living donors from a social media campaign. The survey questions were a mix of multiple-choice questions, select all that apply , categorical questions, Likert scales, and some allowed participants to provide qualitative answers. Disclosing center name and OPTN region were optional to maximize survey response. The survey was electronically distributed to the OPTN Living Donor, Transplant Administrator, and Transplant Coordinator Committee members, posted publicly on the OPTN and UNOS websites, directly electronically mailed to program directors, transplant administrators, and quality directors of living donor programs, and a link to the survey was included in the September 2019 issue of the UNOS Transplant Pro eNewsletter. An IRB exemption was obtained from the US Department of Health and Human Services Health Resources and Services Administration (HRSA).N = 63) survey responses and 23.6% (N = 41) did not provide an OPTN region. Based of those disclosing their transplant program, the estimated response rate was at least 38.7% (91/235 active living kidney and/or liver donor programs at the time of the survey). Participants were predominantly from kidney transplant programs  and frequently used digital questionnaires to collect health information during the initial screening . Those using digital questionnaires felt these enhanced the process efficiency . Most kept record of the donor referral source  with 62% (N = 97) receiving referrals from social network sites  and 11.6% (N = 18) from donor membership sites. The referral volume through social media was variable with most respondents indicating 50 or fewer of such referrals in the past year and only a handful (N = 6) received more than 100 social media referrals in the past year. Yet, the number of social media referrals appeared to be on the rise with 61% reporting greater number of these referrals over the past year.There were a total of 174 survey responses from at least 91 unique transplant programs, representing all OPTN regions (Table N = 136) but practices, timeline, and format were variable. Findings are summarized in Table N = 88) and was reinforced at multiple timepoints and upon request , social workers , and the independent living donor advocate (ILDA)  were the most frequent team members educating patients in this topic and typically on a one-on-one setting . Only 14.7% (N = 20) survey participants stated their center included a patient’s family and friends during this education. Survey participants also noted that discussing expectations and possible concerns from patient’s use of social media was important. Concerns about efficacy and efficiency of a given social media campaign, privacy and legal issues, and possible associated cost/expenses were cited  felt that patient-implemented living donor social media campaigns lead to a higher number of unprepared/uneducated potential living donors and/or donor-recipient questionable/inappropriate behaviors  and programmatic challenges  which presumably leads to a more resourceful program when dealing with such challenges. There were eight transplant programs attributes reported to enhance a program’s level of preparedness to handle donors referred through social media that highlighted the importance of having experienced and/or adequate number of staff and streamlining the donor intake processes as key features driving preparedness  and did not correlate with a program’s perceived preparedness to handle these donors (p = 0.3941). A qualitative analysis of 60 responses uncovered four spheres within a potential living donor evaluation process  where specific interventions could improve the process navigation , and setting of the education were highly variable. Only a small fraction (14.7%) of respondents reported including a patient’s family and support network when educating patients about social media in living donation. This finding contrasts with the recommendations from the 2014 AST Consensus Conference on Best Practices in Live Kidney Donation and other data suggesting that effective patient education should include a patient’s “family and friends” , 27, 28.Notably, the OPTN survey found that potential living donors emerging from social media campaigns were often unprepared or insufficiently educated or had questionable/unrealistic expectations from the patient or the transplant program. While the level of donor unpreparedness was not precise, practices that could allow programs to overcome such challenges were offered. These include having experienced staff, automation of the donor intake process, and sensible donor prioritization which included limiting the number of donor evaluations occurring simultaneously. While a number of “manageable simultaneous potential living donor evaluations” was not quoted by survey participants, a recent publication by Habbous et al. discussing the cost-effectiveness of concurrent multiple living kidney donor evaluations for the same patient rather than sequentially, noted that that number may be up to four simultaneous potential living donor evaluations . In HabbSince 2003, several social media sites have been launched offering various types of technology and capacity to share information , 30. SocAs conveyed by survey participants, several ethical concerns arise from the use of social media in the context of living donation. Commonly cited concerns in the literature include the risk of compromising personal privacy and safety, maintaining veracity and truthfulness, interference with legal requirements necessary in living donation, coercive donor and recipient relationships and, inadvertently, worsening existing disparities in access to living donor transplantation , 37. As It is important to mention that data obtained through this national survey had some limitations. Namely, survey participants were not expected to identify their transplant program or OPTN region in an attempt to maintain anonymity. This issue results in the possible inclusion of multiple responses for the same transplant program or obtains information detailing specific program’s practices and regional trends. As responses to all questions were not mandatory, many questions were left unanswered and therefore data is not inclusive. Most importantly, two major events occurred after this survey took place. First, the CMS Conditions of Participation Interpretive Guidelines now mandate that the ILDA must interview potential living donors before a living donor evaluation can be initated . This reRecognizing the influential nature of social media and its capacity to augment living donation rates, many thought-provoking questions remain unanswered. Do we need legislative and regulatory changes as social media gains traction in living donation? Should the transplant community collaborate with industry and communication experts to make this tool effective and accessible? How do we safeguard vulnerable groups from unintentionally disadvantaging them? And, should the use of social media be prioritized in social groups with lower rates of living donation? If so, is this an ethical practice? And, how do we measure its effectiveness and unexpected consequences? We must pay special attention to these challenges and proactively engage in providing guidance for safe and constructive use of this tool. In the end, social media and digital technology are here to stay so we will do well to embrace them in the service of optimizing transplant opportunities for our patients.Supplementary file1 (DOCX 14 KB)Below is the link to the electronic supplementary material.


This research is aimed at investigating the relationship between liver fibrosis in viral hepatitis and macrophage colony-stimulating factor (M-CSF), tissue inhibitor of matrix metalloproteinase (TIMP-1), and ceruloplasmin (CER) in serum level.  Patients were randomly selected among those admitted to our hospital, and 60 healthy volunteers were chosen to serve as control participants. The levels of serum M-CSF, CER, and TIMP-1 were compared. According to the severity of their liver fibrosis, patients with CHB were separated into four groups: S1, S2, S3, and S4. Serum levels of M-CSF, CER, and TIMP-1 were correlated with liver fibrosis and hepatitis markers, and the diagnostic usefulness of the three indices was assessed with liver cirrhosis patients. P < 0.05). Serum levels of M-CSF, CER, TIMP-1, HA, PC-III, C-IV, and LN differed significantly across the four study groups (P < 0.05). Over time, as liver fibrosis worsened, we observed a progressive uptick in M-CSF, TIMP-1, LN, HA, C-IV, and PC-III levels and a progressive downtick in CER levels, with significant (P < 0.05) differences between the groups. There was a significant positive correlation between liver fibrosis and serum M-CSF, PC-III, TIMP-1, HA, LN, and C-IV levels in the CHB group (P < 0.05) and a significant negative correlation between serum CER and these same factors (P < 0.05). The AUC of 0.956 for diagnosing the S4 stage was greater than that of 0.857, 0.851, and 0.817 for M-CSF, CER, and TIMP-1, respectively.  