An important clinical challenge is improving the healing rate of diabetic chronic wounds, and developing new approaches that can promote chronic wound healing is essential. A new biomaterial that has demonstrated great potential for tissue regeneration and repair is self-assembling peptides (SAPs); however, they have been less studied for the treatment of diabetic wounds. Here, we explored the role of an SAP, SCIBIOIII, with a special nanofibrous structure mimicking the natural extracellular matrix for chronic diabetic wound repair. The results showed that the SCIBIOIII hydrogel in vitro has good biocompatibility and can create a three-dimensional (3D) culture microenvironment for the continuous growth of skin cells in a spherical state. The SCIBIOIII hydrogel in diabetic mice (in vivo) significantly improved wound closure, collagen deposition, and tissue remodeling and enhanced chronic wound angiogenesis. Thus, the SCIBIOIII hydrogel is a promising advanced biomaterial for 3D cell culture and diabetic wound tissue repair. Diabetes mellitus (DM) is a serious systemic metabolic disease characterized by high blood glucose levels for a long time, resulting in a series of complications . The higHydrogels are a very promising biomaterial owing to their several beneficial functions, such as excellent moisturization, permeability, biocompatibility, degradability, and drug transportation ,8. Self-In this study, a novel self-assembled peptide, SCIBIOIII, is reported. First, we observed its gel-forming ability. In order to provide a 3D microenvironment for cell growth, we cultured mouse skin fibroblasts, in vitro, in a hydrogel formed by SCIBIOIII and analyzed the growth activity of the fibroblasts in the SAP hydrogel. Subsequently, a diabetic mouse wound model was prepared, and the SCIBIOIII hydrogel was applied to the damaged skin of the mice, and the wound healing rate, wound histomorphological characteristics, collagen formation, and angiogenesis after SCIBIOIII treatment were assessed.The observation of the SCIBIOIII microstructure under AFM shows that after 24 h of assembly, the peptide forms many ordered, dense nanofiber structures that intertwine to form a nanofiber network scaffold .The results of Congo red/aniline blue staining demonstrated that the SCIBIOIII structure without the addition of PBS was quicksand−like and diffusely sparse at 24 h, while after adding PBS to trigger self-assembly for 24 h, it aggregated into a dense and stable fragment-like structure with clear boundaries, similar to a fibrous mesh structure, which is conducive to the construction of a 3D environment for cell growth .Under an ordinary light microscope, the mouse skin fibroblasts can be observed under conventional culture conditions in a long shuttle shape and grow in a single layer against the wall. In contrast, mouse skin fibroblasts grew in a round spherical shape and multi−layered space in the hydrogel formed by SCIBIOIII. The results showed that the SCIBIOIII hydrogel could create a 3D microenvironment for NIH3T3 cell growth .Further, we observed that mast live cells were growing in a circular shape in the 3D environment via live/dead staining assays, and the number of live cells in the 3D environment gradually increased with the increase in culture time. These experimental results indicate that NIH3t3 cells can grow in the 3D environment constructed by SCIBIOIII, which has good biocompatibility and no cytotoxicity .The impact of the SCIBIOIII hydrogel on chronic wounds was first assessed in the diabetic mouse trauma model. By photographing the back wounds of both groups of mice at the indicated times, it was found that from the 5th day after treatment, the SCIBIOIII group had significantly higher wound healing than the diabetic group A. FurtheSubsequently, we performed morphological analysis of wound tissue in diabetic mice. The granulation tissue formation is an important index to evaluate the degree of wound healing. The HE staining results showed that the skin wound in the DM group was significantly wider than that in the SCIBIOIII group on the 7th day after treatment, and no obvious new granulation tissue was observed, while obvious granulation tissue and new epidermis were observed in the hydrogel injection group. Notably, on postoperation day 11, the degree of wound healing was significantly higher in the DSCIBIOIII group than that in the model group, and the skin epidermis and dermis were more intact and orderly .Next, collagen deposition changes in the skin samples treated with and without the SCIBIOIII hydrogel were examined. MASSON staining demonstrated a significant increase in collagen deposition in diabetic skin wounds on days 7 and 11 after SCIBIOIII hydrogel injection compared to the DM group without SCIBIOIII treatment A. MoreovFinally, the effect of the SCIBIOIII hydrogel on diabetic wound angiogenesis was also analyzed. First, IHC was used to stain the wound tissue of each group for the vascular endothelial cell marker CD31. The results revealed that the wound tissue of the SCIBIOIII group displayed an obvious increase in CD31 expression compared to the model group on days 7 and 11 following surgery, and a greater number of new capillaries was observed A. FurtheWound healing is an intricate and dynamically changing process. The wound healing process primarily consists of four overlapping steps: hemostasis, inflammation, proliferation, and, finally, wound remodeling. During these stages, multiple cells and growth factors interact together to participate in wound repair . PatientSAP hydrogels can imitate the natural microenvironment of cell growth in vivo and have been designed as scaffolding materials for 3D cell culture and tissue engineering . The micThe ECM is a noncellular 3D macromolecular network comprising collagen, proteoglycan/glycosaminoglycan, elastin, fibronectin, laminin, and several other glycoproteins . ECM remA major issue in the recovery of chronic diabetic wounds is inadequate blood supply, manifesting primarily as insufficient angiogenesis and decreased granulation tissue formation . NeovascIn summary, our results confirmed that under ion-triggered conditions, the SCIBIOIII used in this study can form a nanofibrous network and has good biocompatibility for 3D cell culture. Moreover, it can spontaneously form a hydrogel to create a microenvironment with high water content at the wound site that mimics the ECM and accelerates diabetic chronic wound healing by promoting ECM remodeling and angiogenesis. However, this study failed to explore the specific molecular mechanism of its promotion of diabetic wound healing; secondly, wound healing in animal experiments was only observed up to day 11, while complete wound healing was not observed. Additionally, inflammation is also a challenge affecting diabetic wound healing; therefore, in future studies, whether SCIBIOIII can control diabetic wound inflammation to further accelerate chronic wound healing must be investigated.In our study, we used a novel self-assembling peptide to investigate its role in chronic diabetic wound healing. Our results demonstrate that this peptide has good gel-forming properties and is able to rapidly change from liquid to gel on the wound surface. Moreover, this peptide possesses good biocompatibility and can be used in cellular and animal experiments. Importantly, in animal experiments, we observed a striking accelerated chronic wound healing effect. Thus, this peptide is a very valuable biomaterial with promising applications in the biomedical field. In conclusion, our study provides a new therapeutic strategy for clinical practitioners.A total of 1 mL of double−distilled water was added to 10 mg SCIBIOIII lyophilized powder with 1 mL disposable syringe in the ultra-clean table, mixed thoroughly, and stored at 4 °C.The surface microscopic topography and structure information of SCIBIOIII was obtained by AFM. Briefly, we added 10 µL PBS into 10 µL peptide solution (10 mg/mL), assembling for 24 h at 37 °C, acquiring images under AFM.A total of 10 mg/mL SCIBIOIII solution was mixed in the ratio of 1:1 with PBS; moreover, Congo red/aniline blue dye was added after assembly at 0 h and 24 h. A microscope was used to observe the morphology of SCIBIOIII.2. The 3rd–5th generation cells were harvested in good growing conditions. The cell concentration was adjusted to 2 × 104 cells, and the cells were resuspended with 1 mL of 10% sucrose solution. The peptide solution was quickly blended with the sucrose/cell mixture at a volume ratio of 1:3 on 96-well plates. A total of 100 µL of high glucose DMEM complete medium was added to each well, cultured at 37 °C in an incubator with 5% CO2. Photographs were recorded under the microscope on days 1, 3, and 5.High glucose DMEM medium with 10% FBS was used to cultivate NIH3t3 cells  in a 37 °C incubator with 5% COThe cells were assayed for growth activity in the hydrogel on days 1, 3, and 5. The complete medium was discarded to add PBS to wash the cells 2–3 times. In the dark, the prepared Calcein−AM staining solution  100 µL was added to each well and incubated at 37 °C for 25 min. The pictures of stained cells were recorded using a fluorescence microscope.n = 5), and each group was treated using 20 µL of PBS or SCIBIOIII (10 mg/mL) for three consecutive days.Wild-type (WT) C57BL/6 male mice , aged 7–8 weeks, were habitually housed in an SPF−grade animal laboratory at a temperature of (21 ± 1) °C, at a relative humidity of 55 ± 5%, with 12 h of translating light and dark, and free drinking water. After dissolving 50 mg/kg Streptozotocin  in citrate buffer , it was injected into mice’s abdomen for five consecutive days. Blood glucose levels were tested by glucometer  after one week. Mice with random blood glucose concentration >16.7 mmol/L and weight loss were diagnosed with diabetes mellitus. Mice with diabetes were anesthetized using Isoflurane after two weeks. After shaving and disinfection, using a skin biopsy perforator  made two 6 mm diameter total excision wounds on the back of each one. Two groups of the model mice were randomly selected  staining was used to evaluate the degree of wound healing; Masson staining was used to evaluate the collagen formation of skin wounds; while immunohistochemical and immunofluorescence stainings were used to analyze new blood vessels, collagen deposition, and wound tissue cell proliferation. The intact skin wound tissue was fixed in 4% paraformaldehyde for HE staining and Masson staining, was dehydrated, embedded in paraffin, sectioned, and stained using HE staining solution  or Masson staining solution according to the kit instructions. For immunohistochemical staining (IHC) staining, skin tissue sections were prepared as before, antigen retrieval was performed on the sections, and target antibodies CD31 and collagen I or fluorescently labeled antibodies Ki67 and a−SAM were added and incubated overnight. Following this secondary antibodies were added to incubate for color development, and then hematoxylin was used to restain the nuclei. Following staining and slide mounting, images were acquired under a light microscope or a fluorescence microscope.Using a purchased commercial kit , total RNA was extracted from the sample and reverse transcribed into cDNA. SYBR Green was used as a fluorescent marker. Target gene primer sequences were provided in t-tests were used to examine the differences between the groups. Data were analyzed using the GraphPad Prism program (version 9.0). All trials in this paper were repeated three or more times. p < 0.05 was regarded as statistically significant.The data were all given as mean SEM. Two independent-sample


