In cervical fluid, the concentrations of FcgammaBP were elevated in the presence of intra-amniotic infection in pregnancies with PPROM only . FcgammaBP in amniotic fluid might be a marker of intra-amniotic infection in women with both PPROM and PTL However, in cervical fluid, it is only observed in women with PPROM.To determine the IgGFc-binding protein (FcgammaBP) concentration in amniotic and cervical fluids in preterm prelabor rupture of membranes (PPROM) and preterm labor with intact membranes (PTL) and to assess the diagnostic indices of FcgammaBP to predict intra-amniotic infection . In this study, we included 170 and 79 women with PPROM and PTL, respectively. Paired cervical and amniotic fluid samples were obtained using a Dacron polyester swab and transabdominal amniocentesis, respectively. The FcgammaBP concentrations in the samples were assessed using an enzyme-linked immunosorbent assay. The presence of intra-amniotic infection was associated with elevated FcgammaBP concentrations in pregnancies with PPROM and PTL [PPROM—presence: 86 ng/mL vs. absence: 13 ng/mL,  The latter form of preterm delivery is more frequent and is responsible for more than two-thirds of all preterm deliveries2. Spontaneous preterm delivery can also be divided into two clinical phenotypes: (i) preterm prelabor rupture of the membranes (PPROM) and (ii) preterm labor with intact membranes (PTL)2.Preterm delivery, defined as delivery before 37 weeks of gestation, is divided into two major subgroups: (i) iatrogenic preterm delivery and (ii) spontaneous preterm delivery11. This condition is termed intra-amniotic inflammation and can be identified in both PPROM13 and PTL14. Based on the presence or absence of microorganisms and/or their nucleic acids in amniotic fluid, intra-amniotic inflammation can be further divided into two clinical subtypes: (i) intra-amniotic infection and (ii) sterile intra-amniotic inflammation15. Clinical relevance of intra-amniotic inflammation, its association with adverse short- and long-term neonatal outcomes, and optimal diagnostic markers, are still a matter of intense debate28.Some pregnancies can be complicated by the elevation of amniotic fluid concentrations of various inflammatory mediators, such as cytokines, chemokines, and antimicrobial peptides38. The ability to identify hundreds of proteins and to quantify changes in their abundance across multiple amniotic fluid samples makes proteomics very appealing, particularly in the discovery phase of the biomarker search process61. Therefore, proteomic analysis of amniotic fluid may reveal new proteins involved in the complex pathogenesis of intra-amniotic inflammation40.The broad availability and recent advances in proteomics, an unbiased technology, bring it within the scope of researchers working in the field of intra-amniotic inflammatory complications42. FcgammaBP is a relatively unknown protein, with limited reports in relation to conditions such as bowel inflammatory disease, autoimmune disease, or thyroid gland tumors64. Nevertheless, FcgammaBP has been identified in amniotic fluid obtained from women with uncomplicated pregnancies42 as well as from women with pregnancies complicated by PPROM36 and PTL37. In pregnancies with PPROM, an elevation of FcgammaBP concentration in amniotic fluid has been observed during microbial invasion of the amniotic cavity and in acute histological chorioamnionitis36. However, there is a paucity of information on whether concentrations of FcgammaBP in amniotic and cervical fluid reflect the presence of intra-amniotic inflammatory complications in both the clinical phenotypes of spontaneous preterm delivery.IgGFc-binding protein (FcgammaBP) is one of the proteins identified in amniotic fluid using proteomicsTo fill this gap in the knowledge, we conducted this study with the following goals: (i) to quantify the FcgammaBP concentration in amniotic fluid samples from pregnant women with PPROM and PTL based on the phenotype of intra-amniotic inflammation; (ii) to quantify the FcgammaBP concentration in cervical fluid samples from pregnant women with PPROM and PTL based on the phenotype of intra-amniotic inflammation; and (iii) to assess the predictive value of FcgammaBP concentrations in amniotic and cervical fluids for intra-amniotic infection in pregnant women with PPROM and PTL.In total, 170 and 79 women with PPROM and PTL, respectively were included in the study. Among women with PPROM, the presence of intra-amniotic infection, sterile intra-amniotic inflammation, colonization, and negative amniotic fluid were observed in 11% (19/170), 5% (9/170), 10% (16/170), and 74% (126/170) of women, respectively. Among women with PTL, intra-amniotic infection, sterile intra-amniotic inflammation, and negative amniotic fluid were found in 15% (12/79), 27% (21/79), and 58% (46/79) of the women, respectively. None of the women with PTL had colonization.The demographics of all the women in this study and clinical data of the women with PPROM and PTL, based on the presence and absence of intra-amniotic infection, are shown in Tables p < 0.0001; Fig. PPROM pregnancies with intra-amniotic infection and sterile intra-amniotic inflammation had higher amniotic fluid FcgammaBP concentrations than did the women with colonization and with negative amniotic fluid . Women with intra-amniotic infection had higher amniotic fluid FcgammaBP concentrations than did women with sterile intra-amniotic inflammation and with negative amniotic fluid . The differences in cervical fluid FcgammaBP concentrations were revealed among the subgroups . Women with intra-amniotic infection had higher cervical fluid FcgammaBP concentrations than women with sterile intra-amniotic inflammation, colonization, and negative amniotic fluid . No difference in cervical fluid FcgammaBP concentrations was found among the subgroups .A weak positive correlation was observed between the concentrations of FcgammaBP in amniotic and cervical fluids (ρ = 0.25(1) FcgammaBP was identified as a constituent of amniotic and cervical fluids from pregnancies complicated by PPROM and PTL; (2) the concentration of FcgammaBP in amniotic fluid was elevated in the presence of both phenotypes of intra-amniotic inflammation, being more pronounced in the presence of intra-amniotic infection in women with PTL; (3) the concentration of FcgammaBP in cervical fluid was elevated in the presence of intra-amniotic infection only in women with PPROM; (4) the FcgammaBP in amniotic fluid might be a marker of intra-amniotic infection in women with PPROM and PTL; and (5) the FcgammaBP in cervical fluid might be a non-invasive marker of intra-amniotic infection in women with PPROM.65. This specific site differed from previously recognized receptors in the Fc region of IgG65. The specific site for the Fc region of IgG was later termed FcgammaBP and identified as a protein primarily localized in the mucosal granules of the small intestinal and colonic epithelia that are secreted into the intestinal lumen66. Based on the current knowledge, FcgammaBP is considered to be a protein that provides immunologic protection of the intestinal tissue and facilitates the interaction between the intestinal mucus and potentially harmful stimuli  with the ultimate goal of protecting the mucosal surface66. However, its exact biological function has yet to be fully elucidated.FcgammaBP was discovered more than 30 years ago as a specific site for the fragment of crystallizable (Fc) region of the immunoglobulin (Ig) G antibody in the small intestinal and colonic epithelia63. However, its expression has not been observed in the brain, heart, kidney, liver, lung, and skeletal muscles62. Interestingly, the ability to produce FcgammaBP was confirmed only in humans and monkeys, but not in mice, rats, rabbits, dogs, bovines, and porcines62.The production of FcgammaBP has been described to occur in the intestinal epithelial cells, placenta, and thyroid tissue64. However, its serum concentrations were elevated in the presence of autoimmune diseases such as Crohn’s disease, ulcerative colitis, rheumatoid arthritis, systemic lupus erythematosus, and progressive systemic sclerosis64. The presence of FcgammaBP has been further proven in amniotic fluid, urine, saliva, and cerebrospinal fluid42. Liu et al. found FcgammaBP to be a constituent of amniotic fluid in the second trimester of uncomplicated pregnancies42. In addition, FcgammaBP was shown to be among the most abundant (35/1624) proteins found in amniotic fluid42. Our group described the amniotic fluid presence of FcgammaBP in pregnancies complicated by PPROM and PTL37. The observation from this study, where we found FcgammaBP as a constituent of amniotic fluid from pregnancies with PPROM and PTL, is in line with the abovementioned findings.FcgammaBP has been found in low concentrations in human serum from healthy individuals36. Interestingly, no differences in the amniotic fluid concentration of FcgammaBP between the presence and absence of the abovementioned complications were identified in women with PTL, where amniotic fluid was obtained from the forewaters at the end of the first stage of labor37.Previously, concentrations of FcgammaBP in amniotic fluid have been shown to be higher in women with PPROM with microbial invasion of the amniotic cavity and acute histological chorioamnionitis than in those without these complicationsIn this study, we found elevated amniotic fluid concentrations of FcgammaBP in the presence of both phenotypes of intra-amniotic inflammation. Interestingly, in women with PTL, the concentrations of FcgammaBP in amniotic fluid were higher in the presence of intra-amniotic infection than in the presence of sterile intra-amniotic inflammation. Collectively, the results from this study clearly show that both infectious and non-infectious stimuli might trigger the production of FcgammaBP.70.In this study, the concentrations of FcgammaBP were measured in paired amniotic and cervical fluid samples obtained from women with both phenotypes of spontaneous preterm delivery. Interestingly, the FcgammaBP concentrations were higher in the cervical fluid samples than in the amniotic fluid