Increases in M-CSF and TIMP-1 in the CHB group but decreases in CER were statistically significant ( In CHB patients, the liver fibrosis degree is associated with the M-CSF, CER, and TIMP-1 levels, and the combined clinical detection of these three markers has better diagnostic significance. The liver is crucial to functioning several vital processes, including metabolism, immunity, coagulation, and many more. Long-term infection with the hepatitis B virus (HBV) is the primary cause of chronic hepatitis B (CHB) fibrosis, a multisystem chronic liver disease. However, HBV is not easy to clear, the patient's liver function inflammation is apparent, and it may progress to liver cirrhosis, which has become a significant public health problem. Proactive and efficient therapeutic approaches will slow the advancement of liver fibrosis to cirrhosis , 2. LiveMacrophage colony-stimulating factor (M-CSF) is composed of fibroblasts, activated macrophages, endometrial epithelial secretory cells, bone marrow stromal cells, vitamin D-activated osteoblasts, and activated vascular endothelial cells. Hepatic inflammatory responses are partly mediated by serum M-CSF, which has a role in the pathophysiology of hepatic fibrosis  by attraFrom March 2017 to March 2020, a total of 115 CHB patients admitted to our hospital were selected. Following were the criteria for inclusion: (1) the pathological diagnosis of the liver biopsy was based on the Protocol for Prevention and Treatment of Viral Hepatitis , and the2 . (2) After enrolling patients, 3 mL of fasting venous blood was obtained, and the upper serum was collected by centrifugation. This allowed us to measure the serum levels of M-CSF, CER, and TIMP-1. Enzyme-linked immunoadsorption (ELISA) was used to measure M-CSF and TIMP-1 in the serum. The kits were purchased from Rapid Bio Company, USA. The serum CER level was detected by the immunoturbidimetric method, and the instrument was an IMMAGE 800 automatic specific protein analysis system produced by Beckman Company in the United States, and supporting reagents were used. (3) Comparison of serum liver fibrosis indexes in the CHB group is as follows: After the patients were enrolled, 3 mL of fasting venous blood was taken, and the upper serum was collected by centrifugation. Enzymatic chemiluminescence immunoassay was used to detect type IV collagen (C-IV), laminin (LN) levels, hyaluronic acid (HA), and type III procollagen (PC-III); the kits were purchased from Roche Germany.(1) General data collection is as follows: weight, height, gender, systolic blood pressure (SBP), diastolic blood pressure (DBP), and age of the CHB group and the healthy group were collected. The changes in SBP and DBP of the subjects were measured by a sphygmomanometer. Body Mass Index (BMI) = weight/heightt-test was employed on measurement data represented as x ± s. The rate (%) was calculated from the count data, and a χ2 test was performed. The association between serum levels of M-CSF, CER, and TIMP-1 and HA, PC-III, C-IV, and LN was analyzed using Pearson's correlation analysis. Serum M-CSF, CER, and TIMP-1 levels were analyzed in connection to liver fibrosis severity using Spearman's correlation. Using a ROC curve, we compared the diagnostic accuracy of serum M-CSF, CER, and TIMP-1 for the S4 stage; a difference at the P < 0.05 was considered significant.For the statistical analysis, we utilized SPSS 20.0. P > 0.05).P < 0.05).P < 0.05). As liver fibrosis progressed, the blood levels of M-CSF, TIMP-1, and CER changed over time, with statistically significant variations across all groups (P < 0.05), as indicated in The levels of serum M-CSF, CER, and TIMP-1 varied significantly across the S1, S2, S3, and S4 groups (P < 0.05). The blood levels of HA, PC-III, C-IV, and LN steadily increased as liver fibrosis progressed, and statistically significant differences existed between all groups (P < 0.05), as shown in Serum C-IV, LN, PC-III, and HA levels varied across the S1, S2, S3, and S4 groups in a statistically significant way (P < 0.05), respectively, as shown in 1The serum M-CSF and TIMP-1 in the CHB group were positively connected with hepatitis A, B, C, and IV, liver fibrosis, and liver nodules severity  activation. Liver fibrosis develops when activated HSCs deposit their ECM-laden extracellular matrix into liver tissues, causing a shift in the matrix's constituent proteins . Since lM-CSF is a cytokine produced by activated macrophages, endometrial epithelial secretory cells, osteoblasts, and other cells. By adhering to its receptors, it can control the proliferation and differentiation of mononuclear macrophage cells, which is crucial for physiological processes such as bone metabolism, mammary gland development, and ovarian ovulation; it can also be used as a proinflammatory cytokine to participate in inflammatory and immune responses and to participate in the pathological process of liver fibrosis , 17. In α2 globulin with oxidase, mainly synthesized and secreted by the liver. When hepatocytes are immune-damaged by HBV replication in the body, the synthesis of CER will be reduced [CER is a single-chain  reduced . Studies reduced  individu reduced  have sho reduced  showed tTIMP-1 was firstly isolated from the skeletal muscle of rabbits. The encoding gene is located in the P11 region of the X chromosome and is secreted by HSCs. The TIMP levels were proportional to the number of activated HSCs . There wIn conclusion, liver fibrosis is associated with M-CSF, CER, and TIMP-1 levels in CHB patients. The diagnostic significance of the clinical combination detection of these three indications is higher, which aids in clinical prevention and treatment.


Additionally, we could not find out contributing risk factors for postoperative CSF leak and meningitis. ADM and banked homologous fascia lata are non-inferior to autologous fascia lata for endoscopic skull base reconstruction in water-tight reconstruction or safety without additional donor site morbidities.Cerebrospinal fluid (CSF) leak is a crucial complication after endoscopic skull base surgery. Therefore, multilayer reconstruction with grafts is as essential as a reconstruction with pedicled flaps. Although widely used, the multilayer technique with autologous fascia lata has drawbacks, such as additional wound and donor site complications. We compared acellular dermal graft and banked homologous fascia lata graft  with autologous fascia lata graft for high-flow CSF leak repair. We retrospectively enrolled 193 subjects who underwent endoscopic skull base reconstruction with multilayer fascial grafts and nasoseptal flap for high-flow CSF leaks from November 2014 to February 2020 at a single institution. Acellular dermal matrix (ADM), banked homologous fascia lata, and autologous fascia lata were used in 48 (24.9%), 102 (52.8%), and 43 (22.3%) patients, respectively. Postoperative CSF leaks occurred in 23 (11.9%) patients and meningitis in 8 (4.1%). There was no significant difference in postoperative CSF leak ( Postoperative cerebrospinal fluid (CSF) leak is a possible complication of the transsphenoidal approach (TSA). In particular, when removing tumors involving the suprasellar area, posterior fossa, or anterior skull base through the endoscopic endonasal approach (EEA), high-flow CSF leak is inevitable . The nasWe retrospectively investigated the medical data of 1395 consecutive patients who underwent EEA procedures with skull base reconstruction between November 2014 and February 2020 in a single institution. EEA was performed to remove the various types of tumors invading the sellar to suprasellar portion, posterior fossa, or anterior skull base. Among them, 193 patients who had an intraoperative high-flow CSF leak and underwent multilayer reconstruction using fascial grafts with NSF were included in this study. High-flow CSF leak was defined as the occurrence of large diaphragmatic/dural defect during resection through suprasellar, transclival or transcribriform approaches according to Esposito’s grade 3 [The three types of grafts were chosen depending on the time of surgery, the tissue bank situation or the patient’s preferences. We preferred the autologous fascia lata graft in the early study period (until 2015). After then, homologous fascia lata was chosen as the first option to avoid additional donor site morbidities. If there were not enough banked fascia lata in the tissue bank in our hospital, we used ADM and, if a patient had a preferred graft type, this was selected. The selection of graft material was chiefly determined after preoperative counselling, and informed consent was obtained from the patients prior to surgery regarding the availability of one of three types of grafts.