Menstrual problems affect as high as 90% of adolescent females and are the main reason for gynecology visits. Dysmenorrhea was the most frequent menstrual disorder for which adolescents and their parents were referred to a physician. Many undergraduate students are adolescents who undergo several hormonal changes that affect menstrual patterns. This study aimed to determine the prevalence of menstrual disorders and to determine the effect of menstrual disorders on quality of life (QOL) of female undergraduate students at Makerere University college of health sciences.P < 0.05 was considered statistically significant.A cross sectional study design using a self-administered questionnaire. WHO  QOL-BREF (QOL-Best Available Reference document) questionnaire was used to assess QOL of participants. Collected data was double entered into EPIDATA, and transferred to STATA for analysis. Data was presented using tables, and analyzed using percentages, frequencies, medians, interquartile range, means and standard deviations, t-test and ANOVA were used to establish statistical significance. N = 258) followed by dysmenorrhea (63.6% (95% CI: 57.7–69.1), N = 175), irregular menstruation (20.7% (95%CI: 16.3–25.9), N = 57), frequent menstruation  and infrequent menstruation (3.3% (95% CI:1.7–6.2), N = 9). Dysmenorrhea and premenstrual symptoms significantly reduced the QOL scores of participants.Of the participants, 275 were included in the data analysis. The median age of the participants was 21 years with range of 18–39 years and interquartile range of 20–24 years. All the participants had attained menarche. Of the participants, 97.8% (95%CI: 95.2–99.0) (269/275) reported some form of menstrual disorder. Premenstrual symptoms were the commonest disorder (93.8% (95%CI: 90.2–96.1), Menstrual disorders were highly prevalent with negative effects on QOL and class attendance. Efforts should be made to screen and possibly treat menstrual disorders among university students as well as to conduct further studies to elucidate more on the effects of menstrual disorders on quality of life.The online version contains supplementary material available at 10.1186/s12905-023-02290-7. Menstrual disorders refer to abnormalities of menstruation that include menstrual cycle irregularities; irregular menstrual cycle, heavy menstrual bleeding, infrequent menstruation, frequent menstruation, intermenstrual bleeding, amenorrhea , dysmenoMany undergraduate students are adolescents who undergo several hormonal changes that affect menstrual patterns. Menstrual disorders can be confused with menstrual pattern variation caused by these hormonal changes. This variability in menstrual patterns poses a diagnostic challenge to clinicians and may lead to delay of treatment of underlying conditions . InvestiIn a study of the prevalence and patterns of menstrual symptoms among Lebanese nursing students, the most common menstrual disorders were irregular frequency of menstruation (80.7%), premenstrual syndrome (54.0%), irregular duration of menstruation (43.8%), dysmenorrhea (38.1%), polymenorrhoea (37.5%) and oligomenorrhoea (19.3%) .Whereas most studies in Uganda focused on menstrual hygiene management, one study reported that 19.7% of participants from selected secondary schools in Entebbe missed school at least once during their most recent menstrual period and those who used a menstrual diary reported 28% school absenteeism on period days compared to 7% on non-period days and that missing school during the most recent period was associated with physical symptoms; headache, stomach pain, back pain and with changing protection 4 or more times per 24 h period . This stThis study aimed at determining the prevalence of menstrual disorders and how they impact on Quality of life of undergraduate students at Makerere University College of health sciences and generate information to foster further research as well as policy on menstrual management.A cross sectional study.The study was conducted in Makerere University, the largest public University in Uganda located about 2 km north of Kampala City with a student population of 39,546, 44%  being females as of 2017, specifically in Makerere University College of health sciences. The college of health sciences has school of medicine, school of biomedical sciences, school of health sciences and the school of public health, and has a diverse population of students from all over the country as well as international students that makes it a representative sample population. Female undergraduate students who consented to the study were included while those on hormonal contraception or those who were pregnant or lactating were excluded from the study.The sample size obtained using the Kish Leslie (1965) formula basing on a previous study in Nigeria by Amu & Bamidele, 2014 was 265, assuming withdrawal of consent or incomplete data of 10%, we aimed at a minimum of 265/0.9 = 295, thus a sample size of 295 was used. Study participants were selected by consecutive sampling of available female undergraduate students in Makerere University College of health sciences.Data was collected using a pre-tested self-administered questionnaire from a population of female undergraduate students at Makerere University College of health sciences who enrolled for the study having met the eligibility criteria. The questionnaire contained questions on sociodemographic characteristics such as age, course, year of study, number of pregnancies, education sponsor, etc. menstrual characteristics such as presence or absence of menstruation, age at menarche, length of cycles, regularity of cycles, presence or absence of painful menstrual, duration of menstruation, number of sanitary pads used each menstruation to quantify the amount of bleeding, the severity of menstrual pain , premenstrual symptoms and participants’ perception of menstrual pain based on their experience were collected. Menstrual disorders were defined as follows: Normal menstrual frequency was defined as menstrual cycles repeated about once every 24–38 days, regular menstrual cycle referred to cycles in which the shortest to longest cycle variation is ≤ 9 days, irregular menstrual cycle referred to cycles in which the shortest to longest cycle variation is ≥ 10 days, infrequent menstruation referred to a menstrual cycle repeated about once every > 38 days, frequent menstruation referred to a menstrual cycle repeated once every < 24 days, intermenstrual bleeding referred to bleeding between cyclically regular onset of menses, dysmenorrhea referred to painful menstruation, premenstrual symptoms were symptoms preceding menses and resolving with onset of menses and included painful breast on touching, difficulty concentrating, mood changes and depression, primary amenorrhea referred to no menses by age 16 years, secondary amenorrhea referred to no menses for an interval of time equivalent to a total of at least three months in students who had menstruated previously.QOL was measured using the WHO QOL BREF   which coThe WHOQOL-BREF  producedPretesting questionnaire was done by collecting data from a set of 30 students who were not part of the final study participants and the questionnaire was found to be appropriate. The WHO QOL BREF has been validated in multiple studies as a tool for measuring QOL , 17 and The research assistants were trained on the research protocol, ethical issues and data collection procedures. Completed questionnaires were double-checked for completeness and accuracy and stored under key and lock by the principal investigator who was also the only person with permission to access the data for confidentiality purposes. There was double entry of data into EPIDATA manager version 4.6.0 and the two entries compared and contrasted to eliminate errors in data entry. Data cleaning to ensure consistency and data integrity was done before export to STATA version 14.1 .P < 0.05 was considered statistically significant. The mean domain scores of QOL in participants with menstrual disorders were compared with those without. The strength of the associations between menstrual disorders and QOL was tested using Cohen’s d effect size calculated as the difference in means divided by pooled standard deviation. Cohen’s d > 0.25 was considered to indicate practical Independent variables were dysmenorrhea, premenstrual symptoms, heavy menstrual flow, amenorrhea, frequent menstruation, irregular menstruation, intermenstrual bleeding, infrequent menstruation while the dependent variables were the quality-of-life Domains in the WHO QOL BREF i.e., physical, psychological, social and environmental domains.Participant characteristics such as age, menarche, were described using percentages, frequencies, tables, medians and their interquartile range. The prevalence of menstrual disorders was calculated and presented as proportions. The domains of the QOL BREF were summarized using means and 95% confidence intervals because the data were normally distributed. ANOVA test and t-test were used to establish the relationship between the reported menstrual characteristics and the mean domain scores in the QOL BREF, Informed consent was obtained from participants and only those who agreed by signing in the consent form were recruited in the study. Protocol was handed to the department of obstetrics and gynecology Makerere University for review after which it was forwarded to the Makerere University School of medicine research and ethics committee (SOMREC) for approval . Copies of permission letters and attached copies of the protocol were distributed to the principle of the Makerere University College of health sciences seeking his permission to collect data. Participation in the study was completely voluntary and participants were free to withdraw from the study any time they wished. Information obtained from the participants were kept confidential and anonymous by assigning code numbers for participants’ questionnaires that were used on all research notes and documents and keeping questionnaires and any other identifying participant information in a locked file cabinet in the personal possession of the researcher.Participant response rate was 96% (295/306). Table The mean age of menarche was 13.07 ± 1.37 years. Table Table Menstruation is a normal physiologic process but is frequently complicated by pre-menstrual and menstrual disturbances . DisordeIn this study we established the prevalence of menstrual disorders and their effect on the quality of life of female undergraduate students in Makerere University college of health sciences. The participants comprised mainly young people. Of the 275 participants, 269  reported one of the different forms of menstrual disorders considered in this study including irregular menstrual cycle, frequent menstruation, infrequent menstruation, intermenstrual bleeding, premenstrual symptoms and dysmenorrhea. A similar high prevalence of 91% was reported among students studying health sciences in Saudi Arabia . It is wThe prevalence of dysmenorrhea in this study was 63.6% (95% CI 57.7–69.1) with 46.8% reporting dysmenorrhea in all menses and 40.6% reporting it in some menses. The prevalence of dysmenorrhea in this study is lower than that reported in northern Ghana (83.6%) , Spain  of participants. This prevalence is lower than that reported among university students in Turkey (31.2%) . A DanisOf the participants, 93.8% (95% CI 90.2–96.1) reported at least one premenstrual symptom. The most prevalent symptoms were depression/mood changes (60.7%) and painful breast on touching (40.4%) This prevalence is higher than the 79% reported among Japanese College students  and amonNone of the participants reported missing menses in the 3 consecutive months preceding the study indicating no cases of primary or secondary amenorrhea.p < 0.001, effect size d = 0.504), psychological  and social domains , and environmental domain  respectively. These effect sizes demonstrate practically significant associations. 10.9% (30/275) of participants reported ever missing class in the 2 months preceding the study. 100% (30/30) of participants who missed classes had dysmenorrhea while 93.3% had premenstrual symptoms. Intermenstrual bleeding, frequent menstruation, infrequent menstruation and irregular menstruation did not significantly reduce quality of life of the participants.Dysmenorrhea and premenstrual symptoms had statistically significant negative effects on the quality of life of participants in the environmental (45.9 vs 50.3, In India dysmenorrhea reduced the QOL WHO BREF scores in all domains  similar Dysmenorrhea was reported to negatively affect the health related quality of life of university students in Western Turkey . This stIn studying the relationship between premenstrual symptoms, menstrual pain, irregular menstrual cycles, and psychosocial stress among Japanese college students, Yamamoto et al. reported that students who reported dysmenorrhea, irregular menstrual cycles and premenstrual symptoms had higher scores perceived stress than those who did not . Since hQOL being an individual’s perception of their position in life in the context of culture and value system where they are inserted, which also involves their goals, perspectives, standards and concerns  could beWe believe these study findings provide a reasonable representation of the prevalence of menstrual disorders and their effects on quality of life of female undergraduate students studying health sciences in Uganda, however more studies with emphasis on the effect of confounders and effect modifiers of QOL in this population would be valuable.Confounding factors can affect QOL scores other than menstrual disorders –37 and wMenstrual disorders were highly prevalent among female students in the college of health sciences. Premenstrual symptoms and dysmenorrhea were the most prevalent menstrual disorders and had significantly negative effects on quality of life of the affected students.Efforts should be made to screen and possibly treat menstrual disorders among University students to ameliorate their negative effects on quality of life of the affected students. Further studies are necessary to elucidate more on the effects of menstrual disorders on quality of life.Additional file 1.