samples, despite the fact that cervical fluid samples obtained with a swab were diluted in 1.5 mL of the buffer. These observations suggest that epithelial cells and/or immune cells in the endocervical canal are able to produce FcgammaBP. This finding supports the key role of the cervix during pregnancy, which is its immunologic protection against the ascension of microorganisms from the vagina and the cervix toward the upper genital tractIt is obvious that the protein composition of a cervical fluid sample from pregnancies with PPROM may be substantially affected by amniotic fluid that has leaked from the amniotic cavity. Therefore, the cervical fluid samples of PPROM pregnancies may reflect such a situation in both the intra-amniotic and cervical compartments. This is a possible explanation as to why women with intra-amniotic infection had higher cervical fluid FcgammaBP concentrations than those without intra-amniotic infection in PPROM but not in PTL pregnancies. However, a weak positive correlation between amniotic and cervical fluid FcgammaBP protein concentrations was also found in PTL.21. Comparing diagnostic indices among amniotic fluid FcgammaBP, MMP-8, and IL-6 to identify intra-amniotic infection in pregnancies with PTL, amniotic fluid FcgammaBP has the highest likelihood ratio 21.This study suggests that FcgammaBP might be a new biomarker for intra-amniotic infection in both phenotypes of spontaneous preterm delivery. This finding is clinically very relevant since confirmation of intra-amniotic infection represents a challenge for clinicians. The necessity to rule in or rule out the presence of microorganisms in amniotic fluid makes the diagnosis of intra-amniotic infection time-consuming and more expensive when the techniques used to identify either non-culturable or difficult-to-culture microorganisms are employed. Therefore, from a clinical point of view, there is an urgent need to discover a single marker of intra-amniotic infection that has reliable sensitivity and specificity. In this study, FcgammaBP in amniotic fluid was identified as a potential marker of intra-amniotic infection in pregnancies with PPROM and PTL. Previously, Chaemsaithong et al. has described diagnostic indices of rapid matrix metalloproteinase (MMP)-8 and interleukin (IL)-6 point-of-care test  to identify intra-amniotic infection in pregnancies with PTL71. In addition, cervical fluid sampling can be safely repeated during the latency period of pregnancy complicated by PPROM. Therefore, FcgammaBP assessment in cervical fluid might be used to monitor the development of secondary intra-amniotic infection during the latency interval in women with PPROM.In addition, in women with PPROM, FcgammaBP in cervical fluid was also revealed as a potential marker of intra-amniotic infection. Particularly, cervical fluid FcgammaBP can be a clinically relevant marker given the non-invasive nature of cervical fluid sampling. Moreover, its diagnostic indices are better than previously published diagnostic indices of IL-6 in cervical fluid  in PPROM pregnanciesThe strength of this study is the relatively large cohort of paired samples of amniotic and cervical fluid. Second, the fluid samples were collected from well-defined phenotypes of spontaneous preterm delivery (PPROM and PTL). Finally, the thorough assessment of microbial invasion of the amniotic cavity, by a combination of culture and non-culture methods, provided an opportunity to precisely distinguish the subsets of women with intra-amniotic infection and sterile intra-amniotic inflammation.62, the questions of which part of the placenta is a source of FcgammaBP and whether fetal membranes produce FcgammaBP still remain unanswered. A body of evidence has shown that intestinal epithelial cells produce FcgammaBP , but no data are available on whether amniotic epithelial cells can produce FcgammaBP. Given the importance of the amniotic epithelium as a barrier against the ascension of microorganisms into the amniotic cavity74, some similarities between intestinal and amniotic epithelial cells might be identifiable such as, (1) to serve as mechanical barriers76; (2) to have spatially expressed toll-like receptors78; and (3) to indicate that the expression of toll-like receptors changes when inflammation is present79. Therefore, we hypothesize that the amniotic epithelium might be involved in FcgammaBP production.This study also has some limitations that are worth mentioning. For example, there was a small number of women with intra-amniotic infection (n = 19 and n = 12). To confirm whether the concentration of FcgammaBP in amniotic fluid and cervical fluid is a reliable marker of intra-amniotic infection, the results need to be replicated in independent cohorts. Next, despite the FcgammaBP expression in the placenta that was describedThe concentrations of FcgammaBP observed in amniotic and cervical fluid were elevated in women with intra-amniotic infection. Thus, after replication in an independent cohort, FcgammaBP in amniotic fluid might be a potential marker of intra-amniotic infection in pregnancies with PPROM and PTL. Moreover, FcgammaBP in cervical fluid could be a marker of intra-amniotic infection in pregnancies with PPROM.This retrospective cohort study included pregnant women who were admitted to the Department of Obstetrics and Gynecology at the University Hospital Hradec Kralove in the Czech Republic between March 2017 and May 2020. The inclusion criteria were the following: (1) singleton pregnancy, (2) maternal age ≥ 18 years, (3) gestational age between 22 + 0 and 36 + 6 weeks, (4) PPROM or PTL, and (5) the performance of transabdominal amniocentesis at the time of admission to determine intra-amniotic inflammation. In contrast, the exclusion criteria were the following: (1) pregnancy-related and other medical complications such as fetal growth restriction, gestational or pre-gestational diabetes, gestational or chronic hypertension, and preeclampsia; (2) structural or chromosomal fetal abnormalities; (3) signs of fetal hypoxia; and (4) significant vaginal bleeding.The gestational age was determined via the use of first-trimester fetal biometry. PPROM was diagnosed by examining the women, using a sterile speculum, for pooling of amniotic fluid in the posterior fornix of the vagina. In the case of clinical uncertainty in diagnosing PPROM, amniotic fluid leakage was confirmed by the presence of insulin-like growth factor-binding proteins  in the vaginal fluid.80.PTL was diagnosed as the presence of regular uterine contractions (at least two contractions every 10 min), along with cervical length, measured using transvaginal ultrasound, shorter than 15 mm or within the 15–30 mm range with a positive PartoSure test Women with PPROM were treated with antibiotics. Those with intra-amniotic inflammation received first-line treatment with intravenous clarithromycin for seven days. Unless delivery occurred earlier, the antibiotic treatment was eventually modified under the condition of microbial invasion of the amniotic cavity; the women without intra-amniotic inflammation received benzylpenicillin . Women with PPROM below the gestational age of 35 + 0 weeks received corticosteroids (betamethasone) to accelerate fetal lung maturation and reduce neonatal mortality and morbidity. Women with PPROM were managed expectantly, except those with intra-amniotic infection beyond the gestational age 28 + 0 weeks wherein labor was induced or an elective cesarean section was performed within 72 h of admission.Women with PTL received a course of corticosteroids (betamethasone) and tocolytic therapy with either intravenous atosiban  or with nifedipine, which was administered orally, for 48 h. Patients with proven intra-amniotic inflammation received treatment with intravenous clarithromycin for seven days, unless delivery occurred earlier. Antibiotic treatment was eventually modified under the condition of microbial invasion of the amniotic cavity. Women with PTL that were positive for group B Streptococcus (GBS), as determined from the vaginal-rectal swab, or with an unknown GBS status received intravenous benzylpenicillin  during an active labor.All participants in this study provided informed written consent prior to the collection of amniotic and cervical fluid samples. Sample collection for this research was approved by the Institutional Review Board of the University Hospital Hradec Kralove . All experiments were performed in accordance with relevant guidelines and regulations. All participants were Caucasian.g for 15 min at room temperature. The supernatant was divided into aliquots and stored at − 80 °C until further analysis.Paired cervical fluid and amniotic fluid samples were collected at the time of admission from all women included in this study, prior to the administration of antibiotics, tocolytics, and/or corticosteroids. Each cervical fluid sample was obtained by placing a Dacron polyester swab in the cervical canal for 20 s to achieve saturation. Once collected, the polyester swab was inserted into a polypropylene tube containing 1.5 mL of phosphate-buffered saline; the tube was then shaken for 20 min. Upon removal of the polyester swab, the tube was centrifuged at 300×Ureaplasma species, Mycoplasma hominis, and Chlamydia trachomatis; (iii) sequencing of the 16S rRNA gene; and (iv) aerobic and anaerobic cultivation.Ultrasonography-guided transabdominal amniocentesis was performed after cervical fluid sampling. Approximately 2–3 mL of amniotic fluid was aspirated, and the amniotic fluid was immediately divided among polypropylene tubes. The samples of amniotic fluid were used for (i) the assessment of amniotic fluid interleukin (IL)-6; (ii) polymerase chain reaction (PCR) analysis of 22.IL-6 concentrations were assessed using the immuno-analyzer Cobas e602, a part of the Cobas 8000 platform . The measurement range was 1.5–50,000 pg/mL. The coefficient of inter- and intra-assay precision was < 10%C. trachomatis/Ureaplasma/M. hominis-FRT kit  to detect the DNA from Ureaplasma species, M. hominis, and C. trachomatis in the same PCR tube (multiplex format). We included a PCR run for beta-actin, a housekeeping