®  or Alloderm® , made by processing donated human skin. Homologous fascia lata from the tissue bank in our hospital was used from harvesting cadaver donors’ tensor fascia lata. None of the donors had any transmissible viral disease based on screening tests. All tissues were sterilized by gamma irradiation (25 × 103 Gy) and frozen at −70 °C in liquid nitrogen. An autologous graft was harvested at the patient’s lateral thigh for tensor fascia lata.This study used cross-linked ADM as an allograft material, Megaderm®  or fibrinogen collagen sponges . If the intradural dead space was large, in case of olfactory groove meningioma or large posterior fossa defects, an abdominal fat graft was used to avoid dead space. Double-layered dura reconstruction using ADM, homologous fascia lata or autologous fascia lata was performed in an inlay–onlay fashion. The inlay graft was designed to be larger than the defect size in a rectangular shape and placed on the subdural surface. Then, the onlay graft was inserted into the epidural surface and under the skull base bone. An NSF was then applied to sufficiently cover the skull base defect and direct contact with the bare bone. Compressive packing was performed with synthetic materials, such as gelatin foams and Merocel®  to reduce dead space under the NSF and control bleeding. Postoperative lumbar drainage was performed when large posterior fossa defect was created, or when the onlay fascial graft was not tightly placed within the bone margins.All the reconstruction was performed by single ENT surgeon and two different neurosurgeons with two layered graft and nasoseptal flap. After the tumor was resected, the intradural space was gently covered with SurgicelThe demographics assessed and extracted from medical records included age, sex, body mass index (BMI), pathologic diagnosis, tumor size at diagnosis, duration of the surgery, history of radiotherapy or gamma knife radiosurgery, revision cases, postoperative lumbar drainage, and follow-up period. Depending on the origin of the tumor, the skull base defect locations were also checked as suprasellar, posterior fossa, and anterior skull base. The primary endpoints in this analysis were the rate of postoperative CSF leak and meningitis.p < 0.05. Continuous variables are presented as mean  and categorical variables as numbers . The Kruskal–Wallis test, Chi-Square test, and Fisher’s exact test were conducted. In addition, Mann–Whitney test, Chi-Square test, and Fisher’s exact test were performed to compare according to the presence or absence of postoperative complications as a reference for risk factors that are more likely to cause CSF leak and meningitis. To determine the relevant predictors for postoperative complications, multivariate analysis using logistic regression analysis was performed.All statistical analyses were performed using IBM SPSS statistics version 25, and statistical significance was set at 2. The most common pathology was craniopharyngioma , followed by tuberculum sellae meningioma . Other pathologic diagnoses included 37 pituitary adenomas (19.2%), 11 chordomas (5.7%), 5 olfactory groove meningiomas (2.6%), and 28 types of other tumors (14.5%), including 10 olfactory neuroblastomas, 3 epidermoid cysts, 2 chondrosarcomas, 2 sinonasal squamous cell carcinomas, 2 schwannomas, and 1 each of immature teratoma, mature teratoma, arachnoid cyst, sinonasal adenocarcinoma, medulloblastoma, xanthogranuloma, metastatic adenocarcinoma, gauzoma, and retinoblastoma. The mean tumor diameter at diagnosis was 30.4 ± 10.0 mm, and 24 (12.4%) patients underwent revision surgery. Nine patients (4.7%) had previous radiation therapy and 10 patients (5.2%) had previous gamma knife radiosurgery.A total of 193 patients who had high-flow CSF leaks during EEA and underwent multilayer reconstruction with an NSF for skull base reconstruction were analyzed in this study. The baseline characteristics, variables related to surgery, and outcomes are shown in The highest location of the defect opening to the CSF space after tumor removal was in the suprasellar region . Thirteen (6.7%) defect openings were in the posterior fossa and 18 (9.3%) in the anterior skull base, such as in olfactory neuroblastoma or meningioma at the olfactory groove. An autologous fat graft was inserted into the intradural dead space in 40 patients (20.7%). Banked fascia lata was used in 102 (52.8%) patients, ADM in 48 (24.9%) and autologous fascia lata in 43 (22.3%) patients. Postoperative lumbar drainages were placed in 111 (57.5%) patients and maintained for 4.8 ± 2.2 days. The mean follow-up period was 3.5 years (from 1 month to 8.0 years).p = 0.95), sex (p = 0.33), BMI (p = 0.10), tumor size (p = 0.42), time taken for surgery (p = 0.63), previous radiation (p = 0.31) or previous gamma knife (p = 1.00) history, revision cases (p = 0.15), and diaphragmatic/dural defect location (p = 0.52) among the three groups (p = 0.36) or meningitis (p = 0.17) was observed among the three groups  patients, and 8 (4.1%) developed postoperative meningitis . Postopee groups . As a sue groups .Variables were compared according to the presence or absence of postoperative complications as a reference for risk factors that are more likely to cause CSF leak and meningitis (Endoscopic skull base surgery is associated with a relatively lower morbidity than craniotomy and easier access to the skull base lesions. However, postoperative CSF leak and meningitis have been the most-criticized complications. The pathway from the sinonasal cavity to the subdural or subarachnoid space must be water-tightly sealed. High-flow CSF leaks commonly occur during resection of craniopharyngiomas, tuberculum sellar/olfactory groove meningiomas, giant pituitary adenomas, clival chordomas, and olfactory neuroblastomas. Many reconstruction methods have been introduced for high-flow CSF leak repair. Previous international consensus statement strongly recommended use of vascularized reconstruction in the presence of large dural defects and high-flow CSF leaks to reduce postoperative CSF leaks . Among vVarious techniques using graft materials are available for water-tight skull base reconstruction. “Gasket-seal” method or button-type closure can be performed to provide rigid buttress or to prevent the migration of graft material into intracranial space ,25. A diAs reported in one meta-analysis , there wFurthermore, this study only included high-flow CSF leaks following EEA, and the incidences of postoperative CSF leaks and meningitis were 11.9% and 4.1%, respectively; these figures were relatively higher than those reported in previous studies ,12,26. I® or Megaderm® used in this study was flexible and its thickness was variable, so it was easy to implant into dural defects. However, in our institution, the Alloderm® and Megaderm® are 2.7 and 2.2 times more expensive than banked fascia lata, respectively. Although ADM can be relatively expensive and has slower tissue growth than autologous grafts [ADM, being a homologous dermal graft, does not require autologous donor tissue harvesting, so there is no worry about additional wounds or donor-site complications. In addition, since the viable cells are removed, there is little risk of infection, and because the cellular components of the epidermal and dermal layers are removed, the risk of cell-mediated rejection is small ,27,28. As grafts , the outBanked fascia lata has been widely used in various fields, such as general, orthopedic, gynecologic, or ophthalmologic surgery ,32,33. TIn this study, we analyzed the clinical outcomes of three different sheet-type graft materials when a high-flow CSF leak was encountered. There have been no studies comparing the outcomes of three types of grafts in cases of high-flow CSF leaks. The complication rate of each group seems different, but since the number of meningitis and postoperative CSF leaks in each group is tiny, there does not seem to be a statistical difference . HoweverSeveral limitations of the study should be reviewed. First, there could be selection bias because this was not a randomized trial. However, graft selection was chiefly determined before the beginning of surgery and there was no significant difference among the groups regarding demographic data that could influence the surgical results. Therefore, the risk of selection bias could be lowered. Second, tumor pathology and defect location heterogeneity may bias the surgical outcome. However, there was no difference in defect location among the three graft groups. Additionally, there was no significant difference in postoperative CSF leaks according to defect location, but 13.0% in suprasellar lesion, 7.7% in posterior fossa lesion, and 5.6% in anterior skull base defect. However, since the number of tumors located in the posterior fossa and anterior skull base is relatively small, 13 and 18, respectively, further large-scale studies for these defect sites will help to evaluate the topics related to this study.This large-series study in a single institution suggests that ADM or homologous banked fascia lata could be used instead of autologous fascia lata for multilayer reconstruction of high-flow CSF leaks in the EEA. Furthermore, these alternative non-autologous materials did not increase the postoperative complications and had no additional donor-site morbidities.