The remaining necessary agronomic practices and crop management activities were undertaken uniformly. The data presented under this dataset article includes phenological Parameters , growth parameters  and yield and yield component parameters . All the collected data were subjected to analysis of variance (ANOVA) and the analysis was carried out using the SAS version 9.0 software computer program's General Linear Model (GLM) procedure Maize is one of the most important cereal crops in Ethiopia. However, its yield is lower than its potential mainly due to low soil fertility problems. Therefore, this field experiment was conducted during 2021 main rainy season with the objective of improving maize production through application of brewery sludge and blended NPS fertilizers in North Mecha district, Northwestern Ethiopia. Factorial combination of three levels of brewery sludge (0, 10 and 20 t ha Specifications Table1•In Ethiopia, maize production and productivity is below its potential even though higher than other major cereal crops produced in the country. This is due to a combination of several production constraints, the most significant of which is nutrient deficiencies that cause declining soil fertility. Hence, to address this problem of soil fertility and enhancing maize productivity, blended fertilizer sources  were applied in blanket recommendation. But, currently its cost is not affordable by smallholder farmers. Therefore, this dataset gives information on the response of maize growth, yield and yield components to different rates of blended NPS fertilizer.•Currently, the number of brewery factories in Ethiopia is increasing. Dashen Beer Limited Company is one among those, based in Gondar, Amhara region. It produces a huge amount of sludge every year and too costly to dispose of and pollutes the environment. But, its reuse and application to agricultural land has unrestricted importance as an organic fertilizer and substitute mineral fertilizer to increase crop productivity without any adverse environmental effects. And also it is relatively less cost for application to agricultural land by smallholder's farmers in Ethiopia. Because, it contains 20-30 % more nutrients than commonly used organic fertilizer and reduce 40-50 % application of chemical fertilizers. Thus, this dataset also provides valuable information on the effects of brewery sludge (a byproduct of brewery factory) on maize productivity. As, it supplies both macro and micronutrients and improves soil physical (structure) and biological (microorganisms) activity as to increase its nutrient retention capacity. This information, therefore, can help maize producing farmers to recycle brewery sludge to their agricultural land instead of chemical fertilizer as their cost is now a days increased and not affordable by smallholder farmers.•However, sole application of chemical (Blended NPS) or organic (brewery sludge) fertilizer has its own limitations. Hence, integrated application of brewery sludge and Blended NPS fertilizer has been emerged as source of alternative that reduces production costs while increasing maize productivity. As a result, agronomists can utilize this dataset article to run trials on different crops or the same crop in different soil types and seasons. This could result in increased crop output at cheap or no cost to farmers, as well as improved research operational efficiency among scholars. 2•To give information on the effects of brewery sludge and blended NPS fertilizer rates on yield and yield components of maize in North Mecha district, Northwestern Ethiopia•To show the optimum rates of brewery sludge and blended NPS fertilizer in harnessing maize economic production in North Mecha district, Northwestern Ethiopia•To give valuable data on the economic feasibility of using brewery sludge and blended NPS fertilizer rates in maize production of North Mecha district, Northwestern Ethiopia3−1) of maize. 2), leaf area index) of maize are indicated in −1 in −1) in 1 in 1 in 1 in https://osf.io/42wn5).This dataset was collected in a field experiment conducted during the main cropping season from June to December 2021 at Kudmi village of North Mecha district, Northwestern Ethiopia . The dat4−1) and four levels of blended NPS fertilizer rate  were laid out in RCBD2) and a gross plot size of 3.75m width x 3m length (11.25m2). A 1.0 m wide open path separated the adjacent blocks and a 0.5m wide path separated plots within a block from one another. Using a random table number, experimental treatments were assigned to experimental plots in each block. As a result, there were 5 rows totaling 3m in length, with 10 plants per row and 50 plants per plot. To avoid possible border effects, the net plot size (harvestable area) was 2.25m x 2.4m (5.4m2) (i.e. the middle 3 rows from each plot) by excluding 1 outermost row on both sides of each plot horizontally and 0.3m row segment from both ends of the plot vertically. Then, all the remaining necessary agronomic practices and crop management activities  were undertaken uniformly.Factorial combinations of three levels of brewery sludge ; L: Length of leaf (cm); W: Width of leaf (cm); K: correction factor (0.75) 2) to the area of land occupied by the plant i.e. ear leaves, one above and one below the ear leaf as described by Where, LA: Leaf area per plant : It was calculated as the ratio of grain yield to total above ground dry biomass yield multiplied by 100 at harvest from the respective treatments 4.2The collected data were subjected to analysis of variance (ANOVA) and the analysis was carried out using the SAS version 9.0 software computer program's General Linear Model (GLM) procedure 4.3−1 during harvest. Prices of blended NPS fertilizer and brewery sludge and their transportation and application cost were taken as fixed price during sowing.A partial budget analysis was performed to investigate the economic feasibility of the treatments (brewery sludge and blended NPS fertilizer). A partial budget, dominance and marginal analysis were used. Partial budget and marginal analysis of economic concepts were used to analyze the economic data. Partial budget is a method of organizing experimental data and information about the costs and benefits of various alternative treatments. Marginal analysis is a method for comparing the costs that vary with the net benefits for selecting the best technology for recommendations from the experiment. The market cost of maize was taken from local market prices in Ethiopian Birr (ETB) kgPartial budget averaged of the twelve (12) treatments calculated from income and expenses based on variable cost . The meaThe dataset collected in this study did not involve animals and humans.Fenta Assefa: Conceptualization, Methodology, Software, Writing – original draft, Investigation, Formal analysis. Zenebe Gebremedhin: Supervision, Writing – review & editing. Teferi Alem: Supervision, Writing – review & editing.