gene that served as the control, to examine the presence of polymerase chain reaction inhibitors.DNA was isolated from amniotic fluid using the QIAamp DNA Mini Kit , according to the manufacturer’s instructions. Real-time PCR was conducted on a Rotor-Gene 6000 instrument (Qiagen) using the commercial AmpliSens 82. Each reaction contained 3 μL of target DNA, 500 nM forward and reverse primers, and Q5 High-Fidelity DNA polymerase  in a total volume of 25 μL. Amplification was carried out on a 2720 Thermal Cycler . The products were visualized on an agarose gel. Positive reactions yielded 950 bp products that were subsequently analyzed by sequencing. The 16S rDNA PCR products were purified and subjected to sequencing with the above-mentioned primers and the BigDye Terminator kit v.3.1 . The bacteria were then typed via searches for the obtained sequences using BLAST and SepsiTest BLAST.Bacterial DNA was identified by PCR targeting the 16S rRNA gene with the following primers: 5′-CCAGACTCCTACGGGAGGCAG-3′ (V3 region) and 5′-ACATTTCACAACAC-GAGCTGACGA-3′ (V6 region)Gardnerella vaginalis selective medium, MacConkey agar, a Neisseria-selective medium (modified Thayer–Martin medium), Sabouraud agar, or Schaedler anaerobe agar. The plates were cultured for 6 days and checked daily. The species were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using MALDI Biotyper software .The amniotic fluid samples were cultured on Columbia agar with sheep’s blood, Microbial invasion of the amniotic cavity was determined based on a positive PCR analysis of Ureaplasma species, M. hominis, C. trachomatis or a combination of these species or positivity for the 16S rRNA gene, aerobic/anaerobic cultivation of the amniotic fluid, or a combination of these parameters. Intra-amniotic inflammation was defined as amniotic fluid IL-6 concentrations ≥ 3000 pg/mL22. Intra-amniotic infection was defined by both microbial invasion of the amniotic cavity and intra-amniotic inflammation. Sterile intra-amniotic inflammation was defined as the presence of intra-amniotic inflammation without the concomitant microbial invasion of the amniotic cavity. Colonization was defined as the microbial invasion of the amniotic cavity without intra-amniotic inflammation. Negative amniotic fluid was defined as the absence of microbial invasion of the amniotic cavity and intra-amniotic inflammation.The concentrations of FcgammaBP were assessed in the amniotic fluid and cervical fluid samples using an enzyme-linked immunosorbent assay (ELISA), the Human FCGBP ELISA Kit , according to the manufacturer’s instructions. The amniotic fluid and cervical fluid samples were diluted tenfold and 50-fold, respectively. The sensitivity of the kit was 0.117 ng/mL. The absorbance values were read at 450 nm on a Multiskan RC ELISA reader (Thermo Fisher Scientific).U test for continuous variables and are presented as median values (interquartile range [IQR]). Categorical variables were compared using Fisher’s exact test and are presented as a number (%). The normality of the data was tested using the Anderson–Darling test. Because the FcgammaBP concentrations in the amniotic fluid were not normally distributed, the nonparametric Kruskal–Wallis and Mann–Whitney U tests were performed for statistical analyses, and the results are presented as a median value (IQR). Spearman’s partial correlation analysis was performed to adjust the results for gestational age at sampling. Spearman’s correlation was used to assess the relationship between the concentrations of amniotic fluid FcgammaBP and cervical fluid FcgammaBP and gestational age at sampling. Receiver operating characteristic (ROC) curves were constructed to assess the predictive value of amniotic fluid and cervical fluid FcgammaBP for the presence of intra-amniotic infection. Cutoff values were determined based on the highest positive likelihood ratio. Differences were considered significant at p < 0.05. All p values were obtained using two-tailed tests, and all statistical analyses were performed using GraphPad Prism, version 8.1.1. for Mac OS X  or the Statistical Package for Social Sciences (SPSS), version 19.0 for Mac OS X .The women’s demographic and clinical characteristics were compared using the nonparametric Mann–Whitney


A diagnostic biomarker for the detection of breast cancer remains an unmet clinical need despite decades of intensive research efforts. Herein, we describe, for the first time, the use of nuclease activity as a biomarker to discriminate between healthy and cancer biopsy samples. We have identified a panel of three nucleic acid probes able to target nucleases derived from breast cancer tumors with high sensitivity and specificity. These results are in good agreement with histopathological analysis as the diagnostic gold standard. Moreover, these findings support nuclease activity as a potential adjacent diagnostic tool and shed light on the use of nuclease activity as a detection biomarker in breast cancer.Breast cancer is one of the most common pathologies diagnosed in the clinical practice. Despite major advancements in diagnostic approaches, there is no widely accepted biomarker in the clinical practice that can diagnose breast malignancy. Confirmatory diagnosis still relies on the pathological assessment of tissue biopsies by expert pathologists. Thus, there is an unmet need for new types of biomarkers and novel platform technologies that can be easily and robustly integrated into the clinic and that can assist pathologists. Herein, we show that nuclease activity associated to malignant tumors can be used as a novel biomarker in breast cancer, which can be detected via specific degradation of nucleic acid probes. In this study we have identified a set of three chemically modified nucleic acid probes that can diagnose malignancy in biopsy samples with high accuracy (89%), sensitivity (82%) and specificity (94%). This work represents a breakthrough for the potential clinical use of nuclease activity as biomarker, which can be detected via nucleic acids probes, for the clinical diagnosis of malignancy in breast tissue biopsies. This platform technology could be readily implemented into the clinic as adjunct to histopathological diagnostic. Breast cancer is one of the most frequently diagnosed malignancies and the most common cause of cancer death in women all over the globe. In the last two decades, the clinical management of breast cancer patients has been greatly facilitated by the availability of several biomarkers with prognostic and predictive values, such as human epidermal growth factor receptor 2 (HER2/neu) gene amplification, elevated levels of estrogen receptor (ERs) or progesterone receptor (PR) and proliferation-related genes, such as the proliferation index Ki67), that helped stratify patients for receiving appropriate therapy and predict the likelihood of therapy response . These b7, that hBRCA1/2 genes [In the last decade, several emerged biomarkers, such as serum carcino-embryonic antigen (CEA) , carbohy/2 genes  circulat/2 genes , tumor i/2 genes ,12. The /2 genes ,14,15. UAmong proteins with catalytic activity, nucleases are enzymes that degrade nucleic acids by hydrolyzing the phosphodiester bonds that join the sugar residues . They arThe availability of several chemically modified nucleotides  offers tWe have previously demonstrated that we can differentiate healthy cells from cancer cells by detecting their associated nuclease activity, using chemically modified nucleic acid probes as substrates . SpecifiWe hypothesized that we can use nuclease activity to identify breast cancer tumors. We focused our efforts in developing nucleic acid substrates or probes that can specifically detect nuclease activity derived from breast tissue biopsies. We designed and performed a proof-of-concept study for the screening and identification of nuclease activity as biomarker in breast cancer tumors. In this study, we used computational analysis to identify the best performing nucleic acid probes. Thus, we report on the identification of a panel of three nucleic acids probes that can correctly diagnose 54 out of 61 patients (89% accuracy) with high sensitivity (82%) and specificity (94%). To the best of our knowledge, this is the first report on the use of chemically modified nucleic acids probes for the detection of malignancy in breast tissue biopsies via a specific tumor-associated nuclease activity.We sought to screen the profile of nuclease activity derived from healthy and breast-tumor tissues. Nuclease activity of a total of 102 samples from 51 patients (paired samples) was evaluated by a two-step screening method. Clinical information for all patients in this study (retrospective and prospective cohorts) is provided in We screened the 12 generic probes (p01–p12) against 58 paired tissue samples  obtained from 29 patients. Detailed probe sequences are included in Forty-four retrospectively collected paired tissue samples  were screened for the nuclease activity, using 24 probes designed as described in the Materials and Methods section. To identify which probes can better discriminate between healthy and tumor tissues, we again performed computational analysis on the raw fluorescent data from the nuclease activity assay and predicted the overlap of the healthy and tumor probability distribution functions . We idenTo prove the efficiency of our approach based on nuclease activity, we designed a prospective study, using fresh biopsy samples derived from 61 patients. Clinical information for all patients in the prospective cohort is provided in We performed a pre-analysis by using the six probes identified in the first-step retrospective screening  and second-step retrospective screening . We searched, from all possible 63 combinations of these six probes (predictors), for the optimal combination of probes that could discriminate healthy from tumor tissues in the prospective study. We preprocessed the measurements of the six probes, as in the retrospective case. Next, we estimated the probability of a prospective sample to be classified as healthy or tumor with all the possible combinations of predictors. While