Adipokines are signaling molecules involved in the integration of metabolism. Changes in their concentrations were observed in obesity, metabolic syndrome, diabetes mellitus and cardiovascular diseases, as well as endocrine disorders. Cushing’s syndrome is associated with metabolic dysregulation, but the significance of adipokines in this entity and related complications is largely unknown. The aim of our study was to determine the concentrations of adipokines: fetuin A, fatty acid binding protein 4 (FABP4) and retinol binding protein 4 (RBP4) in Cushing’s syndrome and to assess their relation to established cardiovascular and diabetes risk markers.We examined 21 subjects with Cushing’s syndrome and 24 healthy controls in a cross-sectional manner. Venous blood samples were analysed for adipokines, cortisol, adrenocorticotrophin, glucose, insulin, glycated haemoglobin (HbA1c), triglycerides, cholesterol fractions, thyrotropin and free thyroid hormones concentrations. Patients’ body mass index (BMI) was evaluated, homeostatic model assessment-insulin resistance and Systematic Coronary Risk Evaluation (SCORE) were calculated.We found that the concentration of fetuin A was lower, while FABP4 and RBP4 concentrations were higher in Cushing’s syndrome compared to controls . Fetuin A correlated inversely, while FABP4 and RBP4 positively, with the concentrations of urinary free cortisol and adrenocorticotrophin. Fetuin A was positively related to LDL-cholesterol, and negatively to SCORE and HbA1c. FABP4 was associated positively with BMI, HbA1c and triglycerides, while RBP4 correlated positively with triglycerides and systolic blood pressure.Adipokines’ concentrations change in hypercortisolism. Further research is needed to ascertain whether adipokines are involved in the development of metabolic complications accompanying Cushing’s syndrome or secondarily reflect metabolic dysregulation. Adipokines are the cytokines secreted from adipocytes, that participate in signalling between tissues involved in the regulation of body metabolism. Altered adipokine production reflects adipose tissue dysfunction, that has been linked to obesity and associated complications . RecentlThe aim of this study was to determine whether hypercortisolism affects the concentrations of fetuin A, FABP4 and RBP4 and to assess their association with established biomarkers of cardiovascular disease and diabetes, including lipid profile, glycated haemoglobin, homeostatic model assessment-insulin resistance (HOMA-IR) and Systematic Coronary Risk Evaluation (SCORE and SCORE2).The study participants were recruited between October 2019 and June 2022 at the Department of Endocrinology, Metabolism and Internal Medicine, Poznan University of Medical Sciences. The study group comprised 21 subjects newly diagnosed with endogenous CS in the course of Cushing’s disease (CD) (10 patients), ectopic ACTH production due to neuroendocrine tumours (5 patients) and small cell lung cancer (2 patients) or cortisol-secreting adrenal tumours (4 patients). The diagnosis of CS was made based on clinical features and laboratory findings according to clinical practice guidelines . The conAll enrolled subjects underwent a full clinical examination. Blood samples were taken after an overnight fast. The following biochemical measurements were performed: creatinine, uric acid, fasting glucose, insulin, total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, thyroid stimulating hormone (TSH), free triiodothyronine (fT3), free thyroxine (fT4), dehydroepiandrosterone sulphate (DHEA-S), sex hormone binding globulin (SHBG), serum and urinary free cortisol (UFC) using Cobas 8000 modular analyser , glycated haemoglobin (HbA1c) using high performance liquid chromatography - D10 system . For statistical analyses, we used average UFC calculated from three consecutive measurements. The samples for determination of serum fetuin A, FABP4 and RBP4 were frozen in minus 80 degrees Celsius and stored. After recruitment completion, the analyses were performed using commercially available enzyme linked immunosorbent assay kits: Human fetuin A ELISA kit (BioVendor Laboratory Medicine Cat# RD191037100), Human adipocyte FABP ELISA kit , Human RBP4 ELISA kit (Immundiagnostik AG Cat# K 6110).Cardiovascular risk was estimated using the SCORE system for Polish population , as wellBioethics Committee of the Poznan University of Medical Sciences approved the project (Resolution no. 118/21). All participants gave written, informed consent to participate in the study. The project was conducted in accordance with the Declaration of Helsinki.t test or Mann-Whitney U (exact) test were applied. For correlations Pearson product-moment correlation or Spearman’s rank-order correlation tests were used. A P-value<0.05 was considered statistically significant. The acquired data were analysed using PQStat Software (2022). PQStat v.1.8.4.136.Data are expressed as mean ± standard deviation or median [quartiles] as appropriate. Normality was verified with Shapiro-Wilk test, while equality of variances was analysed using Fisher-Snedecor test. For comparisons between groups either Student’s Clinical characteristics and laboratory results of the study groups are presented in the r=-0.810, p=<0.001), while FABP4  and RBP4  positively, as shown in the The comparison of the analysed adipokines’ serum concentrations between the study groups is shown in the p= 0.005). We found no correlations between adipokines and fasting glucose or HOMA-IR. Moreover, adipokines’ concentrations did not differ significantly between CS subjects with normal glucose tolerance and prediabetes. Fetuin A correlated negatively with HbA1c, while FABP4 - positively. The lipid panel measurements were not related to adipokines’ concentrations, with the exception of LDL cholesterol, which correlated positively with fetuin A and triglycerides, which were positively related to FABP4 and RBP4. Only fetuin A correlated with SCORE. Fetuin A correlated positively with TSH, fT3, fT4 and SHBG. Conversely, FABP4 and RBP4 correlated negatively with TSH, thyroid hormones and SHBG.Associations between fetuin A, FABP4, RBP4, metabolic and hormonal parameters are summarised in the Adipokines were also related to each other Figure 3In this study, we found that fetuin A concentration is significantly lower, while FABP4 and RBP4 concentrations are significantly higher in CS compared to healthy controls. Adipokines correlated with 24-hour UFC and ACTH.This is the first study to evaluate fetuin A and RBP4 concentrations in CS, which makes comparisons with the literature data challenging. Previously, Lewandowski et al. investigated the influence of two-day oral administration of dexamethasone on circulating RBP4 in healthy subjects and observed no change in RBP4 during the trial . Their rAtherosclerotic cardiovascular complications and thromboembolic events are the commonest causes of death in patients with CS, accounting for almost 45% of mortality . Underlyvia inducing endothelial dysfunction and foam cell formation . The participants provided their written informed consent to participate in this study.DD, ES-P and MR contributed to conception and design of the study. DD acquired, analyzed and interpreted the patient data and was a major contributor in writing the manuscript. EW performed the ELISA determinations of analyzed adipokines. ES-P and MR revised the manuscript. All authors contributed to the article and approved the submitted version.


Bilobed testis is an uncommon congenital malformation with only eight cases reported up to now. It seems that bilobed testicle is a form of polyorchidism which is not yet thoroughly divided.This report could provide information about diagnosing minor lobulation on ultrasound and MRI for the first time.In this report, a 13-year-old boy presented with extreme Epididymo-orchitis on the right testis, without any history, which showed itself on ultrasound as type A3 polyorchidism or bilobed testis.Recent studies have not shown an apparent association between bilobed testis with testicular torsion and malignancy. In our case, because the minor lobulation is small, it probably has no association with torsion. The bilobed testis seems benign, so there is no requirement to check tumor markers. An inflamed testicular appendix and epididymitis can appear similar to a major lobulation which must be accurately found and evaluated on ultrasound as separate entities.Inflamed minor lobulation of the testicle can demonstrate itself as polyorchidism or bilobed testicles; thus, Ultrasound and MRI can assist in diagnosing minor lobulation. Serial examination and imaging are recommended for managing minor lobulation. •How to detect bilobed testis in ultrasound•MRI was used for the first time to diagnose minor lobulation of the testis.•The bilobed testis seems benign, so there is no requirement to check tumor markers.•There is probably no association between minor lobulation of testicles and testicular torsion.•Bilobed testicle can be evaluated by serial examination and imaging. Ultrasound results revealed that the right testicular appendix is prominent, but it also has a hypoechoic structure that resembles the testicular parenchyma. This structure is above the right testicle and the medial part of the right epididymis, is wholly attached to the testicle, and has 17 × 12 mm dimensions. Furthermore, in color Doppler, the coloration is sharply increased .Fig. 1UlThe above findings suggest extreme Epididymo-orchitis on the right and maybe a type A3 polyorchidism with adjacent extra testis or a bilobed testis.After treatment of the Epididymo-orchitis, Magnetic resonance imaging (MRI) was performed one month later. In axial and coronal T2 FISP images, a Hemi-testicular structure of similar signal intensity to that of the adjacent normal testis is noted on the right side, albeit with significantly decreased size due to the resolution of the inflammatory phase .Fig. 2MR3It is essential to differentiate the bilobed testis from polyorchidism because polyarchidism can be associated with testicular torsion and malignancy Bilobed testis appears to be a type of polyorchidism. There is a 5.7–7 % chance of malignancy in polyorchidism cases, which is higher than the usual risk of the general population,0006 % There is a greater tendency for infection in minor lobulation due to problems in the drainage of sperm or blood supply. Due to infection and subsequent inflammation, this minor lobulation demonstrates more pronounced swelling and inflammation than the main part of the testicle and hence may seem to be polyorchidism or a major lobulation; the testicular appendix and epididymis can also become inflamed and appear similar to major lobulation.The appendix and testicular epididymis must be accurately found and evaluated on ultrasound as separate entities. After that, if there is another element mimicking the parenchymal structure of the testicle on ultrasound, we should think of minor lobulation that is inflamed and swollen. In these situations, minor lobulation shows itself similar to major lobulation.4Polyorchidism is commonly associated with complications such as testicular torsion and malignancy, while there is probably no association with torsion in minor lobulation of the testis; therefore, distinguishing bilobed testis from polyorchidism through ultrasound and MRI can be beneficial for us.Since Minor lobulation of the testis seems to be benign malformation and the possibility of torsion and other complications are low, serial examinations and imaging are recommended for managing these cases.5This case report has been reported in line with the SCARE Criteria The patient and the patient's family were satisfied with all the work that was done for them, and the patient's family said, “Thank you for the work you have done to find out about our child's illness.”There was no funding.It is not required.Written informed consent was obtained from the parents for publication of this case report and accompanying images. A copy of the written consent in our native language is available for review by the Editor-in-Chief of this journal on request.None.None.The corresponding author is the guarantor of submission.There is no relationship.