HGD) gene. This leads to a deficient HGD enzyme with the consequent accumulation of homogentisic acid (HGA) in different tissues causing complications in various organs, particularly in joints, heart valves and kidneys. The genetic basis of AKU in Egypt is completely unknown. We evaluated the clinical and genetic spectrum of six pediatric and adolescents AKU patients from four unrelated Egyptian families. All probands had a high level of HGA in urine by qualitative GC/MS before genetic confirmation by Sanger sequencing. Recruited AKU patients were four females and two males (median age 13 years). We identified four different pathogenic missense variants within HGD gene. Detected variants included a novel variant c.1079G > T;p. and three recurrent variants; c.1078G > C;p.(Gly360Arg), c.808G > A;p.(Gly270Arg) and c.473C > T;p.(Pro158Leu). All identified variants were properly segregating in the four families consistent with autosomal recessive inheritance. In this study, we reported the phenotypic and genotypic spectrum of alkaptonuria for the first time in Egypt. We further enriched the HGD-variant database with another novel pathogenic variant. The recent availability of nitisinone may promote the need for genetic confirmation at younger ages to start therapy earlier and prevent serious complications.Alkaptonuria (AKU) is a rare autosomal recessive metabolic disorder caused by pathogenic variants in the homogentisate 1,2-dioxygenase ( HGD, OMIM* 607474), commonly causing a complete deficiency of the HGD enzyme (EC 1.13.11.5), with the consequent accumulation of homogentisic acid (HGA) derived from the metabolism of the amino-acids tyrosine and phenylalanine1. HGA in alkaptonuria is commonly excreted in massive amounts in urine, causing its darkening upon air exposure, which is considered a pathognomonic sign of the disease. Furthermore, HGA is also deposited as an “ochronotic” pigment when oxidized within the articular connective tissues causing ochronotic arthritis, a degenerative joint disease2. Extra-articular complications of the disease include cardiovascular manifestations, such as mitral and aortic valve calcification with stenosis or regurgitation, renal and prostatic stones and hypothyroidism3.Alkaptonuria  is a rare autosomal recessive inborn error of metabolism. It is caused by pathogenic variants in the homogentisate 1,2-dioxygenase gene (HGD gene is located on the long arm of chromosome-3 (3q13.33) and contains 14 exons. The HGD protein consists of 445 amino acids, with a protein expression pattern mainly localized to the liver and kidney, but also expressed in other organs, such as gallbladder, gastrointestinal tract, prostate, thyroid and cartilage (https://www.proteinatlas.org/ENSG00000113924-HGD/tissue)4. Darkening of urine upon standing is the earliest sign of alkaptonuria and in our experience can be noticed immediately after birth in some cases. It is commonly the main clinical feature of the disease in pediatric patients and many affected individuals may remain clinically asymptomatic until the third or fourth decades of life5.The 6. However, being a monogenic disease with a relatively short gene, Sanger sequencing of the 14 exons of HGD with their exon/intron boundaries is most convenient and at the same time critical to confirm affected individuals and carriers of the disease1.When AKU is suspected, the presence of homogentisic acid in urine can be assayed by gas chromatography mass spectrometry (GC/MS), which serves as a quick diagnostic marker8. The actual prevalence of AKU may be underestimated due to the subtle signs of the disease, causing many patients to go undiagnosed for most of their lives8. Although the prevalence of AKU in Arab countries is not well studied, it is likely to be higher than in most populations due to the autosomal recessive nature of the disease and the high prevalence of consanguineous marriages in the region9. No data about the disease incidence or genotype in Egyptian AKU patients is yet available.The worldwide incidence of alkaptonuria is approximately 1:250,000–1,000,000 with relative hot spots in Slovakia, the Dominican Republic, India and JordanHGD gene in all suspected individuals with their parents and available siblings in a trial to confirm pathogenicity of the detected variants and capture presymptomatic cases and carriers in siblings of affected individuals.This study was conducted to investigate the genetic makeup of Egyptian patients with suspected alkaptonuria based on clinical manifestations and elevated HGA in urine. We performed Sanger sequencing of the 10. Written informed consent was obtained from all participants prior to inclusion in the study. The study was conducted in accordance with the declaration of Helsinki for studies involving human participants and the study protocol was approved by the Research Ethics Committee at Faculty of Medicine, Cairo University, Egypt .We recruited four unrelated Egyptian families with suspected AKU from different locations within Egypt. Probands and their affected siblings were followed up at the metabolic clinic at Cairo University Children's Hospital over a duration ranging from 1 to 16 years. We sampled a total of 17 individuals from the four families. AKU patients were recruited based on family history, clinical examination and the presence of homogentisic acid in urine using a qualitative GC/MS technique. The urinary organic acid analysis was performed by the Agilent 7890B GC with Agilent 7010 triple quadropole GC/MS and 7693A autosampler  using non-labelled internal standards and following the methodology of La Marca and RizzoEDTA blood was collected from all suspected AKU patients, their parents and all available healthy siblings. Genomic DNA was extracted using QIAamp® DNA Blood Mini Kit . The quantity and quality of the extracted DNA were evaluated by the NanoDrop2000®  and DNA was stored at − 20 °C until further processing.HGD gene of each index case was sequenced using specific primers designed with the help of Integrated DNA Technologies, OligoAnalyzer™ Tool . The sequences of all used primers are listed in Supplementary Table The Six AKU patients from four unrelated Egyptian families were recruited in the current study . Three families were reported consanguineous (parents were first cousins), while in family II, consanguinity was denied; however, both parents originated from the same small village and a distant common ancestor cannot be excluded. Demographic and clinical features of affected individuals are summarized in Table The proband of family I (Patient I-1) is currently a 20-year-old male and his sister (I-2) is 17 years old. The family was from Beni-Suef governorate in Central Egypt  and an unaffected male. The proband (II-3) was diagnosed with AKU through HGA detection in urine when she was 6-month-old. Her only manifestations so far are darkening of urine upon standing and hyper pigmentation and brown discoloration of buttocks Table . Her sibFamily III consisted of four siblings, three females including two AKU patients and one male sibling. The proband (III-4) was diagnosed with AKU at 6 months of age. She recently developed hyperpigmentation of buttocks and a long-standing bluish-brownish patch on the arm. Consequently, due to the experience gained by the mother, her sister (III-5) was spotted at one month of age with the same blackish discoloration of urine Table . The othThe proband (IV-6) of family IV was the youngest male and the only affected member among three older siblings. He had normal motor and mental development. Parents noticed pink to light red urine and black ear cerumen immediately after birth. At the age of 2 months, he was misdiagnosed as having urinary bleeding, although all his renal functions were normal. At the age of 6 years he started having joint pains and recurrent abdominal pain Table . AlkaptoHGD gene revealed the existence of four different missense pathogenic/likely pathogenic variants in exons 8, 11 and 13 in the four recruited families including a novel pathogenic variant in exon 13 detected in family III  consistent with the autosomal recessive inheritance nature of the disease Fig. .HGD exon 13; NM_000187.4:c.1078G > C, NP_000178.2:p.(Gly360Arg). The variant is recurrent and has been reported previously in the HGD variant database and predicted to be pathogenic according to the American College of Medical Genetics (ACMG) criteria for the classification of pathogenic variants11 . It is also predicted as pathogenic according to ACMG and ClinVar . This novel variant was detected in homozygousity in the proband (III-4) and her affected sister (III-5), while it was heterozygous in their parents. It is predicted to be pathogenic based on the ACMG criteria PM1, PM5, PM2, and PP311. The variant belongs to a hot-spot region of 17 amino-acids in the HGD enzyme, which has 13 missense/in-frame variants including 11 pathogenic variants, 1 uncertain variant and 1 benign variant, thus qualifying as pathogenic strong (PM1). Three alternative variants at the same amino-acid location are also reported pathogenic in ClinVar , c.1078G > C; p.(Gly360Arg) and c.1079G > C; p.(Gly360Ser)) (PM5). Furthermore, it is extremely rare in population databases  (PM2) and the absolute majority of predictor tools evaluate the variant as pathogenic (PP3).Family III had a novel missense pathogenic variant in exon 13 of HGD gene NM_000187.4:c.473C > T; NP_000178.2:p.(Pro158Leu). This variant was detected in homozygous state in the proband (IV-6) and heterozygous in both parents. It is predicted to be likely pathogenic according to ACMG criteria11; however, it was reported as variant of uncertain significance in ClinVar (Table 17.The fourth family (IV) had a missense likely pathogenic variant in exon 8 of In the current study, we evaluated the clinical and genetic characteristics of four unrelated Egyptian families suffering from alkaptonuria. All patients presented with isolated dark urine at a median age of 6 months and were confirmed to have a high level of homogentisic acid in urine by qualitative GC/MS evaluation of organic acids in urine.18, Algeria19, UAE20, Iraq21, Lebanon22 and Morocco23. Furthermore, the prevalence and genetic makeup of the disease are completely unknown in Egypt, although expected to be more prevalent than developed countries due to higher rates of consanguineous marriages that propagate the autosomal recessive inheritance of the disease. We conducted this study to identify the underlying HGD pathogenic variants within Egypt causing AKU and to raise awareness among Egyptian health care professionals about the disease. Furthermore, we wanted to establish the routine genetic confirmation of AKU in Egypt before the authorization of the specific therapeutic agent, nitisinone in Egypt.AKU is a rare metabolic disorder that is almost forgotten in Egypt and in the Arab region, as only a few cases were reported from Jordan15.In our study, we identified four different variants causing AKU, including three recurrent variants reported in several populations as disease causing: c.1078G > C; p.(Gly360Arg) detected in family I, has been reported in patients from Italy, Australia, UK, France, Spain, India and the USA. The variant c.808G > A; p.(Gly270Arg) detected in family II, has been previously reported in patients from Italy, Slovakia, France, Armenia, Brazil, USA, Dominican republic, Turkey, the UK, Russia, Germany, Peru and India, while c.473C > T; p.(Pro158Leu) detected in family IV in our study, has been previously reported in patients from USA, Canada, Macedonia, and TaiwanHGD gene, which contains over ten missense pathogenic variants including the detected variant at the same amino acid location in family I, c.1078G > C;p.(Gly360Arg), indicating that this domain of the HGD protein is very sensitive to amino-acid alterations, and non-synonymous missense variants most likely will result in a deleterious effect on the protein function. All four variants were detected in homozygous state in affected patients and segregated properly in their families.The last detected variant in our study is a novel missense pathogenic variant c.1079G > T; p. that was encountered in family III. This variant is not reported in ClinVar; however, it lies within a mutational hotspot in exon 13 of the 24. They also didn't have any noticeable teeth discoloration characteristic of alkaptonuria25, and repeated clinical examination didn't pick up any cardiac or renal complications, which is mainly related to the relatively young age of most of our patients.Although many of our patients had arthralgic pain in weight bearing joints, especially adolescents, they did not have evident radiographic abnormalities commonly detected in adult AKU patients, such as spine osteoporosis, calcification of the nucleus pulposus, osteophytosis or reactive sclerosis of the articular surfacesThe recent availability of the new medication nitisinone (2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedioneor, NTBC), which inhibits the enzyme 4-OH phenylpyruvate dioxygenase, thus blocking the formation of HGA, enhances the need for genetic confirmation and carrier frequency detection in families before starting this rather expensive therapy.26. This drug has been used for more than 20 years for the treatment of children with tyrosinemia type I27. An earlier start of nitisinone together with a diet restricting phenylalanine and tyrosine intake may lead to an improved long-term functional outcome. At the same time, nitisinone treatment should be cautiously monitored to avoid the development of mental and cognitive disturbances30. The low dose used should also reduce the possibility of increased plasma tyrosine levels, which may cause nitisinone keratopathy. In a recent study conducted by Ranganath et al., to compare the effects of nitisinone 2 mg and 10 mg in the treatment of alkaptonuria, they reported that nitisinone 10 mg was more efficient in slowing the disease progression; however, it simultaneously increased tyrosine levels and the incidence of corneal keratopathy (14.5% vs 4.9%)31. Furthermore, ntisinone is still a very expensive drug, so one of the major advantages of the low-dose therapy will be its economic benefit, especially in a resource-limited country, such as Egypt.Low dose nitisinone in the range of 0.2–2 mg daily reduces urinary HGA excretion by > 90%, which may prevent AKU-related complications in the long runThe early development of remarkable and progressive joint complaints in the majority of pediatric and adolescent Egyptian AKU patients in our study indicates that diet restriction of phenylalanine and tyrosine and supportive therapeutic strategies are not efficient in controlling HGA deposition in tissues and that nitisinone, which is not yet covered by insurance in Egypt, is needed.Identifying the underlying genetic variants of families with AKU in Egypt should have an impact on health community policy towards the disease. Since heterozygous variants have a major role as one of the liability genetic determinants in subsequent generations, raising awareness regarding premarital genetic counseling at least in affected families may break the line of inheritance in such disorders. Furthermore, biochemical analysis of HGA in urine and genetic confirmation should be routine screening steps for all family members of affected individuals to detect presymptomatic cases, especially that a specific therapy that can minimize long term complications is currently available.Concerning family I, we further recommend whole exome or genome sequencing, as their complex genetic trait requires a more extensive genetic sequencing technique to fully elucidate the etiology behind their apparently syndromic intellectual disability and sensorineural hearing loss.HGD gene with a novel pathogenic variant detected in two AKU siblings.In conclusion, in this study we explored the phenotypic and genotypic spectrum for alkaptonuria for the first time in Egypt. Although we revealed the genetic makeup of only a limited number of Egyptian patients, this study may form a base to be built upon for establishing clinical genetic screening and family counseling of AKU in Egypt and surrounding populations. We further enriched the mutational spectrum of the Supplementary Information.