searching for clusters of combinations of predictors, we observed, in the map of distances of prediction probabilities , that a Finally, to find which members of the clusters reported in The three probes identified by computational analysis   were useTo verify the suitability for clinical use, the three cancer probes identified in this study  were further tested in human serum from a healthy donor. Nuclease assay was performed on the serum sample, along with healthy and tumor tissue homogenate samples. We have previously proposed nuclease activity as a novel biomarker for cancer using in vitro cell cultures . Herein,Given the high cell heterogeneity of the tumor environment and the documented presence of altered nuclease expression in cancer, we hypothesized that there exists a global and differential nuclease activity associated to tumors versus healthy tissues. To test our hypothesis, we designed a library of nucleic acids probes (substrates), to screen for a specific nuclease activity associated to tumors but not to healthy tissues. Since endogenous nucleases rapidly digest natural nucleic acids, such as DNA and RNA, we designed these nucleic acids probes with various chemical modifications, to increase their resistance towards non-specific nucleases. Moreover, we combined various sequences and chemical modifications, to increase the stringency of the screening process. We have identified DNases as the main type of nucleases with high activity in breast tumor tissues, based on the efficient degradation observed for the DNA-based probes. These findings are in very good agreement with previous work in our group on breast cancer cell lines . After tWe found two false positives and five false negatives by using our probe combination approach. The histopathological assessment of these seven samples (H&E staining) is shown in This approach could help pathologists to reach the best conclusions and provide the right diagnosis for the borderline cases. This is very relevant especially in those situations when misdiagnosis could lead to over-treatment or under-treatment of the patients.In this study, we developed a workflow  to identThe retrospective screening was divided in two steps: (a) an initial screening, using the 12 general probes, to identify the degradation profile or blueprint of the tumor associated nucleases; and (b) a follow-up screening, using a new set of probes, designed based on the information obtained in the initial screening. Thus, the second screening was intended to target tumor nucleases in a more specific manner. The size of the retrospective study was not pre-specified. A total of 102 frozen tissues  from 51 patients, stored in 2 mL vials, at −80 °C, were obtained from the Biobank of the Basque Country, Spain. The tissues were thawed over ice and weighted. The buffer for tissue homogenization (HB) was prepared by adding a protease inhibitor cocktail (10 µL/mL) to PBS containing Ca2+ and Mg2+. Tissues were then homogenized in HB to a final concentration of 0.5 g/mL. Next, the homogenates were centrifuged at 14,000 rcf, for 15 min, at 4 °C. The supernatants were then collected in a fresh 1.5 mL Eppendorf tube and were either kept on ice for immediate nuclease assay experiments or stored at −80 °C for further use.Patients were enrolled in this study after signing a written informed-consent form. The size, N, of the prospective study was calculated by using normal approximation in Z statistics ,47, usin0 = 0.58, the proportion of subjects in Group 1 (tumor) is q1 = 1 − q0 = 0.42. Thus, using equation (2), A = 4.105. By imposing a threshold for the probability for rejecting the null hypothesis, α = 0.2, and another threshold for the probability of failing to reject the null hypothesis under the alternative hypothesis, β = 0.2, we obtained, using the two-tailed Z statistics, the standard normal deviates for α and β, Zα = 1.282 and Zβ = 0.842, respectively. Thus, using equation (3), B = 4.508. Finally, by applying equation (1), we determined that the total group size N is 61. After sample collection, 3 of the 64 samples  were not tested, due to the low amount of tissue available for processing. Thus, these patients’ samples were excluded from this study, and we analyzed a total of 61 samples.E/S is the standardized effect size; for an effect size E = 0.55 and assuming that the one standard deviation of the outcome is S = 1, E/S = 0.55. Since the proportion of subjects in Group 0  is qBreast tissues from 61 patients were collected during surgical biopsy or breast surgery. Tissues were stored on ice and processed within 1 hour, to preserve as much as possible the integrity of the nucleases. Typically, the biopsy pieces weighted between 0.060 and 0.150 g. Tissues were washed in cold PBS and homogenized in homogenization buffer (HB), using a handheld homogenizer  in round-bottom 2 mL tubes. Samples were kept on ice for the entire period of processing, and the resulting supernatants were then collected in a fresh 1.5 mL Eppendorf tube, as previously indicated for the retrospective study. Subsequently, the nuclease activity assay was performed.The initial library of 12 probes was designed to cover a broad spectrum of known or suspected types of nuclease activities  that are present in tumors during the malignant transformation. Thus, this probe library comprises sequences containing natural nucleic acids, DNA and RNA, as well as sequences containing nucleic acids fully modified with 2’-Fluoro and 2’-O-Methyl chemistries. These 4 probes were named according to the nature of their nucleic acid makeup, as follows: DNA probe, RNA probe, All-2´-Fluoro probe and All-2´-O-Methyl probe. To simplify the nomenclature for the data analysis, all probes were also given a probe number, from p01 to p12 . We alsoOligonucleotide probes were synthesized and purified by Biomers.net , as we previously described . BrieflyNuclease activity assay was performed as previously reported , with smThe retrospective measurements were used as a training set to estimate the heuristic probability distribution functions of healthy and tumor conditions used to select the best probes. To correct the background signal, during the preprocessing stage for each patient, we subtracted the buffer control probe measurement from the healthy- and tumor-sample probe measurements. To equalize the corrected signal, we applied a log2 transform after setting to 1 the corrected values lower than 1. To analyze the clustering of combinations of predictors, we used Principal Component Analysis (PCA), the map of distances of probabilities between combinations of predictors based on the 1 minus the Pearson correlation (ρP) metric, MρP, and the hierarchical clustering using the MρP metric and the unweighted average distance (UPGMA)  linkage method. The heuristic probability distribution functions of healthy and tumor conditions were predicted by fitting the corresponding log2 transformed signals, using the generalized extreme value model.To estimate the discrimination capability of each probe, we calculated the overlap of the healthy and tumor probability distribution functions. The probes with best discrimination capabilities are those with smaller overlap between healthy and tumor probability distribution functions. To estimate the healthy and tumor probabilities of the samples from the prospective studies, first we preprocessed the measurements of the probes, as in the retrospective case. Next, we selected the set of best predictors from the two screenings of the retrospective study and we calculated the performance of all the possible combinations of selected probes, using performance metrics of the Receiver Operating Characteristic (ROC), namely the distance to the optimal point  of the ROC space (D01), the accuracy and the specificity. Finally, we chose the combination of probes with the minimal distance to the optimal point  of the ROC space. Data processing and graphics were performed with in-house developed functions in Matlab .Fresh surgical biopsy tissues were randomly fragmented into smaller pieces. Fragments with a surface area of 5–8 × 4–6 mm and with a thickness of approximately 4 mm were divided into 2 halves. One half was fixed in 4% formaldehyde, for further histopathological examination, and the other half was immediately transported in ice, for nuclease activity assessment in the prospective screening. The fixed tissues were embedded in paraffin overnight, and sections of 3 or 4 microns were cut and mounted on slides, followed by staining with Hematoxylin–Eosin (H&E). All samples slides were imaged with a digital light microscope for standard histopathological analysis. The process was carried out blindly, without knowledge of the nuclease activity assay results.In summary, we reported on a panel of three probes that have the capability to differentiate between breast cancer tumors and normal healthy tissues, with an accuracy of 89%, sensitivity of 82% and high specificity of 94%. Moreover, the detection of nuclease activity derived from breast cancer tissues offers a robust and easy platform that could readily be implemented in the clinic as an adjunct method to the standard histopathological interpretation of tissue biopsies. As a future direction, nucleic acids–based probes could open the doors for the development of alternative and more efficient diagnostic approaches for other types of cancer and even other diseases. We envision that, once a specific set of probes is identified, the sequences can be incorporated into MRI probes that can work as contrast agents, thus adding diagnostic value to the non-invasive detection of cancer through the use of MRI machines, which are widely implemented into the clinic. We and others are currently exploring the translation of enzymatic-based probes into MRI probes for diagnostic purposes ,51,52,53