Hydrocephalus is a severe complication of intracerebral hemorrhage with ventricular extension (ICH-IVH) and causes cerebrospinal fluid (CSF) accumulation. The choroid plexus epithelium plays an important role in CSF secretion and constitutes the blood–CSF barrier within the brain–immune system interface. Although the NLRP3 inflammasome, as a key component of the innate immune system, promotes neuroinflammation, its role in the pathogenesis of hydrocephalus after hemorrhage has not been investigated. Therefore, this study aimed to investigate the potential mechanism of NLRP3 in hydrocephalus to discover a potential marker for targeted therapy.−/− rats. By studying the features and processes of the model, we investigated the relationship between the NLRP3 inflammasome and CSF hypersecretion in the choroid plexus.A rat model of hydrocephalus after ICH-IVH was developed through autologous blood infusion in wild-type and Nlrp3+- and K+-coupled water transport, via MCC950 or Nlrp3 knockout. In combination with the in vitro experiments, this experiment confirmed the involvement of the NLRP3/p-NKCC1 pathway and Na+ and K+ flux.The ICH-IVH model rats showed ventricular dilation accompanied by CSF hypersecretion for 3 days. Based on the choroid plexus RNA-seq and proteomics results, we found that an inflammatory response was activated. The NLRP3 inflammasome was investigated, and the expression levels of NLRP3 inflammasome components reached a peak at 3 days after ICH-IVH. Inhibition of NLRP3 by an MCC950 inflammasome inhibitor or Nlrp3 knockout decreased CSF secretion and ventricular dilation and attenuated neurological deficits after ICH-IVH. The mechanism underlying the neuroprotective effects of NLRP3 inhibition involved decreased phosphorylation of NKCC1, which is a major protein that regulates CSF secretion by altering NaThis study demonstrates that NKCC1 phosphorylation in the choroid plexus epithelium promotes NLRP3 inflammasome-mediated CSF hypersecretion and that NLRP3 plays an important role in the pathogenesis of hydrocephalus after hemorrhage. These findings provide a new therapeutic strategy for treating hydrocephalus.The online version contains supplementary material available at 10.1186/s12974-022-02530-x. Intracerebral hemorrhage (ICH) occurs in 10–15% of all strokes worldwide each year , 3. Exte+/K+/2Cl− cotransporter (NKCC1), which is expressed in the luminal membrane of the choroid plexus, contributes to approximately half of the production of CSF [+, K+, and Cl− transmembrane coupled water, which permits water to be transported independently of, and even against, an osmotic gradient [CSF circulation failure is the major mechanism of hydrocephalus after hemorrhage. Most relevant studies have aimed to develop techniques for controlling the flow of CSF by targeting the obstruction. Hydrocephalus after hemorrhage becomes aggravated by damage to ependymal glia and dysfunction of arachnoid granulations, which can block the outflow of CSF , 10. Then of CSF . NKCC1 cgradient . As a chgradient . Intrace−/−), the secondary brain injury caused by ICH was alleviated, and most relevant studies have aimed to investigate the NLRP3 inflammasome in immune cells, such as microglia, macrophages and astrocytes [As a critical function of the innate immune response to tissue injury, the NLRP3 inflammasome is increasingly being considered to be a driver of injury after hemorrhage; this inflammasome senses cellular deviation from homeostasis as a danger signal and subsequently initiates inflammatory responses –19. The trocytes , 23. HowDespite partial elucidation of the underlying mechanism of hydrocephalus after hemorrhage, its pathogenesis is not well understood. To achieve a comprehensive understanding of the biological processes of this difficult-to-treat condition, based on transcriptomics and proteomics of the choroid plexus, we hypothesized that NLRP3 would aggravate hydrocephalus after ICH with ventricular extension (ICH-IVH) by altering NKCC1 phosphorylation to enhance CSF secretion in the choroid plexus. The present study investigated the function and molecular mechanism of the NLRP3 inflammasome in the pathogenesis of hydrocephalus with the aim of discovering a new therapeutic target for hydrocephalus patients.Nlrp3−/− rats were purchased from Cyagen Biosciences . The rats were housed in a temperature-controlled room under specific pathogen-free conditions and a standard 12-h light/dark cycle, with ad libitum access to food and water. All experimental procedures involving animals were performed in compliance with the Guidelines for the Care and Use of Laboratory Animals, approved by the Laboratory Animal Welfare and Ethics Committee of Third Military Medical University (AMUWEC2020762), and reported according to the ARRIVE  guideline.Adult male Sprague-Dawley rats (220–250 g) were purchased from the Animal Experimental Center of Third Military Medical University . The surgical procedures used in this study for rats subjected to hemorrhage followed the procedures that are described in detail in our previous study , 24. In The rats were evaluated for neurological dysfunction using the mNSS method, as described previously . The mNSA rotarod test was performed to evaluate sensorimotor deficits as previously described . BrieflyThe grip strength of the front limbs was measured by a grip strength meter , which consisted of a steel wire grid connected to an isometric force transducer following a method described earlier . Each raAll behavioral tests were performed by an investigator who was blinded to the treatments.x, − 0.6 cm; y, − 1.6 cm). Next, the rat’s head was rotated on the ear bars by 90° so that it was oriented nose down, and the suboccipital muscles were dissected to the cisterna magna to expose the atlantooccipital ligament. The ligament was punctured, and a 29-gauge needle was advanced 5 mm through the foramen of Magendie to the fourth ventricle. Sterile molecular-grade oil  was infused through the tube to occlude the aqueduct of Sylvius, thereby creating a closed system of CSF circulation in the lateral ventricle. With the rat in the same position, a glass capillary tube  was advanced through the burr hole into the lateral ventricle . The volume of CSF collected at a given time (20 min) was calculated as V (mm3) = π·r2·d, where r is the radius of the glass capillary tube and d is the distance that CSF traveled within the capillary tube. The rate of CSF formation (μl/min) was then calculated from the slope of the volume–time relationship.The rates of CSF production were measured using a method as previously described . BrieflyWestern blot analysis was performed as previously described . The braRats were anesthetized with pentobarbital . CSF was collected through the cisterna magna; then, the rats were decapitated to obtain the choroid plexus. The CSF was centrifuged at 10,000 rpm to separate the supernatant. The choroid plexus was homogenized in ice-cold phosphate-buffered saline with protease inhibitor cocktails . Total protein concentration was quantified using a bicinchoninic acid protein assay . The CSF and choroid plexus cytokine levels were measured using inflammation cytokine assay kits  according to the manufacturer’s instructions.Immunofluorescence staining