Bioactive fatty acids possess several benefits for human health; however, these molecules show a reduced oxidative stability and consequently reduced bioavailability. This work aimed to develop novel bigels as a strategy to protect bioactive fatty acids present in three different vegetable oils with nutritional attributes  during passage through the gastrointestinal tract (GIT). Bigels were prepared using monoglycerides-vegetable oil oleogel and carboxymethyl cellulose hydrogel. These bigels were analyzed in terms of structure and rheological characteristics. According to the rheological properties, bigels exhibited a solid-like behavior since G’ was higher than G”. The results showed that the proportion of oleogel was essential to the viscosity of the final formulation as an increase in this fraction was responsible for an increase in viscosity. The fatty acids profile was evaluated before and after simulated GIT. The bigels protected the fatty acids against degradation; in the case of coconut oil, the reduction of key fatty acids was 3 times lower; for avocado oil, 2 times lower; and for pomegranate oil, 1.7 times lower. These results suggest that bigels can be used as part of an important strategy for bioactive fatty acid delivery for food applications. Bioactive lipids  provide many health benefits when consumed in appropriate amounts. They may act as antioxidants, as immunomodulators, and can improve bone health, eye and brain function, and reduce coronary diseases . Unfortu10, β-carotene, and lycopene and thus show potential for the delivery of bioactive fatty acids [Considering these facts, the food industry is seeking new methods to fortify foods with more stable and bioavailable forms of these bioactive lipids, particularly fatty acids. In this context, bigels appear to present interesting strategy, because they combine the characteristics of organogels and hydrogels, which make them capable of delivering both hydrophilic and lipophilic bioactive compounds . Technicty acids ,9,10,11.The incorporation of bioactive fatty acid sources in functional foods faces several challenges due to the high susceptibility of fatty acids to oxidation, which causes rancidity (off-flavors), consumer rejection, and a decrease in nutritional value . In thisp values are related to the importance of gelling agents in oil binding capacity. For coconut oil, Tween 80 had a higher p-value (0.001) than geleol (0.000), which means that a higher percentage of oil binding capacity can be obtained using a higher quantity of Tween 80. In the case of avocado oil, both gelling agents contributed equally to the oil binding capacity as both presented the same p-value. Regarding the pomegranate oil, high percentages of oil binding capacity can be achieved when increasing the Tween 80 quantity  of CMC [−1 observed in the coconut and pomegranate bigel spectra can be associated with the C-O stretching vibration in the ester group [The FTIR spectra of bigels were analyzed to assess the interactions between the oleogel and the hydrogel within the bigel system. As shown in  oleogel ,17. The er study . The higycerides ,19. The ycerides  and sunfycerides  bigel syer group ,19. AccoThe results  showed t1 increases in the instantaneous viscosity (ƞ’), after which point a decrease was observed. With the increase in the shear rate, the ƞ’ decreased. In contrast, the increase in shear rate increased the ƞ’ of avocado and pomegranate oil bigels. This difference in behavior of coconut oil bigel can be attributed to the high oleogel fraction. A previous work using polymer fish oil bigels demonstrated that an increase in the oleogel fraction resulted in a decrease in the apparent viscosity [For the viscometry measurements results , coconutiscosity .In vitro digestion was used to study the potential of produced bigels to reduce the degradation of bioactive fatty acids during passage through the gastrointestinal tract (GIT). The fatty acids profile of the bigels and respective oils before and after GIT is presented in p < 0.05) were obtained for all tested samples. In the case of coconut oil, the fatty acid content was reduced by 87.9% after GIT (this percentage was much lower when coconut oil was delivered as a bigel (27.7%)). Regarding avocado oil, there was a degradation of 86.7% of fatty acid content during GIT. As verified for coconut oil, the use of bigel led to a lower degradation percentage of 44.2%. The polyunsaturated nature of pomegranate oil makes it more susceptible to oxidation, as can be seen from the 96.3% reduction in total fatty acid content. The formulated pomegranate oil bigel reduced this percentage to 56.6%. According to the fatty acids profile results, the release percentages of the main bioactive fatty acids were calculated. As shown in Significant differences (c9) is the main fatty acid present in this oil, and it is responsible for its main putative potential health benefits [c9c12) and linolenic acids (C18:3) whose release percentages in bigels were 2 and 3 times higher, respectively, when compared to avocado oil.Regarding the formulation with avocado oil, a positive effect of the bigel was also observed A. Oleic benefits . In the c9t11c13 (the main bioactive CLNA present in this oil) reached the intestine. In contrast, only 2% of this pure oil fatty acid reached the intestine after digestion. In addition, higher release percentages were also observed for other important CLNAs .Pomegranate oil is an important source of polyunsaturated fatty acids, particularly conjugated linolenic fatty acids (CLNAs), which have several beneficial effects on human health; however, this compound has reduced stability ,26. The The evolution of the release of the main bioactive fatty acid from each oil during digestion  verifiedAccording to these results, bigels can be a valuable strategy to protect different bioactive fatty acids during passage through the GIT. Although there are no similar studies using bigels as protective delivery systems for such oils, several studies point to the positive effect of bigels as delivery systems for lipophilic compounds such as lycopene, β-carotene, vitamin E, and Coenzyme Q10, given their composition and unique structure ,10,11,13Bigels can be a valuable delivery system for different classes of bioactive fatty acids with high nutritional value. In the current study, a bigel delivery system was used for the first time for delivery of bioactive fatty acids from coconut, avocado, and pomegranate oils. The oils structured in bigels showed high GIT release percentages when compared to pure oils, which can enhance their biological potential and allow for targeted delivery in the intestine. Moreover, the high release percentages were also traduced in the enhanced amount of essential fatty acids after GIT. At this point, it is important to understand the bioactivity of the loaded bigels in the intestine in order to provide a blueprint for the future development of bioactive lipids delivery and protection structures. The rheological characteristics of produced bigels can be easily modulated by alterations in the organogel/hydrogel ratio, presenting different opportunities to protect and deliver lipids in food industry applications.Geleol was kindly donated by Gatefossé . Avocado oil was provided by Fula , pomegranate oil was obtained from All Organic Treasure , and coconut oil, from Origens Bio . Carboxymethylcellulose was obtained from Merck . Tritridecanoin and punicic, catalpic, α and β- eleostearic acids standards were obtained from Larodan ; HPLC grade methanol, hexane, and dimethylformamide (DMF) were obtained from VWR ; and sodium methoxide was procured from Acros Organics . The standards Supelco 37 and CRM-164, Tween 80, and sulfuric acid were obtained from Sigma .In this work, a response surface methodology was used to evaluate the effect of gelling agents on the oil binding capacity of bigels containing bioactive oils. The selected factors were the amount of geleol and Tween 80. The experimental design was based on a central composite design with two factors with two levels (coded as −1 and 1) composed of 8 cube points and 6 center points in the cube. The design was performed using Minitab software. w/v) was added under continuous mixing (2 min). After this step, 5 g of vegetable oil were added, and the mixture was homogenized  in an ultra-turrax  and then sonicated (Sonics Vibra-Cell™ VCX 130) at 60% amplitude for 1 min. Following this, the bigels were cooled and stored at room temperature until use.Geleol was melted at 60 °C using a hotplate before being mixed with pre-heated (to 60 °C) Tween 80 . Carboxymethylcellulose (2% m1). The tubes were then centrifuged at 4 °C and 15,000 rpm for 20 min, and the excess oil was decanted. The total mass of the tube with the remaining bigel (m2) was weighed. The oil binding capacity was calculated according to the following equation:Oil binding capacity was determined as follows. A total of 1 g of bigel was placed in pre-weighed centrifuge microtubes and weighed  was performed using a Spectrum 100 FTIR Spectrometer  equipped with the attenuated total reflectance (ATR) mode. The spectra were recorded from 4000 to 500 cm−1) using a 150 µm gap were conducted to study the shear behavior of the bigel samples. All samples were analyzed in triplicate.Analyses were performed to determine the rheological behavior of the samples. Measurements were conducted on a Gemini Advanced Rheometer  coupled with a Peltier unit for temperature control, using a stainless-steel cone-and-plate geometry probe CP . Samples were placed on a sampling plate, and analysis was performed, using a 1000 µm gap for coconut oil bigel and 150 µm for avocado and pomegranate oil bigels. Elastic and viscous moduli  and complex viscosity (η*) were determined as a function of frequency, which varied between 0.1 and 100 Hz, using a strain of 0.1%, which was previously determined to be within the linear viscoelastic region (LVER). Viscometry measurements  and correspondent oils’ (15 mg) fatty acid profile was evaluated via gas chromatography (GC) after transesterification according to the method described by Machado et al. 2022 . GC analAll results are presented as mean ± standard deviation. The data normality was assessed using the Shapiro–Wilk test. Statistical significances between samples were tested at a 0.05 significance level using a one-way analysis of variance (ANOVA) followed by a multiple comparisons test (Tukey) using Minitab software .