Dengue fever prevalence is rising globally and it causes significant morbidity and mortality. Fluid extravasation during the critical phase of dengue haemorrhagic fever (DHF) leads to shock, multi-organ failure and death if not resuscitated appropriately with fluids. The mainstay of management is judicious fluid replacement using a guideline based, calculated fluid quota of maintenance (M) fluid plus 5% deficit (M +  5% deficit) to prevent organ hypoperfusion.We conducted an observational follow-up study in Sri Lanka from January–July 2017 to identify the fluid requirements of DHF patients and to identify whether features of fluid overload are present in patients who exceeded the fluid quota. Patients who developed DHF following admission to the place of study, were recruited and the amount of fluid received during the critical phase was documented.n = 80, 69.6%) received fluid in excess of the recommended maintenance + 5% deficit and this group had higher body mass index  and a lower white cell count at the onset of the critical phase . The highest fluid requirement was seen within the first 12 and 24 h of the critical phase in patients requiring fluid M +  5%–7.5% deficit and ≥ M +  7.5% deficit respectively. Patients exceeding M + 5% deficit had narrow pulse pressure and hypotension compared to the rest. DHF grades III and IV were seen exclusively in patients exceeding the fluid quota indicating higher amount of fluid was given for resuscitation. Fluid overload was detected in 14 (12.1%) patients and diuretic therapy was required in 6 (5.2%) patients.A total of 115 DHF patients with a mean age of 30.3 (SD 12.2) years were recruited to the study. There were 65 (56.5%) males and the mean fluid requirement was 5279.7 ml (SD 735) over the 48 h. Majority of the study participants (The majority of patients received fluid in excess of the recommended quota and this group represents patients with narrow pulse pressure and hypotension. Although, fluid overload was infrequent in the study population, clinicians should be cautious when administering fluid in excess of M +  7.5% deficit.The online version contains supplementary material available at 10.1186/s12879-021-05971-6. The incidence of dengue fever has increased dramatically in the tropical and subtropical regions of the world during the recent decades with an estimated 390 million infections occurring per year, spanning across 128 countries . Surveil3), and evidence of plasma leakage as shown by the presence of pleural effusion, ascites, or an increase in packed cell volume (PCV) by 20% [Dengue infection is caused by one of the four distinct dengue virus serotypes, DENV 1–4. Many patients infected with dengue virus remain asymptomatic while symptomatic infection ranges from undifferentiated fever, dengue fever (DF), dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) . Hallmar) by 20% . Due to ) by 20% .Early detection of dengue and proper medical care has lowered the fatality rate below 1%. In the absence of specific antiviral therapy, meticulous fluid administration is the mainstay of management of DHF and judicious fluid administration during the “critical phase” is vital in reducing mortality . The curThe maintenance fluid required depends on the caloric expenditure of an individual. Holliday et al. in their landmark paper published in 1957, have described the 100/50/20 rule of calculating this volume based on the body weight , 9. A feThere are no randomized controlled trials comparing rates and amount of fluid to be given during the critical phase of the illness. The current practice guidelines are based on consensus of expert opinions, some assumptions and studies performed on paediatric populations . This inThis observational follow-up study was carried out in the University Medical Unit of Colombo South Teaching Hospital, Sri Lanka from January 2017 to July 2017. Adults above the age of 18 years who had DHF according to WHO criteria and who have not received fluid therapy before recruitment were enrolled to the study  with adjustments made according to the vital parameters, urine output and the PCV. Patients were examined at the end of the critical phase (48 h later) by the research team for evidence of fluid overload. Presence of facial puffiness, shortness of breath, large pleural effusions, fine basal crepitations or moderate ascites was considered as evidence of fluid overload.www.graphpad.com. Continuous variables were presented as mean (standard deviation) in normally distributed data and median (IQR) in skewed data. Comparisons were performed using either t-test, Mann-Whitney U test, ANOVA or Kruskal-Wallis test as appropriate with Bonferroni correction for multiple comparisons. Chi squared test or the fisher’s exact test was used to compare categorical variables. A subgroup analysis was performed in patients who received fluid in excess of M + 5% deficit as discussed in the results section.Data were entered into a MS EXCEL database and analysed using SPSS  and GraphPad Prism version 7.0.0 for Windows, GraphPad Software, San Diego, California USA, n = 108, 93.9%) were admitted to the hospital within 4 days of onset of fever. Severity of DHF in the patients revealed that 88 (76.5%) had stable DHF (DHF Grade I &II) while 25 (21.7%) had compensated shock (DHF grade III) and 2 (1.7%) patients developed DSS (DHF grade IV). There were no deaths in the study population. Fluid received by the patients ranged from 3920 ml to 9280 ml during the 48 h of the critical phase. There were 98 (85.2%) patients who received crystalloids only while 15 (13%) patients received colloids in addition to crystalloids. The crystalloid used for fluid resuscitation was 0.9% saline and the colloid used in the study was dextran-40. There were 2 (1.7%) patients who received blood transfusion during the study period. The mean fluid requirement was 5279.7 ml (SD 735) over the 48 h in the study group. There was one outlier who received 9280 ml fluid over the 48 h and this participant was removed from analysis when comparing fluid received among groups during the 48-h period to overcome the undue effect from the extreme value. Further analysis revealed that the highest fluid requirement was within the first 24 h. The mean fluid requirement over the 0–12 h was 1395.4 ml (SD 326.2) and it was 1401.2 ml (SD 424.7) over 13–24 h. The requirement was 1298.4 ml (SD 308) and 1196.5 ml (SD 185.5) in the 25–36 h and 37–48 h of the critical phase respectively.A total of 115 DHF patients diagnosed according to the WHO criteria were enrolled to the study. One participant with missing data was removed from the analysis. Serological confirmation was available in 48 (41%) of patients; NS1 antigen was positive in 46 and IgM positive in 2 patients. Patients had a mean age of 30.3 (SD 12.2) years and there were 65 (56.5%) males. The day of fever on admission ranged from day 1 to day 6 and majority of patients (Patients who received fluid within the recommended quota (M + 5% deficit) and patients who received fluid in excess (>M + 5% deficit) were compared Table . The basDiagnosis of DHF in the study population was established by detection of third space fluid accumulation  either clinically or ultrasonically, by a rise in PCV and/or haemodynamic instability Table . AlthougAll patients were monitored regularly and managed according to the local dengue management guideline after the onset of the critical phase. The parameters at the onset of the critical phase in the 2 groups are given in Table p < 0.01) or patients who required M + 5%–7.5% deficit of fluid (p < 0.05)  showed that patients requiring fluid ≥M +  7.5% received significantly higher amounts of fluid than patients in the ≤M + 5% group (5) Table . Figure p < 0.0001). Patients who required fluid within the recommended quota of ≤M + 5%, did not receive isotonic saline boluses, dextran (colloid) or blood transfusion. The study population requiring fluid ≥ M + 7.5% needed saline boluses, dextran and blood transfusion more frequently than patients who received fluid M + 5%–7.5% deficit  patients in the study population. Mild fluid overload as evidenced by facial puffiness was observed in 2 (3.6%) patients who received M+ 5% -7.5% deficit amount of fluid and 6 (24%) who received ≥M+ 7.5% deficit amount of fluid. Moderate to severe fluid overload  was rare and observed in 6 out of 24 patients  who received ≥M+ 7.5% deficit amount of fluid. Diuretic therapy was required in 6 (5.2%) patients to relieve symptoms of fluid overload.This study is a single centre experience in management of DHF. The management of DHF could vary between units and it is important to have a multicentre study to understand the patterns of fluid therapy prior to generalising the data. The observational nature of the study poses limitations on predicting causality and a prospective randomised controlled trial on different fluid regimens would be useful in this aspect to improve existing guidelines. Any observer bias that could occur due to investigators being involved in patient management was minimised by data collection and analysis done by researchers external to the treatment team. Also, dengue serology and/or NS1 antigen testing was not performed in all patients and this is a minor drawback in the study. A further limitation of the study was not excluding malaria in the study population by specific investigations. However, malaria has been eradicated from Sri Lanka and few exported cases are detected in the country.This study has shown that fluid requirement during the critical stage of DHF is highly variable and majority of patients require fluid in excess of the currently recommended M + 5% deficit to maintain the clinical parameters. Patients requiring fluid in excess of ≥M+ 7.5% deficit represented a group with more severe fluid extravasation as evidenced by haemodynamic instability and more severe disease. Clinically significant fluid overload and the need for diuretic therapy are infrequent in adult DHF patients but clinicians need to be cautious when exceeding fluid amount beyond M+ 7.5% deficit. The rate of infusion should be increased instead of giving fluid boluses in patients with reduced urine output in the absence of hypovolaemia. However, restricting the amount of fluid in fear of fluid overload may lead to impaired organ perfusion and tissue hypoxia.Additional file 1: Supplementary Table 1: Calculation of the total fluid requirement in the critical phase (48 h): M+ 5% deficit.Additional file 2.