of the brain tissue was performed on fixed frozen sections, as previously described . Rats wen = 6 per group), with a 40-μm space in between used for cell counts. Three high-power images were used for cell counting. NLRP3- and p-NKCC1-positive cells were counted in the choroid plexus. Cell counts were performed by two researchers in a blinded manner. All measurements were repeated three times, and the mean values were used.Cells were counted 3 days after ICH-IVH. For quantification of the positive cells in the choroid plexus, consecutive slices were made in two sections per animal  with a T2*gradient-echo sequence and a T2 fast spin-echo sequence using a view field of 35 mm × 35 mm and 17 coronal slices (1.0 mm thickness). There are two classical methods for evaluating hydrocephalus based on MRI, i.e., the Evans index and whole-ventricle volume assessments . Consideg for 5 min at 4 °C to remove any tissue debris. Inductively coupled plasma-optical emission spectrometry (ICP-OES) was used for K+ and Na+ quantification. All tests were performed with 5–10 μl of CSF.CSF was collected by inserting a syringe into the cisterna magna, and the collected CSF was centrifuged at 10,000×n = 6 in each group, 3 replicates). Total RNA of the choroid plexus was isolated using TRIzol reagent . The quantity and purity of total RNA were then detected by a NanoDrop ND-1000 instrument . Furthermore, RNA integrity was detected by an Agilent 2100 instrument, and a RIN > 7.0 was taken as the qualified standard. Then, the qualified RNA was provided to LC-Bio Technology Co., Ltd. , for subsequent library preparation and sequencing using a HiSeq 4000 platform as described previously [p < 0.05 and FPKM > 1. The GO and KEGG databases were used to explore the functions and biological pathways in which the differentially expressed genes were involved.The rats in the sham group and ICH-IVH group (3 days) were anaesthetized on ice and dissected longitudinally to obtain the choroid plexus (eviously . The exphttp://www.lc-bio.com/). Differentially expressed proteins were defined as proteins with a fold-change greater than 1.2 and a p value (t-test) less than 0.05. Functional classification of differentially expressed proteins was conducted by mapping with GO terms, and the highest bit score sequence was selected. Pathway analysis was performed using the KEGG database.The choroid plexus was rapidly frozen and ground in liquid nitrogen and then treated as previously reported to obtain the purified protein concentration . Briefly2 at a 1:5 ratio) and incubated at 37 °C for 20 min; the tubes were tapped every 5 min. CPEC media included 10% FBS and 10,000 units/ml Pen-Strep in DMEM. Tissue was dissociated using a pipette and then plated on poly-d-lysine and laminin-coated cell culture plasticware. Choroid plexus epithelial cells were cultured for 14 days, with the media changed every 2–3 days, and then used for experiments. Cells were treated with 1 μg/ml LPS or 1 μg/ml lysis-RBCs for 12 h. Thirty minutes after incubation with lysis-RBCs, MCC950 (5 μM) was added.Primary choroid plexus epithelial cells from rats were cultured as previously described . In brieA = 0.05; two-tailed; and desired power, 80%. The results indicated that a minimum of 3 rats per group were needed. All data were analyzed using GraphPad Prism and satisfied a normal distribution and the homogeneity of variance. Immunostaining, cytokine measurement data, and behavioral experiments were subjected to one-way ANOVA. Student’s t-test was used for single comparisons, and analysis of variance with post hoc Bonferroni–Dunn correction was used for multiple comparisons, depending on the experiments. A p value < 0.05 was considered to be statistically significant. The number of different experimental groups and statistical methods is shown in the figure legends, and statistical analysis data are detailed in the Additional file The data were analyzed and plotted by GraphPad Prism and presented as the means ± SEMs. Sample sizes were calculated using an a priori sample size calculator with the following assumptions: According to a previous posthemorrhagic hydrocephalus animal model, intraventricular hemorrhage (IVH) and ICH-IVH rat models were constructed . Both ofp < 0.05). According to transcriptome and proteomic analysis of the choroid plexus, we found some change trends at the RNA and protein levels  group  could influence its function [−/− rats, NKCC1 phosphorylation was downregulated compared with that in the control group after ICH-IVH according to p-NKCC1 positive counting and quantitative analysis , Na+-coupled bicarbonate transporters, and Na+/K+-ATPase are the main ion transporters that adapt to the formation of CSF, and NKCC1 plays an important role among these transporters [+, K+, and Cl− altered by NKCC1, we detected Na+ and K+ concentrations in primary choroid plexus epithelial cells. The main regulatory molecule NKCC1 was selected in this study, and it was briefly proven that NKCC1 is a molecular component in NLRP3-mediated CSF hypersecretion. Interestingly, the ion concentration results confirmed our previous results; however, Na+ and K+ showed different change trends, and the degree of Na+ outflow was more severe than that of K+ outflow. K+ efflux and inflammation stimulators (LPS or lysis-RBC) can activate the NLRP3 inflammasome [+ and K+ outflow, and K+ efflux activates the NLRP3 inflammasome, which results in a positive feedback cascade amplification effect. Due to the limited evidence, further studies are still needed to clarify the mechanism of NLRP3-mediated CSF hypersecretion after ICH-IVH.After we discovered that the NLRP3 inflammasome became activated after ICH-IVH in the choroid plexus, we attempted to elucidate the molecular mechanisms occurring between the NLRP3 inflammasome and CSF formation. Approximately 500 ml of fluid is produced by the choroid plexus in the mammalian brain. CSF production is assumed to take place by the transport of osmotically active ions, the passive movement of water and the water channel aquaporin. NKCC1, Ksporters , 64–68. sporters . AQP4 alsporters . NKCC1 iammasome , 72. AftIn this study, we observed that ICH-IVH induced CSF hypersecretion by activating the NLRP3 inflammasome, and the ion cotransporter NKCC1 regulating CSF secretion also participated in this process. In addition, we found blood–CSF barrier disruption and lipid droplet (LD) formation in the choroid plexus by accident.The most widely recognized role of the choroid plexus is in the formation of the blood–CSF barrier, whereby it serves as a controller of the internal CNS microenvironment . Blood–C+ and K+) efflux to transmembrane water transport in primary choroid plexus epithelial cells after ICH-IVH, which also contributed to aggravated hydrocephalus. This study provides evidence that inhibiting NLRP3 may be a potential therapeutic approach for preventing hydrocephalus after ICH-IVH.Our results showed that activation of NLRP3 in the choroid plexus contributed to increased CSF secretion and aggravated hydrocephalus after ICH-IVH, and NLRP3 impacted CSF secretion by regulating the NKCC1 phosphorylation level. In addition, NKCC1 phosphorylation mediated more coupling of ion (NaAdditional file 1. Statistical Results in Figures.