Rhodocyclaceae, Nitrosomonadaceae, and Nitrospiraceae families declined throughout exposure, likely explaining reductions in wastewater treatment performance. Pharmaceuticals were temporarily removed in the first 12 days of the test via both sorption and degradation; both removal processes declined sharply thereafter. This study demonstrates that aerobic granular sludge may successfully remove pharmaceuticals in the short term, but long-term tests are necessary to confirm if pharmaceutical removal is sustainable.This study characterizes the effects of three commonly detected pharmaceuticals—diclofenac, erythromycin, and gemfibrozil—on aerobic granular sludge. Approximately 150 μg/L of each pharmaceutical was fed in the influent to a sequencing batch reactor for 80 days, and the performance of the test reactor was compared with that of a control reactor. Wastewater treatment efficacy in the test reactor dropped by approximately 30–40%, and ammonia oxidation was particularly inhibited. The relative abundance of active  A naturAerobic granular sludge (AGS) is an emerging wastewater treatment biotechnology that may be capable of enhancing pharmaceutical removal from wastewater . AGS is The pharmaceuticals diclofenac (DCF), erythromycin (ERY), and gemfibrozil (GEM) were selected for use in this study because, though each is frequently detected in the environment , few stuThere were three objectives of this study: (1) Identify how the three common, but relatively unstudied, pharmaceuticals listed above impact conventional wastewater treatment by lab-grown AGS; (2) investigate the fate of each pharmaceutical by tracking aqueous and solid phase parent compounds and degradation products; and (3) track microbial community and activity changes throughout exposure. To our knowledge, no studies have confirmed pharmaceutical biodegradation by AGS using detection of degradation products. Notably, the byproducts formed during degradation of pharmaceuticals may be more toxic than the parent pharmaceuticals, and therefore it is vital to improve understanding of the intermediates and products formed. Abiotic removal of the dosed pharmaceuticals was monitored in this study but is not discussed in order to allow sufficient discussion of biotic processes here. In particular, this study sought to link shifts in the active microbial community in pharmaceutical-exposed granules with changes in wastewater treatment efficacy and pharmaceutical fate.22.1® treatment plant in Utrecht, The Netherlands and operated at steady state  for over 300 days prior to beginning experimentation. Immediately before starting experimentation, both SBRs were emptied and AGS was combined, mixed, and redistributed so that granule qualities would be as similar as possible in both reactors.AGS sequencing batch reactor (SBR) operation is detailed in For 80 days, the test reactor received 46 mL of pharmaceutical media with the influent medium, resulting in an influent concentration of approximately 150 μg/L of each pharmaceutical. Individual stock solutions of each pharmaceutical were prepared first in methanol at 10 g/L, then diluted into nanopure water to obtain two pharmaceutical media: one solution (“DG”) containing 17.86 mg/L each of diclofenac sodium (Acros Organics) and gemfibrozil (Acros Organics), and a second solution (“ERY”) containing 35.8 mg/L erythromycin . Twenty-three mL of each solution  were delivered with the influent medium throughout the anaerobic feed period. Approximately 0.13 mg/L methanol was also present in the combined influent stream. The pharmaceutical media were protected from light to prevent photolytic degradation and prepared fresh every 8–10 days.Results discussed in 2.2HT Total Organic Carbon analyzer system.Influent and effluent samples from both reactors were regularly taken and filtered through 0.45 μm regenerated cellulose filters prior to analyses for ammonia, nitrite, nitrate, phosphate, and dissolved organic carbon. Ammonia was quantified with Hach kit TNT 832 and a Hach DR 3900 spectrophotometer, equivalent to US EPA Method 350.1. Other anions listed were quantified with a Dionex ICS-1100 anion chromatography system equipped with an IonPac AS22 RFIC column (4 × 250 mm) and IonPac guard column (4 × 50 mm). Dissolved organic carbon (DOC), defined as that which could pass through a 0.45 μm filter, was measured with a SKALAR Formacs2.3Aqueous influent and effluent samples were prepared for mass spectrometry (MS) analyses by solid phase extraction (SPE) as detailed in Influent and effluent pharmaceutical samples were periodically taken in duplicate to account for possible pharmaceutical losses during the extraction process, as detailed in Measured pharmaceutical concentrations in unspiked samples were thereby corrected for recovery of each analyte. Recovery was 97 ± 9% for DCF, 117 ± 17% for ERY, and 98 ± 11% for GEM . TriplicA calibration curve relating each pharmaceuticals’ peak area with its nominal concentration in analytical standards was used to determine parent compounds’ concentrations. Standards were prepared in both methanol and water. The instrument limits of quantification were under 10 μg/L for all compounds. The open-source software MZmine and R were used to analyze and compile mass spectra data .All samples were also screened for DCF, ERY, and GEM biodegradation products using a personal compound database and library (PCDL) developed from the literature . DegradaPharmaceuticals were also extracted from granules to quantify solid phase concentrations per methods adapted from Aqueous and solid samples were periodically analyzed from the control reactor for pharmaceutical parents and degradation products. Except when noted otherwise, pharmaceuticals were not detected in any phase in the control reactor. Likewise, degradation products were generally not found in the influent to the test reactor.Lastly, the toxicities of detected degradation products were estimated using the US Environmental Protection Agency’s Toxicity Estimation Software Tool (TEST). TEST estimates chemical toxicity using quantitative structure activity relationships .2.42.4.1Granules from both reactors were periodically sampled for molecular characterization of the prokaryotic microbial community using high throughput sequencing of 16S rRNA genes and transcripts. Granules were collected during the aeration phase to ensure samples were fully mixed and representative of communities in the entire reactor. Samples were stored at −80°C prior to extraction. Nucleic acids were extracted from approximately 10 granules at each time point. Extraction and analyses of replicate nucleic acid samples were beyond the scope of this study. All samples were extracted at once by bead beating in DNA/RNA Shield (Zymo Research), then DNA was purified from the lysate with the DNA Clean and Concentrator Kit (Zymo Research). RNA samples were extracted from the same lysate with the Direct-zol RNA Miniprep Kit (Zymo Research), digested with the TURBO DNA-free Kit (Invitrogen), and purified with the RNA Clean and Concentrator Kit (Zymo Research). RNA was then reverse transcribed to cDNA using the ProtoScript II First Strand cDNA Synthesis Kit (New England Biolabs). DNA and RNA concentrations were quantified with Qubit dsDNA HS and RNA HS kits (Invitrogen), respectively. For all kits listed, the manufacturer-provided protocols were followed.2.4.2Metagenomic sequencing of granules was conducted on granular inoculum approximately 300 days before beginning the experimentation described herein; therefore, metagenome data provides an approximation of microbe functionality within granules (given the time between metagenome sampling and the onset of the experiment). DNA was extracted using the Zymobiomics DNA Miniprep kit and quantitated with the Qubit dsDNA kit (Invitrogen). Libraries were prepared for shotgun metagenomic sequencing on the Illumina NovaSeq platform using paired end 150bp sequencing at Novogene with a target of 15Gb of data. To quality filter fastq files, prinseq was used to remove sequences with more than 10 Ns, mean quality scores below 20, sequences shorter than 50 nt, and to trim ambiguous bases from the ends of reads. Paired end sequences were then assembled using metaSPades  with the program KOFAM scan . Pathway2.4.3PRJNA985155).To characterize the prokaryotic community, Phusion Hot Start II DNA polymerase (Thermo Scientific) was used to target the V4 region of the 16S rRNA gene with the primers 515F-A (GTGYCA GCMGCCGCGGTAA) and 806R-b (GGACTACVSGGGTATC TAAT). Reactions were 20 μL each with final concentrations of 0.4 mM dNTPs, 0.2 mM primers, and 1 U polymerase. Thermocycling conditions consisted of an initial denaturation at 98°C for 10 seconds followed by 30 cycles of denaturing at 98°C for 20 seconds, annealing at 60°C for 10 seconds, and elongating at 72° C for 30 seconds. A final extension was then performed at 72°C for 5 minutes. PCR products were purified with Mag-Bind TotalPure NGS beads (Omega), and samples were then barcoded with the Nextera XT Index Kit v2 Set D (Illumina). Barcoded samples were again purified with Mag-Bind beads, quantified, and pooled at equimolar concentration into a sample library containing approximately 30 ng DNA from each sample. Sequencing was performed onsite at Montana State University with the Illumina MiSeq platform using the v3 600 cycle kit. Raw sequence files were deposited to GenBank  and construct an OTU table. To classify ZOTUs, we used SINTAX against a reference database of 16S sequences from the Genome Taxonomy Database  was to examine both the total microbial community, defined as the community recovered in DNA reads, and the active microbial community, defined as ZOTUs with a ratio of 16S rRNA transcripts to rRNA genes greater than or equal to 1 . RarefieTo link shifts in the active microbiome with changes in nitrogen and phosphorus levels, as well as pharmaceutical degradation, we used vector fitting of effluent concentrations of DCF, ERY, and GEM using the function envfit in vegan . More sp33.1Immediately after pharmaceutical dosing to the test reactor began, nitrogen removal dropped to approximately 70% . There wPhosphate removal also declined sharply in the test reactor and remained noisy for the entire experimental duration . Despite3.2All pharmaceuticals were partially removed in the first 10–12 days of dosing, evidenced by lower effluent than influent concentrations . Removal3.2.114H11Cl2NO3, “DCF1”) and 1-(4-hydroxyphenyl)-2-,3-dihydro-1H-indol-2-one  were also present in the aqueous and solid phases in the first 12 days  via reductive dechlorination and amidation, and DCF3 is then formed via further reductive dechlorination  and 7,12-dyhydroxy-6-deoxyerythronolide B , respectively . Desosamine and ERY3 were also not detected, again likely because both are readily metabolizable, or because the mass spectrometry method used was not suitable for these compounds. Although ERY removal peaked at 18% in the first 12 days of dosing, the low levels of both ERY1 and ERY2 detected in that period suggest that intermediates completed the degradation pathway and end products were fully metabolized.ERY degradation pathways potentially used by wastewater bacteria and proposed in The TEST-predicted