This report describes an unusual mesenchymal differentiation in a canine benign mixed mammary tumour. A 13-year-old crossbreed female dog was submitted to surgery to remove an inguinal mammary nodule. The tumour was composed of mammary epithelium and mesenchymal populations, not only of cartilage and bone but also of myoid cells. PTAH demonstrated cross striation of striated muscle, and immunohistochemistry highlighted striated muscle expressing desmin and calponin, and smooth muscle expressing desmin, SMA, and calponin. The tumour was diagnosed as a benign mixed tumour with leio- and rhabdomyoid differentiation. There was no tumour recurrence after one year of clinical follow-up. In conclusion, the well-differentiated features of leiomyocytes and rhabdomyocytes and the growth pattern define the benign origin of the mesenchymal component, which has been confirmed by a benign outcome; therefore, the knowledge of this kind of differentiation is helpful to avoid misdiagnoses. Canine benign mixed tumour and carcinoma-mixed type are neoplasms with more than two cellular populations . The tumThe aim of this report is to histologically describe and histochemically and immunohistochemically characterize the unusual feature of myoid differentiation in a benign mixed mammary tumour and its correlation with the biological behaviour.A 13-year-old crossbreed female dog was submitted to surgery to remove multiple nodules: one nodule of 1.5 × 1 cm in the right cranial thoracic mammary gland, a second nodule of 1 cm in the right inguinal gland region, and a third nodule of 10 × 7 cm in the left inguinal gland.Sections were obtained from all the nodules and stained with haematoxylin and eosin (HE), Masson's trichrome, and phosphotungstic acid haematoxylin (PTAH). Immunohistochemistry (IHC) was performed on serial sections using antibodies to vimentin, cytokeratin (CK) 19, CK 14, p63, desmin, alpha-smooth muscle actin (SMA), and calponin. All specifications are reported in Histologically, the left inguinal mammary mass was well-demarcated, not encapsulated, expansive, and moderately cellular. It was composed of well-differentiated mesenchymal components  and less than 5% of mammary epithelium. The lesion was multilobular with round nodules composed of poorly organized hyaline cartilage that surrounded bone tissue. Around and between the nodules, there were bundles of mesenchymal cells embedded in a loose myxoid stroma Figures . The mesAdditionally, the neoplasm in the right inguinal mammary gland was a mixed-type carcinoma and the right inguinal mass as a cutaneous lipoma.All observed mesenchymal components of the tumour  were well-differentiated and organized in nodules, with no signs of cellular atypia, and therefore were considered of metaplastic origin. Moreover, the morphologic features of epithelial cells were consistent with a benign neoplasm. Based on these findings, we suggest the diagnosis of a canine benign mixed mammary tumour with smooth and striated muscle differentiation.One year after surgery, the dog was in good health and showed no recurrences.The presence of neoplastic epithelial cells, even if minimal, ruled out the diagnosis of a purely mesenchymal neoplasm. In the latter case, the most likely diagnosis would have been benign mesenchymoma, based on the identification of three or more differentiated mesenchymal cell phenotypes in the same neoplasm . Benign Metaplastic carcinoma of the human breast encompasses a group of tumours characterized by differentiation of the neoplastic epithelium into squamous cells and/or mesenchymal-looking elements, including but not restricted to spindle, chondroid, osseous, and rhabdomyoid cells. These neoplasms may be either entirely composed of metaplastic elements or a complex admixture of carcinoma and metaplastic areas . MalignaThe treatment of choice for mammary tumour is surgical excision, and one year after surgery, the dog is in good health, without any disease relapse.To the best of our knowledge, this is the first report of leio- and rhabdomyoid differentiation in a canine benign mixed tumour. The features of leiomyocytes and rhabdomyocytes and the growing pattern define the benign origin of the mesenchymal component, which is confirmed by a benign biological behaviour; therefore, the knowledge of this kind of differentiation is helpful to avoid diagnostic pitfalls.


Cooking is the act of turning nature into the culture, which has enabled the advent of the omnivorous human diet. The cultural wisdom of processing raw ingredients into delicious dishes is embodied in their cuisines. Recipes thus are the cultural capsules that encode elaborate cooking protocols for evoking sensory satiation as well as providing nourishment. As we stand on the verge of an epidemic of diet-linked disorders, it is eminently important to investigate the culinary correlates of recipes to probe their association with sensory responses as well as consequences for nutrition and health.https://cosylab.iiitd.edu.in/recipedb) is a structured compilation of recipes, ingredients and nutrition profiles interlinked with flavor profiles and health associations. The repertoire comprises of meticulous integration of 118 171 recipes from cuisines across the globe , cooked using 268 processes , by blending over 20 262 diverse ingredients, which are further linked to their flavor molecules (FlavorDB), nutritional profiles (US Department of Agriculture) and empirical records of disease associations obtained from MEDLINE (DietRx). This resource is aimed at facilitating scientific explorations of the culinary space  linked to taste (flavor profile) and health (nutrition and disease associations) attributes seeking for divergent applications.RecipeDB (Database URL:  https://cosylab.iiitd.edu.in/recipedb Homo sapiens . FoodBase provides a resource of annotated food entities , essentially making them computable. Among data resources that remotely related to the objectives of RecipeDB, FooDB focusses on the compilation of food chemicals  and AllRecipes . While we reviewed a large number of recipe repositories as a potential source of data beyond these two , The Spruce Eats (https://www.thespruceeats.com), Epicurious (https://www.epicurious.com), Food Network (https://www.foodnetwork.com) and taste.com.au (https://www.taste.com.au)), we chose these based on the uniformity in structure, availability of geocultural mapping and number of recipes. Each recipe was divided into two parts, the ingredients section and the cooking instructions section to extract relevant information out of them.To begin with, recipes were aggregated from the following sources: GeniusKitchen , e.g. 1 teaspoon, 10 g; Size—Portion sizes mentioned, e.g. small, large; Temperature—Temperature applied prior to cooking, e.g.—hot, cold; Dry/fresh—Is the ingredient dry or fresh?While extracting ingredients and their corresponding attributes, we primarily faced following three challenges. Unknown Attributes: There is no known list of attributes, which we could use to extract the required information. Moreover, our database of recipes is immensely varied, which further complicates the task. Identification of Attributes: Homograph attributes are common in recipes. For example, ‘clove’ may refer to the ingredient itself or a unit of measurement. Variation in Lexical Structure of Ingredient Phrases: A phrase in the ingredients section may be written in several different ways. The lexical structure of the sentence may be as simple as ‘1 cup sugar’ or a more complex structure such as ‘(8 ounce) package cream cheese, softened’.et al.  across all the models Table , many efhttps://github.com/cosylabiiit/Recipedb-companion-data.We also compared the class-wise performance of the best model Table . The claSimilarly, in the instructions section, an NER model was trained to identify cooking processes, ingredients and utensils using the Stanford NER . The recEvery recipe was mapped to its geocultural correlate at three levels of hierarchy: Continent, Region and Sub-region (country). The protocol followed for geocultural mapping of recipes was consistent with the one used by Singh and Bagler in their data-driven investigation of recipes . ContineEach of the ingredients was manually mapped to one of the 29 categories  exclusively. The list of ingredient categories available from FlavorDB was used as the baseline . Each inEach recipe was tagged with one of the following commonly accepted and widely practiced dietary styles: Vegan, Pescetarian, Lacto-Vegetarian, Ovo-Vegetarian and Ovo-Lacto-Vegetarian. Rules were created for designating a dietary style based on the ingredient category and/or ingredient(s) used in a recipe. For each dietary style, the rules were formed to specify categories of ingredients that were restricted and the ones mandatory. For a recipe to be associated with a dietary style, it needed to have no ingredient belonging to the dietary style’s list of restricted categories while having at least one or more ingredients belonging to each of the dietary style’s list of mandatory categories. As a precaution, any recipe containing an ingredient under the category ‘bakery’ was assumed to have eggs in it. Also, a recipe was not mapped to any dietary style if it had an ingredient(s) of the ‘dish’ category. Following were the rules used: Vegan ; Pescetarian ; Lacto-Vegetarian ; Ovo-Vegetarian ; and Ovo-Lacto Vegetarian .https://fdc.nal.usda.gov) (whenever possible) by matching the ingredient information (name and state) with the ‘long description of food’ using the ‘Jaccard similarity’ algorithm .Recipe Network visualizes the relationship between ingredients and ingredient categories to provide an interactive way of exploring RecipeDB. The ingredient categories are represented as boxes in the middle of the graph , while the ingredients are laid out along the circumference to make it easier to observe the relationships among entities. To address the dense pattern of relationships, the six most frequently used