Galdieria sulphuraria, for bioaccumulation/removal of REEs from hazardous industrial wastes of CFL bulbs and the physiological response of a synchronized culture of G. sulphuraria. A CFL acid extract significantly affected growth, photosynthetic pigments, quantum yield, and cell cycle progression of this alga. A synchronous culture was able to efficiently accumulate REEs from a CFL acid extract and efficiency was increased by including two phytohormones, i.e., 6-Benzylaminopurine (BAP - Cytokinin family) and 1-Naphthaleneacetic acid (NAA - Auxin family).In recent decades, a shift has been seen in the use of light-emitting diodes over incandescent lights and compact fluorescent lamps (CFL), which eventually led to an increase in wastes of electrical equipment (WEE), especially fluorescent lamps (FLs) and CFL light bulbs. These widely used CFL lights, and their wastes are good sources of rare earth elements (REEs), which are desirable in almost every modern technology. Increased demand for REEs and their irregular supply have exerted pressure on us to seek alternative sources that may fulfill this demand in an eco-friendly manner. Bio-removal of wastes containing REEs, and their recycling may be a solution to this problem and could balance environmental and economic benefits. To address this problem, the current study focuses on the use of the extremophilic red alga,  Rare earth elements (REEs) are a group of 17 chemical elements that comprise yttrium (Y), scandium (Sc), and a series of 15 lanthanides. REEs have practically identical physical and chemical properties although they have rather unique magnetic and catalytic properties . These pChlamydomonas reinhardtii, Chlorella sp. or Arthrospira sp. improved in the presence of REEs , and algae as REE accumulators, bio-removers, and potential bio-miners have been studied extensively . Among t of REEs  whereas, control .G. sulphuraria.In addition, extensive research has been conducted to investigate heavy metal (HM) bioaccumulation/bioremediation by algae employing different chemical agents and phytohormones. However, the underlying mechanisms controlling the effect of phytohormones on bioremediation are elusive , 2018. UGaldieria, has been proposed as suitable models that have the potential to accumulate or adsorb different REEs . The Fv/Fm index also indicates stress conditions such as temperature, light, pH, nutrient concentration, or toxic metals  and Sc3+ (scandium) significantly reduced levels of photosynthetic pigments in Parachlorella kessleri .In the present study, we describe the effect of a CFL acid extract on a synchronous culture of G. sulphuraria  Merola, 002 was acquired from the Algal Collection of the University “Federico II” of Naples, Italy.−1): 1.31 (NH4)2SO4, 0.27 KH2PO4, 0.25 MgSO4·7H2O, 0.02 C10H12O8N2NaFe, 0.14 CaCl2·2H2O, and microelements diluted 500x from the stock solution (mg L−1): 31 H3BO3, 1.25 CuSO4·5H2O, 22.3 MnSO4·4H2O, 0.88 (NH4)6Mo7O24·4H2O, 2.87 ZnSO4·7H2O, 1.46 Co(NO3)2·6H2O, 0.014 V2O4(SO4)3·16H2O, 0.3 Na2NO4·7H2O, 1.19 KBr, 0.83 KI, 0.91 CdCl2, 0.78 NiSO4, 0.12 CrO3, 4.74 Al2(SO4)3K2SO4·24H2O  in distilled water, autoclaved for 20 min. The synchronization of the cultures was carried out by changing the light and dark periods (16 l/8D) according to −2 s−1. The photobioreactors, in the shape of glass cylinders (300 ml) or flat cuvettes (2.5 l), were placed in a thermostatic water bath and illuminated by a panel of dimmable fluorescent lamps . Algal culture suspensions were supplied with a gas mixture of air and CO2 (2% v/v), at a flow rate of 15 L h−1. The experiments were carried out in a batch culture regime under controlled light and temperature conditions.The experimental organism, unicellular red alga 3 and it was insoluble in water or Galdieria-nutrient medium. Based on the preliminary study with different acids, acid mixtures, and their concentrations, the best solubility of CFL particles was observed in 10% HNO3 acid. To prepare the CFL acid extract with a final concentration of 40 μg mL−1, 4 g of CFL powder was extracted into 100 ml of 10% HNO3. The solution was shaken on a horizontal shaker (at 150 rpm) for 1 h and then extracted at room temperature for 12 h, followed by 3 h of shaking. The suspension was centrifuged at 4000 rpm for 3 min and the supernatant was filtered through a 0.45 μm filter to avoid the remaining solid particles. The resulting clear CFL acid extract solution has an approximate pH of 0.5. The stock solution was used in all experiments.Luminophore powder from e-waste of CFL light bulbs was provided by RECYKLACE EKOVUK a.s. . The particle size of CFL powder was 25 μm4OH to pH 3. For CFL treatment, the synchronous cultures of G. sulphuraria, at an initial concentration of 1×106 cells L−1, were cultivated in 4% of CFL extract for 24 h. Samples of the algal culture were collected at desired time intervals for further analyses. Along with CFL–treated culture, a control (untreated culture) was also inoculated with the same initial culture concentration and incubated for 24 h.To study the effect of CFL extract on various parameters of the cell cycle and the accumulation of REEs, 4% CFL acidic extract (v/v) was used to treat the algal culture. After adding the CFL extract to nutrient medium, the pH dropped to 2, and was adjusted with approximately 0.5 ml of NHG. sulphuraria was cultivated with CFL and hormones for 24 h. For this study two synthetic plant hormones, 6-Benzylaminopurine (BAP-Cytokinin family) and 1-Naphthaleneacetic acid (NAA-Auxin family) (Sigma-Aldrich) were used at a final concentration of 5 mg L−1 for this study. At the end of the experiment, cultures with and without plant hormones were harvested by centrifugation , freeze dried and analyzed by ICP-MS.To study the effect of hormones on REEs accumulation, one set of asynchronous cultures of Dry matter was determined from 5 ml of algal suspension centrifuged at 4000 rpm for 5 min in dried and pre-weighed 5 ml test tubes. The pellet was dried at 105°C for 24 h and weighed on a Sartorius TE214S-0 CE analytical balance.G. sulphuraria based on dry matter (DM) according to the equation t is the time of the end of the light phase, T0 is the starting time, and DMt is the value of dry matter at Tt and DM0 is the value of dry matter at T0.Doubling time (DT) was calculated for both the control and treated cultures of a (Chl a) and carotenoids (car), 10 ml of homogenized suspension of both the control and CFL–treated cultures were centrifuged at 4000 rpm for 5 min. Harvested pellets were suspended in 1 ml of phosphate buffer  and 10 μg of MgCO3.The pellets were then each mixed with 500 μl of 0.75–1.00 mm glass beads  and vortexed for 5 min to break the cell walls. For pigment extraction, 4 ml of 100% acetone was added, mixed well, and centrifuged at 4000 rpm for 5 min. After the first round of extraction, the supernatant was transferred to a calibrated tube with a stopper and placed in a dark block. The extraction process was repeated with 80% acetone and the supernatant was transferred to the same calibrated tube. The final volume of extract was made up to 10 ml using 80% acetone. The absorbance of the solution was recorded at 750, 664, 647, 470, and 450 nm by UV-1800 spectrophotometer, Shimadzu Corporation . The content of chlorophyll a and carotenoids were calculated according to To determine the content of chlorophyll −1,was carried out by following the equations of Phycobiliproteins were extracted in 10 ml of homogenized control and CFL–treated culture samples. Each culture was centrifuged at 4000 rpm for 10 min at room temperature and the pellet was washed twice with distilled water. Finally, the culture was re-suspended in 20 mM acetate buffer (pH 5.1) containing 40 mM NaCl and 0.02 M sodium azide, followed by bead beating and repeated freeze-thawing until the phycobiliproteins were released into the supernatant. The collected supernatant was measured in a UV–VIS spectrophotometer . The estimation of phycobiliproteins, expressed in mg mLv/Fm). Two milliliters aliquots were withdrawn from the culture and placed into 10 × 10-mm plastic cuvettes for 30 min in the dark. Quantum yield was measured using an AquaPen-C 100 PAM fluorimeter .Photosynthetic activity was evaluated by fluorimeter as the quantum yield  for 5 min at 3,200 rpm for cell breakage. For depigmentation of algae, 1 ml of 80% ethanol was added to the sample then vortexed and incubated at 68°C for 15 min in a water bath. This process was repeated 3–4 times until the pellet was clear (green color free). The glycogen-containing cell pellets were hydrolyzed with 1.5 ml of 30% perchloric acid for 15 min at room temperature, then centrifuged and supernatants were collected into pre-prepared calibration test tubes. This process was repeated twice more, and the extracts were combined and made up to 5 ml using 30% HClO4. The colorimetric reaction was then carried out by mixing 500 μl of ice-cold extract with 2.5 ml of anthrone reagent (2 g of anthrone in 1 l of 72% (v/v) ice-cold sulphuric acid). The mixture was boiled at 100°C for 8 min followed by cooling and quantification at 625 nm (A625) using a Shimadzu UV-spectrophotometer UV-1800 . The same procedure was followed for the blank (500 μl of 30% HClO4) and standard tubes (500 μl of glucose (100 mg L−1) in 30% HClO4). Glycogen calibration was carried out simultaneously using glucose as the standard. To obtain the calibration curve for glycogen determination, the values measured for glucose were multiplied by 0.9. The data were expressed as picograms (pg) of glycogen per cell.Glycogen content was determined by the anthrone method  