toxicities of ERY1 and ERY2 are summarized in 3.2.315H22O4, “GEM1”) and 2-[(4-carboxy-4-methylpentyl)oxy] benzoic acid , respectively -2,2-dimethylpentanoic acid (GEM2), which is then further oxidized to GEM3. According to the degradation pathway proposed in 3.3Competibacter and Contendibacter. Phosphorus accumulating organisms (PAOs) were also present, such as species within the Accumulibacter. A MAG identified as a species in the Accumulibacter was the most abundant organism detected, at 19% of the population based on the number of raw reads that mapped to the MAG.Metagenomic sequencing generated approximately 25 million high quality reads, resulting in a total of 19 bins with greater than 90% completeness and less than 5% contamination, and an additional 38 with completeness greater than 50% with less than 10% contamination. All MAGs were classified as members of the bacteria, including known members of glycogen accumulating organisms (GAOs) such as species in the Nitrosospira and Nitrosomonas, this pathway was not uncovered in the KEGG analysis of constructed MAGs, though the complete ammonia oxidation pathway was found in the total shotgun community (amo) genes  were relatively short (< 6000 bp). One scaffold contained all three amo genes, and another scaffold contained just amoA and amoC. Based on BLAST , including ommunity . The scaon BLAST , both wet plants . The absRhodocyclaceae, a Competibacter, and a MAG classified as a member of the Thermoanaerobaculia. Only a single MAG (Granule56) had the pathway for dissimilatory nitrate reduction to ammonia (DNRA), identified as a species in the Thermoanaerobaculia in the phylum Acidobacteriota, which also contained the dissimilatory nitrate reduction pathway. An organism related to the Thermoanaerobaculia was also detected in the active community, but at very low levels (< 0.1% relative abundance).Nitrate reductase genes were widespread in the granule community, as seen in previous metagenomic studies of wastewater . Twelve 3.4Shifts in the active microbial community were evaluated using 16S sequencing of rRNA genes and transcripts throughout pharmaceutical exposure. As described in Competibacteraceae, Rhodocyclaceae, and Chitinophagaceae were most abundant (Rhodocyclaceae family includes PAOs (Candidatus Accumulibacter) and a potential denitrifying genus (Zoogloea). The role of bacteria in the Chitinophagaceae family is unclear—multiple OTUs matching Niabella, Terrimonas, and Flavipsychrobacter genera were detected, but to date, the function of these genera in AGS is unknown.At day zero, the distributions of active families in both the control and test reactors were similar: abundant . NotablyRhodocyclaceae spiked in abundance at day five, from 14 to 46% .Despite large shifts in the test reactor community, significant correlations between microbial community composition and effluent pharmaceutical concentrations were generally not found. A significant, though small, correlation between effluent GEM concentrations and the microbial community was observed (RRhodocyclaceae (Azonexus (family Rhodocyclaceae), which made up 40% of the active community at day 5 but was absent by day 17. Large changes in relative abundance were also observed for two members of Accumulibacter, which were present at over 1% of the active community on day 5 but were not detected by day 17.Despite the lack of overall correlations, when times with the largest differences in pharmaceutical removal  increased from under 0.1% to 6% over the same time period. Positive shifts were also observed for members of the Chloroflexota, Gammaproteobacteria, and Acidobacteriota , was not detected in the assembled MAGs; however, this is likely due to the length of time elapsed between metagenome sampling and the onset of experimentation as well as the presence of pharmaceuticals. Four ZOTUs were classified as Competibacter A denitrificans with varying levels of confidence. The remaining ZOTUs with a close relative in the metagenome included a Xanthomonadaceae (genus SCMT01), a species of Accumulibacter, and an Azonexus species.We had limited success linking the ZOTUs to the organisms identified in the metagenome assemblies. Of the top ten most abundant ZOTUs in the active community, three had no close relatives in the metagenome. The most abundant active ZOTU in the test reactor ; however, some Methylophilaceae species can also consume acetate  were not detected for most timepoints in both the control and test SBR. The lack of active AOB data, however, is contradicted by complete ammonia oxidation in the control reactor and partial ammonia oxidation in the test reactor, as well as active Nitrospiraceae abundance patterns over the dosing period. Several active AOA genera were also detected, which may further explain why ammonia oxidation continued throughout dosing. AOA abundance is discussed in more detail in The relative abundance of active nitrifying families exposure , which mexposure . Nitrifiexposure . The pri4.2Nitrosomonas, were detected in both the shotgun metagenomic and metabarcoding datasets, we were unable to construct a MAG with over 90% completeness, which limited our ability to determine the role of these organisms. However, despite their limited abundance overall, it is likely that Nitrosomonas is the main driver of ammonia oxidation in the reactors, given their presence in seed granules and the homology of the identified functional genes.The addition of pharmaceutical compounds led to strong shifts in the microbial community and concomitant reductions in nitrogen and phosphorus removal. Based on the assessment of potential functional pathways, no high-quality MAGs were found with the ammonia oxidation pathway. Although AOB, such as Thermoproteota, including members of the order Nitrosophaerales. Nitrosophaerales contains numerous AOA, such as Nitrosotenuis and Nitrosocaldus genera. AOA have been posited to be important for the oxidation of ammonia to nitrite in commercial wastewater treatment plants (Several archaeal taxa were also found in the metabarcoding dataset though archaea were not detected in the granule metagenome. Archaea detected were primarily species within the t plants , though Rhodocyclaceae and Methylophilaceae (Although archaea made up less than 1% of the sequences identified, 47 ZOTUs were identified as active taxa. We focused primarily on abundant taxa, but the ability of rare organisms to drive ecosystem function is well documented ; for exahilaceae , suggest5Lab-grown AGS was exposed to three commonly found, but relatively unstudied pharmaceuticals at approximately 150 μg/L each. The fate of each pharmaceutical and its degradation products in the aqueous and solid phases were monitored, and pharmaceutical impacts on wastewater treatment performance and microbial communities were evaluated.Rhodocyclaceae declined in abundance in the test reactor, which likely contributed to poor phosphate removal.All pharmaceuticals were partially removed via both biodegradation and sorption in the first 12 days of the study. Biodegradation capacity then declined irreversibly, indicated by washout of degradation products, declining production, and negligible pharmaceutical removal. Exposure to the pharmaceutical mixture negatively impacted wastewater treatment efficacy and the relative abundance of active wastewater treating families. Nitrogen and phosphate removal declined to approximately 73% and 63%, respectively, though carbon removal was not impacted. Declining nitrogen removal was due mainly to inhibited ammonia oxidation and likely also related to the declining abundance of active nitrifiers. Similarly, active Supplementary Material


Bladder cancer is the sixth most incident neoplasm, and the average five-year survival rate drops to 5% for patients with the metastatic disease. Raman spectroscopy probes molecular vibrations by inelastic scattering of laser light. In a retrospective study, control and cancer specimens were prepared from ten human bladder resectates and label-free assessed by Raman microspectroscopic imaging. A data analysis workflow without preprocessing identified the locations and signatures of normal bladder, bladder cancer, necrosis, epithelium, and lipid inclusions. Furthermore, microplastic particles, pigments or carotenoids were detected in 13 out of 20 specimens inside tissue and near tissue margins and their identity was confirmed by spectral library surveys. Hypotheses about the origin of these foreign materials are discussed. Our approach with minimal user interference offers prospects for future clinical translation such as intraoperative tumor detection and label-free material identification in complex matrices.Raman spectroscopy offers label-free assessment of bladder tissue for in vivo and ex vivo intraoperative applications. In a retrospective study, control and cancer specimens were prepared from ten human bladder resectates. Raman microspectroscopic images were collected from whole tissue samples in a closed chamber at 785 nm laser excitation using a 20× objective lens and 250 µm step size. Without further preprocessing, Raman images were decomposed by the hyperspectral unmixing algorithm vertex component analysis into endmember spectra and their abundancies. Hierarchical cluster analysis distinguished endmember Raman spectra that were assigned to normal bladder, bladder cancer, necrosis, epithelium and lipid inclusions. Interestingly, Raman spectra of microplastic particles, pigments or carotenoids were detected in 13 out of 20 specimens inside tissue and near tissue margins and their identity was confirmed by spectral library surveys. Hypotheses about the origin of these foreign materials are discussed. In conclusion, our Raman workflow and data processing protocol with minimal user interference offers advantages for future clinical translation such as intraoperative tumor detection and label-free material identification in complex matrices. Bladder cancer is the sixth most incident neoplasm in the United States, and the disease is four times more common in men than women. Bladder cancer ranges from unaggressive and usually noninvasive tumors that recur and commit patients to long-term invasive surveillance to aggressive and invasive tumors. The average five-year survival in the United States is 77%, and drops to 5% for those with the metastatic disease . BladderEnhanced cystoscopy includes technology used to improve the detection of cancers and can reduce the risk of recurrence. Fluorescence has already been used to perform intraoperative analysis of the bladder wall, which was demonstrated in ten patients with superficial bladder cancer. In photodynamic diagnosis (PDD), fluorescence contrast is measured with a video system allowing quantitative assessment of the 5-aminolevulinic acid (5-ALA) accumulation in tissues. The general condition of the bladder can be assessed, the main foci of the tumor can be determined, and a tumor resection can be performed. The procedure is called transurethral resection (TUR). Further examination and identification of the fluorescing boundaries of the tumor remaining after TUR are performed to make a decision of a re-resection. In addition, small fluorescing tumor foci, not detected