ingredients from each category are depicted. Hovering over an entity shows its association with other entities. One can navigate the network by either clicking on the ingredient category or one of the ingredients. The visual search lands either on the category profile page or on the ingredient profile page. Thus, visual search enables open-ended exploration of RecipeDB. The Flavor Network was implemented with the D3.js JavaScript library , ingredients used/not used and nutritional profile . Structurally similar recipes can be found using the ‘Search Similar in RecipeDB’ button provided on the recipe profile pages. Recipe search yields matching recipes with detailed profiles. RecipeDB also facilitates browsing all 118 171 recipes by doing a null search .Advanced search provides an option for refined search by querying RecipeDB data on the basis of a combination of all the separate search features provided in Recipe Search. Beyond the options provided in the Search panels, advanced search enables querying on 268 cooking process/techniques and 69 utensils.Every recipe is presented with a detailed profile with the following sections: Estimated nutritional profile, Recipe overview, Ingredients, Processes, Utensils and Detailed recipe instructions. Beyond the nutritional profile at the level of the recipe and individual ingredients, the profile provides an overview . Importantly, it provides a temporal sequence of cooking processes/techniques and utensils used in the recipe.Having obtained the recipe structure in its basic constituent elements, one can now seek similar recipes by way of ingredient category composition or the cooking processes/techniques. Using ‘Similar Recipes by Category Composition’ and ‘Similar Recipes by Processes’ button on the recipe profile page, one can obtain recipes that align with the present recipe in the multidimensional space of cooking techniques or ingredients category. Thus, RecipeDB enables searching for ‘similar recipes’ in the vast space of recipes enabling discovery of new recipes and patterns.Each recipe was translated to one-hot encoded vector of dimension (1 × 20 280) and (1 × 268) to create the recipe-ingredient and recipe-process matrices, respectively. Cosine distances between the vectors were calculated and top 50 similar recipes were presented in response to a query. Considering the fact that each recipe consists of around 10 ingredients and 12 processes on an average, both matrices are highly sparse. Therefore, the matrices were converted to condensed sparse row matrices to improve the efficiency of the computation. The cosine distance ranges from 0 to 1 with the former representing to the highest similarity and latter pointing to the least similarity. Accordingly, the similarity index was defined as (1—cosine distance).https://cosylab.iiitd.edu.in/dietrx) and their flavor profile (FlavorDB (http://cosylab.iiitd.edu.in/flavordb). One can explore these in detail to probe the health and flavor correlates of the ingredient, respectively.For each generic ingredient (1636) RecipeDB presents its profile page with details of its category, Wikipedia link and, whenever available, link-out to health impacts  of ingredients from the chosen category.The perception of food happens primarily via sensory mechanisms of taste and odor. RecipeDB provides an exhaustive, structured list of taste and odor receptors. These were curated from Uniprot  by usinghttps://www.sqlite.org/index.html). The webserver has been built using the Python web development framework, Flask (flask.pocoo.org). Flask has agnostic Object Relational Mapper (ORM) for querying the database using any of the ORMs, thus optimizing queries and making it easier to perform complex queries, apart from reducing the development period. The front end has been built using HTML, CSS and JavaScript. The jQuery (https://jquery.com/), MaterializeCSS , D3.js (https://d3js.org/) and plotly (https://plot.ly/) were used to add to the functionality of RecipeDB. An NGINX (https://www.nginx.com/) HTTP Server has been used to route requests to the Flask app and to enable data compression for faster page load times. The site is best viewed in the latest versions of Google Chrome, Firefox, Opera, Internet Explorer and Microsoft Edge.RecipeDB has been designed as a Relational Database using SQLite  present in RecipeDB along with their names, estimated macronutrients, as well as link-outs to the recipe pages and detailed estimated nutritional profiles. Clicking on the recipe name would yield a page with structural details of the recipe, and clicking on the ‘More Info’ tab would open a pop-out a page with estimated nutritional profile for macro- as well as micro-nutrients as available in USDA. One can move across the results using Previous, Next links or by jumping to any page number from the panel presented at the bottom and also search the page using the search box provided at the top.Using the protocol described in the ‘Data Compilation’ section , the nutritional profile of each recipe has been estimated. For example, the ‘Indonesian Pork Satay’ (4 servings) has estimated calories of 1647.39 KCal and protein and fat of 179.12 and 84.59 g, respectively. Clicking on the ‘More Info’ button provides a full estimated nutritional profile of micro- as well as macro-nutrients. A nutritional profile breakdown into its individual elements/ingredients is also provided.From its recipe profile, we observe that ‘Indonesian Pork Satay’ involves, among other things, pouring, mixing in early-stage, marinating in mid-stage and boiling in late stage. The recipe mentions processor and saucepan as utensils being used. The ‘Instructions’ section provides detailed recipe instructions as obtained from the source.Exploring for similar recipes of ‘Indonesian Pork Satay’, the ‘Spicy Indonesian Pork Satay (Or Chicken)’ comes as the closest recipe with a similarity index of 0.64 followed by ‘Grilled Chicken Adobo’ (0.59) using cooking techniques as the measure of similarity. By category composition, the closest recipes are ‘Spicy Pork Baked Ziti With Ragu’ (0.92) and ‘Delicious Bolognese Meat Sauce’ (0.92).RecipeDB facilitates exploring the molecular basis of the flavor and empirically reported health impacts of ingredients of its recipes via out-links to FlavorDB  and DietRecipeDB enables searching for recipes by the presence or absence of an ingredient or ingredient category. For example, searching for all recipes ‘having spinach’ and ‘not having onion’ in them returns 148 pages of results. Similarly, searching for all recipes ‘having dairy’ and ‘not having spice’ in them returns 3198 pages of results. Such queries enable one to seek for recipes across the global repository in a structured manner.Searching for recipes having the desired quantity of macronutrients  is also facilitated by RecipeDB. With default values set around one standard deviation around the mean of all recipes, the interface allows changing the search range.The advanced search enables creating nuanced queries using the individual query elements . For example, one may search for all Indonesian recipes ‘having spinach’, ‘not having onion’ and using cooking process ‘fry’ returns two recipes: ‘Indonesian Sweet and Sour Tofu With Vegetables’ and ‘Nasi Goreng’.Food is a complex subject interwoven with traditional cooking practices (recipes), flavor, nutrition and health. The ‘Complex Systems Laboratory’ at IIIT-Delhi has been putting together the data-driven perspective of these jig-saw pieces of the food puzzle in the form of a ‘Computational Gastronomy’ framework including FlavorDB , DietRx RecipeDB provides one of the most structured repositories of worldwide recipes to integrate cultural, culinary and nutritional aspects with those of flavor and health impacts. While a plethora of (web based as well as printed) recipe resources are available, which archive recipes in a human-readable format, none looks at recipes from a data-centric perspective intended to capture recipe elements for making recipes computable. By creating a curated and structured culinary knowledgebase, RecipeDB fills in this gap and enables open-ended explorations.However, despite an impressive performance of the algorithms to extract relevant information from the recipe instructions, RecipeDB has much scope to improve in gathering the following elements: unit and quantity of an ingredient and the exact process/technique being applied to the ingredient. The former is critical for the estimation of the nutritional profile of the ingredient and, therefore, of the recipe. When encountered with a ‘null unit’, the present algorithm uses a heuristic for assigning the most frequently used unit for the ingredient, which may, at times, be incorrect. The latter is missing in RecipeDB at the moment and is a key factor determining the nutritional profile of the recipes.Other than improving the quantity of the data, there is much scope for improving the quality of the recipes data. The present data compilation strategy relies on the classification of the recipes done by crowd-sourced resources (AllRecipes and Genius Kitchen), which may not be entirely accurate. Compilation of generic traditional recipes is one among the key future directions. Many of the recipes compiled by us could, in fact, be better classified as ‘International Standard Cuisine’. Correspondingly, the estimated nutritional values could change due to use of nonstandard ingredients. Our strategy uses USDA data for nutritional mapping. Going further, it may be more relevant to use national nutritional databases.The data gathered from RecipeDB is a potent source for the analysis of ‘identity’ of cuisine in measurable parameters, similarity among cuisines, quintessential patterns in cuisines, among others, apart from being an excellent resource for asking queries. Thus, RecipeDB provides a quantified resource of the world’s culinary heritage through a structured repository of recipes.baaa077_SuppClick here for additional data file.