accordinConfocal images of treated and control cells were captured with an inverted Zeiss LSM 880 laser scanning confocal microscope  equipped with a Plan-Apochromat 63x/1.4 NA Oil DIC M27 immersion objective. SYBR-Green was excited by Argon laser 488 nm (laser power 0.03%), its emission was captured by GaAs-detector at wavelengths 499–571 nm, Gain 750. Likewise, chlorophyll auto-fluorescence was excited by Argon laser 488 nm (laser power 0.15%) and detected at 695–759 nm . This track was on top of that used to create T-PMT signal (transmission) using Gain 370. The pinhole for this excitation wavelength was kept at 69 μm diameter. MBS was chosen 488 and pixel dwell time was given 16.38 μs. The images were then processed using ImageJ software.n = 3). The presented results are the averages and standard deviations from all three replicates. The data statistics analysis was generated using the Real Statistics Resource Pack software (Release 8.4) for MS Excel 2013. Copyright (2013–2021) Charles Zaiontzv/Fm ratio) multiple linear regression model was used. The fitted regression model was: ß1 + (ß2*time) + (ß3*treatment) + (ß4*time^2) + (ß5*(time*treatment)), where the coefficient values correspond to predictors: ß1 - Intercept, ß2 - Time, ß3 - Treatment, ß4-Time^2, ß5 – Time*Treatment. The comparison of individual REE concentration levels at different time points or between experimental treatments  was performed using the one-way ANOVA test and Tukey’s HSD test. A value of p < 0.05 was considered significant.All experiments were performed in three biological replicates  in a PTFE microwave oven  at 250–600 W for 20 min. Quantitative analysis of REEs was performed using an Elan DRC-e  which is equipped with a concentric PTFE nebulizer and cyclonic spray chamber. Algal samples were passed through a 0.45 μm nylon syringe filter  and diluted 1:10 with distilled water. Values were expressed as micrograms per gram dry weight (μg g−1 DM).Samples of CFL alone and CFL-treated algal biomass were digested with 30% H3, the effect of 10% HNO3 on a synchronous culture of G. sulphuraria was studied. Results of this preliminary study showed that there was no negative effect of 10% HNO3 on the cell shape, size, or growth of this organism , the Chl a content was recorded as 3.0 mg L−1 in the control culture whereas, in the CFL–treated culture, Chl a content was only 2.2 mg L−1. This loss in chlorophyll content could be due to CFL extract stress on the algae. In contrast, carotenoids, which are known to play a role in defense of algae against stress, increased till 4 h to 110 mg L−1 under CFL treatment compared with 78 mg L−1 in the control culture  in the algal biomass  = 132.56, p = 5.43E-08), carotenoids  = 61.55, p = 1.55E-06), and also for phycocyanin  = 125.25, p = 6.98E-08). Both time and time^2 significantly predicted the concentration of all three pigments. Treatment alone did not significantly predict their concentration, however, the interaction term time*treatment did significantly predict the concentration of chlorophyll a, and carotenoids. The treatment influence was therefore significantly time-dependent. There was no time-dependency for phycocyanin, and its concentration in both cultures was not significantly different. For β coefficients and corresponding p-values see Multiple linear regression was used to test if time (hours of cultivation) and treatment (control vs. CFL) significantly predicted the concentration of pigments (chlorophyll  biomass . For fitv/Fm) which is the parameter commonly reflecting reduced function or impairment of the PSII reaction centers. At the beginning of the experiment, the Fv/Fm ratio was recorded as 0.58 in the control culture and 0.55 in the CFL–treated culture  = 12.92, p = 1.84E-05). Both time and time^2 significantly predicted the Fv/Fm ratio. Treatment alone significantly predicted the Fv/Fm ratio, however, the interaction term time*treatment did not. For β coefficients and corresponding p-values see Multiple linear regression was used to test if time (hours of cultivation) and treatment (control vs. CFL) significantly predicted the F biomass . For fitG. sulphuraria was followed throughout the cell cycle and samples were withdrawn every 2 h from both control and CFL–treated cultures . This represents and approximately 8-fold increase in glycogen in the control culture and 5.5-fold increase in the CFL-treated cells during the light phase; around 1/3 of the glycogen content was consumed during the dark phase in both cases  = 75.24, p = 3.84E-12]. Regarding the β coefficients and corresponding p-values in the Multiple linear regression was used to test if time (hours of cultivation) and treatment (control vs. CFL) significantly predicted the concentration of glycogen in the algal biomass . For fitG. sulphuraria was assessed by confocal microscopy using SYBR Green dye for nuclear staining    . In continished) . The mos, and Ce . However, and Ce ; Table 1ll cycle . This suG. sulphuraria. The analysis revealed a statistically significant difference in the concentration of individual REEs between at least two groups, for statistical values see A one-way ANOVA was performed to compare the effect of time  on the concentration of individual REEs in the biomass of G. sulphuraria. Results showed that these hormones did not affect these parameters  and 451 (1.4-fold) μg g−1 DM, respectively, in the presence of NAA  on the concentration of individual REEs in the biomass of G. sulphuraria was selected for the present study due to its ability to grow in a range of diverse habitats including those having thermophilic, acidophilic, halophilic and toxic metal conditions , neodymium (Nd), Eu, Gd, Tb, dysprosium (Dy)) has been studied   . The finnditions , S2. Thunditions  could haChlorella sorokiniana, Nannochloropsis salina and Picochlorum sp. They reported that specific night biomass loss rates were highly variable, and varied between −0.006 and −0.59 μdark day−1  of Se caused a significant decline in the overall content of carotene and chlorophyll a. This result showed that if the level of stress was higher, then no defense mechanism of the cell could protect it. Similarly, Cheng et al., showed that an increasing concentration and exposure time to cadmium (Cd) can cause a decline in Chl a, Chl b and carotenoids in C. vulgaris  are typical for red algae  dark phase  of the PSII reaction center decreased under La exposure in D. quadricauda  were surrounded by the mother cell wall before hatching , chromium (Cr) and copper (Cu), and their growth, cell volumes, and cell divisions were investigated over a period of 72 h  containing environments.In the present study, REE accumulation was concentration-independent and highly selective with the progression of the cell cycle ; Table 1a and Ce , 7. The h medium . Severalh medium . Our resC. vulgaris and Acutodesmus obliquus (Interestingly, almost no research to date has been conducted on the effect of hormones on the bioaccumulation of REEs by microalgae. This is the first study that shows the accumulation of Y and Eu can be doubled in the presence of the plant hormones NAA and BAP ,B. Howevobliquus , 2018.G. sulphuraria can grow in the presence of a CFL acid extract containing 10% HNO3, although growth was slightly slower than the control culture. Photosynthetic pigments such as chlorophyll a, carotenoids and phycocyanin were also decreased under CFL treatment, although carotenoid synthesis minimized the deleterious effects of CFL up to the initial 4th h of the cell cycle. During the light phase of the cell cycle, photosynthetic performance, expressed as the ratio Fv/Fm, was negatively affected by CFL treatment, but later in the dark phase, recovered significantly. In the initial hours of the cell cycle, cell division was delayed by about 2 h with CFL treatment, although later cells were able to complete their 2nd division within 24 h of the cell cycle. The produced daughter cells were of smaller size. Accumulation of REEs by G. sulphuraria was concentration-independent and selective. The REEs most accumulated by G. sulphuraria were Y and Eu, followed by La, Ce, Gd, and Tb, respectively. The plant hormone NAA stimulated the accumulation of REEs and almost doubled Y accumulation. BAP also had a pronounced effect on Y and Eu accumulation, although it did not increase the accumulation of other REEs. Waste luminophores like CFL bulbs could be a great secondary source of REEs, and using the red alga G. sulphuraria may be promising in REE bio-removal/accumulation technology. Recovery of REEs from algal biomass could lead to an economic and eco-friendly solution for the removal of hazardous waste from CFL lights and for the generation of REEs for future use.Being acidophilic in nature, the red alga The original contributions presented in the study are included in the article/MV, DS, and DM: conceptualization and funding acquisition. AS, MČ, and VN: methodology, investigation, and writing—original draft preparation. DS and DM: validation. MR: formal analysis. AS and MČ: resources. MV and DM: data curation, writing—review and editing, and project administration. MV and MR: visualization. MV: supervision. All authors contributed to the article and approved the submitted version.This research was funded by the European fund for regional development, the program Interreg V-A Austria – Czech Republic, the Project ATCZ172 REEgain, the COST Action 19116 – PLANTMETALS and by the institutional support RVO 61388971 and RVO 67985939 of the Czech Academy of Sciences.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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