with white light cystoscopy, can be detected [Raman spectroscopic-based techniques have also been suggested for this application. In contrast to fluorescence, Raman spectroscopy provides a label-free and nondestructive contrast in tissue sections, ex vivo tissue biopsies and in vivo during cystoscopy. The ability to reduce false detection rates in comparison to existing diagnostic tools such as PDD makes Raman spectroscopy particularly attractive as a complementary diagnostic tool. An early in vitro study used a fiber-optic Raman system to collect 220 spectra from 29 fresh frozen bladder samples. A classification algorithm was able to differentiate benign samples from malignant samples with an overall accuracy of 84% . The feaThe current study presents a workflow to collect Raman images from control and cancer tissues of ten human bladder resectates and to process the data by unsupervised hyperspectral algorithms vertex component analysis and hierarchical cluster analysis. Due to the high-quality spectra with more than 40 well-resolved bands, various tissue types and further non-tissue materials were identified. This approach offers prospects for clinical translation for intraoperative tissue assessment as a standalone tool or in combination with other modalities to increase the speed and throughput.Control and cancer specimens were prepared from ten human bladder resectates from the tissue bank (Comprehensive Cancer Center Freiburg) in Freiburg, shipped on dry ice to Jena and stored at −80 °C until use. Their lateral dimensions ranged from ca. 5–15 mm with a thickness of ca. 5 mm. Samples were placed on copper or silver plates in a sample chamber in the frozen state, thawed at ambient temperature, and covered with a calcium fluoride window to protect them from drying . The heiThe Raman spectrometer RXN1 microprobe with a 785 nm laser  was used for the acquisition of confocal microscopic data. A water immersion (WI) objective lens 20×/NA 0.5  focused ca. 130 mW intensity onto the sample. Raman images were registered with an exposure time of 5 s per spectrum in a sequential acquisition mode at 250 µm step size using a motorized stage. The instrument was controlled by the software HoloGRAMS, which automatically performs laser wavelength calibration, wavenumber calibration and intensity calibration .−1 by the hyperspectral unmixing algorithm called vertex component analysis (VCA). Briefly, VCA calculates endmember loadings and scores that represent the spectra and concentrations of the most dissimilar components. The number of endmembers was ranged from 4 to 7. The maximum number was achieved when no further component with significant novel features was detected. If components with cosmic spikes occurred, the affected wavenumbers were corrected and the VCA algorithm was executed once more. Details of the implementation of in-house scripts have been described elsewhere [Raman images were imported to Matlab  using the toolbox HoloMap . Without further preprocessing, Raman data were decomposed in the spectral range 600–1800 cmlsewhere . Loading−1 in −1 and elevated noise level between 1400 and 1800 cm−1 are evident, which might be a consequence of the relatively weak signal intensity of this component. The band at 1630 cm−1 and the location near the tissue margin is typical for spectral contributions of water in 2 band near 1450 cm−1, and shifts of secondary structure sensitive amide III and amide I bands. Furthermore, smaller spectral contributions were found for the phospholipid PC at 718 and 1082 cm−1, and for DNA at 782 cm−1 and the shoulder near 1090 cm−1. These tissue components are almost unaffected by broad spectral glass background, water and OCT medium, and the spectra only show small baseline slopes towards lower wavenumbers. This justifies that the Raman spectral maps could be processed without preprocessing. The high signal to noise ratio and spectral quality enable detailed analysis of biochemical variations of tumors, which will be described next.−1, normalized to equal intensities of the band at 1450 cm−1 and overlaid for comparison. The band positions and relative intensities coincide well with the tumor spectrum 2T (plotted in black), which was already shown in −1, which are assigned to different water content of samples.The tumor spectra of 2T, 3T, 4T, 5T, 6T, 7T and 9T are shown in −1 are less intense relative to the non-fibrous protein bands, e.g., at 1003 and 1450 cm−1. Therefore, this spectrum is typical for the muscle tissue of the bladder wall.The control spectra of 4N, 5N, 6N, 7N, 8N, 9N and 10N are shown in −1 are weak, and bands at 935 cm−1 and amide I band at 1655 cm−1 point to proteins with α helical secondary structures. Furthermore, a new band arises at 839 cm−1.Raman spectra of another tissue type are resolved in 1T, 6N, 7N, and 8N D that ar−1, which point to carotenoids. More intense carotenoid contributions in specimen 10T are presented in Lipid spectra of 1N, 2T, 3N, 4N, 4T, 5N, 5T, 6N, 9N, and 9T are shown in −1 was used as an input for HCA because the weak bands in the range 600–800 cm−1 do not significantly contribute to the variations, the band at 1450 cm−1 shows only small variations, and the range 1500–1800 cm−1 shows unspecific variations due to the water content. The dendrogram in The lipid- and tissue-related spectra were considered for unsupervised segmentation by hierarchical cluster analysis (HCA). The spectral range from 800 to 1380 cm−1 are clearly evident [According to  evident ,17. −1, the lower intensities suggest that the PS particle is located below the surface. In sample 1N, several pixels throughout the specimen are assigned to Raman spectra of a pigment with higher and lower intensity . Here, spectral contributions of tissue are also evident in the lower intense pigment spectrum. The search result identified this pigment as Hostaperm Blue with a hit quality index (HQI) of 58.8 (−1 and CH2 band near 1450 cm−1 overlap with carbon bands. Another polymer was found in sample 1T with a HQI match of 71.2 to poly (phenylene sulfide) (The results of samples 3T and 1N are shown in  of 58.8 . This bl of 58.8 . The pig of 58.8 . The Ram of 58.8 . Residuasulfide) . The bansulfide) , numerousulfide) , which iThe study demonstrates that Raman spectroscopy provides a wealth of information for tissue assessment and Raman images resolve further spectral and lateral details. Consequently, Raman-based methods were reviewed among other optical and spectral technologies for the clinical need of an intraoperative histopathology . Here, RHigher resolution Raman images at 2 µm step size from smaller regions of interest were previously reported for primary brain tumor sections immersed in aqueous buffer  that dem−1, which might point to pre-malignant tissue or another non-normal tissue type.The observation that lipid inclusions were found both in normal and tumor specimens suggests that their presence or absence cannot be considered as tumor markers. Besides the depletion of collagenous proteins, spectral contributions of DNA and the phospholipid phosphatidylcholine were identified as tumor markers. Whereas DNA is not found in non-cancer bladders, increased DNA content is associated with a high density and proliferation rate of cancer cells, which are among the hallmarks of cancer . A revie−1 and differentiated between tumor and non-tumor breast tissue [The assessment of water content in fresh specimens by Raman spectroscopy was proposed as an intraoperative tool. In particular, changes in water content were quantified by measuring the water of the total area of the high wavenumber range between 2800 and 3600 cmt tissue  and oralt tissue . VariatiAn unexpected result was the detection of carotenes, microplastic particles and pigments in bladder samples. The VCA algorithm has sufficient sensitivity and specificity to identify even single pixels with Raman spectra of foreign material. The high spectral quality and signal to noise ratio allowed the direct import of the unknown spectra to a library and gave, in most cases, reasonable assignments. Carotenes has previously been detected by Raman spectroscopy in thin brain tumor sections immersed in buffer using similar unsupervised hyperspectral unmixing approaches . The abiFirst, plastic pollution is taken up via food, drinks or inhalation of air, and accumulates in the bladder while only a fraction is excreted with the urine. The frequent detection of such foreign particles in this work (13 out of 19 specimens from resected bladders!) might point to a factor in the development of malignancies and might have been overlooked so far by state-of-the art modalities such as histopathology, immunohistochemistry and fluorescence that usually administer stains to enhance contrast. Advantages of Raman spectroscopy include that (i) no stains are required, (ii) unprocessed tissue can be analyzed, (iii) algorithms can be applied without complex data pre-processing such as VCA in this and earlier work ,21, and Second, pigments such as phthalocyanine derivatives are used as photosensitizers in PDT, which is also a treatment option in bladder cancer. Photosensitizers are designed so that they accumulate in cancer after administration and degrade upon local illumination using a cystoscope to a new chemical that kills cancer cells. Non-degraded photosensitizers might explain the more frequent occurrence of pigments in non-illuminated control specimens 1N, 4N, 5N, 7N, 8N and 9N than in cancer specimens 6T and 7T.Third, blue, green, red and black pigments are popular tattoo inks, which are injected into the top layer of the skin dermis (ca. 1–2 mm) by needles, and might enter the blood stream and accumulate in organs as already observed in the liver . The potFinally, contaminations during the course of tissue collection at the operating theater and transfer to the Raman spectroscopy lab might occur, e.g., placing samples in Petri dishes made of polystyrene and cellulose fibers originating from papers. Therefore, preparation and isolation protocols that have been developed for microplastics under carefully controlled  conditions from food matrices  need to −1, five tissue components , three non-tissue components , and more than eight additional components . Further studies about the origin, the identity and the quantity of foreign materials in bladder tissues are needed to check the hypotheses with more patients or animal models, anonymous metadata  and careful preparation protocols to minimize possible contaminations. Due to its label-free nature and high specificity, Raman spectroscopic imaging is well suited for such studies, ideally in multiple centers under standardized conditions, and gives unique insights that can complement the established clinical modalities.Our Raman workflow and data processing protocol with minimal user interference and sample preparation offers advantages for future clinical translation such as intraoperative tumor detection and label-free material identification in complex matrices. Here, experimental data from a relatively small cohort of 10 control and 10 tumor samples of resected bladders already revealed a plethora of information encompassing more than 40 Raman bands in the fingerprint range 600–1800 cm