Tumour Virus Research has provided fundamental insights into the mechanisms underlying tumour development. These studies have, in turn, not only driven the field of tumour virology, but have provided unique insights into many of the fundamentals of cell biology and of the events that ultimately lead to tumourigenesis. This has led to the development of new means of prevention and therapeutic intervention in a wide range of different human malignancies, and not only in those with a viral aetiology.Over the last 50 years the field of Papillomavirus Research to Tumour Virus Research, we expect the Journal to be the ideal forum for publishing such studies, resulting in an extension of the scope and impact of the work that is published in the Journal. This, in turn will ensure that Tumour Virus Research will be more broadly applicable to the wider scientific community working on all human tumour viruses and associated model systems.In the revamping and renaming of Tumour Virus Research now has a much broader editorial board, and we welcome submissions from scientists working on all aspects of human tumour viruses.To achieve this, we need to ensure the studies we publish are timely, scientifically and technically excellent, and multidisciplinary. To reflect these new goals,


The characteristics of tumour development and metastasis relate not only to genomic heterogeneity but also to spatial heterogeneity, associated with variations in the intratumoural arrangement of cell populations, vascular morphology and oxygen and nutrient supply. While optical (photonic) microscopy is commonly employed to visualize the tumour microenvironment, it assesses only a few hundred cubic microns of tissue. Therefore, it is not suitable for investigating biological processes at the level of the entire tumour, which can be at least four orders of magnitude larger. In this study, we aimed to extend optical visualization and resolve spatial heterogeneity throughout the entire tumour volume. We developed an optoacoustic (photoacoustic) mesoscope adapted to solid tumour imaging and, in a pilot study, offer the first insights into cancer optical contrast heterogeneity in vivo at an unprecedented resolution of <50 μm throughout the entire tumour mass. Using spectral methods, we resolve unknown patterns of oxygenation, vasculature and perfusion in three types of breast cancer and showcase different levels of structural and functional organization. To our knowledge, these results are the most detailed insights of optical signatures reported throughout entire tumours in vivo, and they position optoacoustic mesoscopy as a unique investigational tool linking microscopic and macroscopic observations. A technique that can image the entire tumour volume with high resolution may help oncologists optimize specific treatments for breast cancer. Jiao Li , Vasilis Ntziachristos , and colleagues have designed a multispectral optoacoustic mesoscope (MSOM) that illuminates millimetre-sized tumours with laser light of various wavelengths, and detects the ultrasound waves generated by internal absorbers, such as haemoglobin, or external nanoparticle probes. By reconstructing the ultrasound signals over multiple frequencies, the team produced 3D images of features that included the vascular network of a tumour with micrometre-scale detail. Experiments with live mice demonstrated that specific tumours could be identified through differences in spatial patterns, such as altered oxygen levels between tumour cores and peripheries. The study highlights the power of MSOM as a tool for preclinical cancer studies. Understanding the tumour microenvironment and its heterogeneity are key to elucidating cancer progression, metastasis and response to treatment. In addition to genetic heterogeneity, spatial heterogeneity may play a vital role in tumour growth, metastasis or treatment, especially if different parts of a tumour respond in different ways2.Tumours contain genetically and phenotypically distinct cell subpopulations exposed to heterogeneous vascularization and signalling microenvironments5 or to radiation therapy6. Nevertheless, optical microscopy is limited by photon scattering7 and can assess small volumes of only a few hundred cubic microns8, so it is not suited for comprehensively studying the morphology, functionality and the overall heterogeneity of an entire tumour volume9. Surgical removal of tissue and the implantation of “optical windows”10 allow microscopic observations of deeper tumour areas but similarly limit the comprehensive visualization of entire tumours. The need to image larger volumes has led to the development of several alternatives. Three-dimensional light-sheet microscopy12 offers the possibility of volumetric imaging. However, it requires chemical processing of specimens to make them transparent and is therefore not suited for in vivo applications or imaging of tissue physiology.Intravital (in vivo) microscopy is commonly used in cancer research and has revealed critical information about cell motility that leads to metastasis, about the function of immune cells within tumours and their role in inhibiting or promoting tumour growth, and about the different responses of cell populations within tumours to the same drug16. There are two major optoacoustic imaging techniques. Optical-resolution optoacoustic (photoacoustic) microscopy uses focused light to excite ultrasound responses, but its depth and resolution are similar to those of optical (photonic) microscopy since both modalities form images limited by optical diffraction. Conversely, acoustic-resolution optoacoustic imaging can be performed using unfocused illumination, in which case the depth and resolution depend strongly on the characteristics of the ultrasound detection arrangement employed and the ultrasound frequency band collected. The combination of deep-seated high-resolution imaging with multi-wavelength illumination gives rise to multispectral optoacoustic tomography (MSOT), which can resolve the spectral signatures of intrinsic chromophores and extrinsically administered absorbers. MSOT has been consequently employed to visualize and quantitate the oxygenation status of tumours19 and resolve angiogenesis and a number of external agents targeting physiological and molecular tumour parameters in animals and humans25. Nevertheless, current optoacoustic imaging systems do not allow high-resolution imaging of tumour heterogeneity. Tomographic systems using detector arrays have allowed three-dimensional imaging that yielded cross-sectional images throughout entire tumours18, but the resolutions achieved have been on the order of 100–500 μm. These resolutions prevent the detailed observation of tumour spatial heterogeneity. Systems that raster scan an ultrasound detector operating at higher frequencies than detector arrays allow higher resolution imaging but offer only limited-view images of superficial vasculature27, typically rendered as two-dimensional maximum intensity projections (MIPs).As an alternative to optical methods, optoacoustic imaging has demonstrated high-resolution imaging of optical contrast through several millimetres to centimetres of tissueHerein, we introduce multispectral optoacoustic mesoscopy (MSOM) with a novel system that combines three-dimensional performance with high-resolution imaging. We identified ~1–30 MHz as the optimal ultrasound detector bandwidth offering the optimal trade-off between resolution and cross-sectional imaging of entire tumours. We implemented this frequency band in a conical geometry enabling optical views of tumour heterogeneity with unprecedented detail. By applying MSOM at multiple wavelengths, we studied, for the first time, patterns of the spatial heterogeneity of tumour oxygenation and validated these patterns through co-registration with patterns of vascularization and permeability throughout the entire tumour mass. By applying the technique to three breast cancer models in mice, we studied intra- and inter-tumour heterogeneity and showed how in vivo MSOM imaging can be applied to quantitatively characterize the spatial heterogeneity in breast cancer based on functional tumour parameters in vivo. We discuss the unique performance achieved by our conical MSOM implementation and the implications for providing new possibilities in cancer research.30. A further novelty of the system is the combination of four-sided illumination and a conical geometry, in which the detector is positioned at a 45-degree angle to the rotation plane, thus allowing the accommodation of tumours of different sizes , and of extrinsic probes such as gold nanoparticles  and high-frequency (4–28 MHz) band  , oxygen saturation (sO2 = HbO2/HbT) and vascular permeability. HbT and sO2 were computed by resolving the distribution of Hb and HbO2 in entire tumours in vivo at resolutions never before possible. The spatial heterogeneity of these parameters was quantitated throughout the volumes of three types of breast tumours: two human breast cancer xenografts  and one mouse mammary tumour allograft (4T1). Vascular permeability in 4T1 tumours in mice following injection of gold nanoparticles was also studied.MSOM was used to interrogate differences in the spatial heterogeneity of total haemoglobin concentration , anti-CD31 antibody for vascular endothelial cell staining 25 Fig. . Inspect 25 Fig. . Profile 25 Fig.  and then 25 Fig.   and that this difference was even more apparent in the tumour centre than on the tumour rim (cf. panels h–i). Although the sO2 contrast and variance in 4T1 tumours were close to those in the two other tumour types, as shown in SI Appendix, Fig. 2 and less hypoxic overall than KPL4 or MDA-MB-231 tumours. SI Appendix, Fig. 2 for the three tumour types. As in the case of sO2, HbT heterogeneity varied substantially across tumour types. 4T1 tumours showed greater heterogeneity than the other two tumour types, and this heterogeneity may be present more in the rim than in the centre. These results provide the first evidence that MSOM can be employed to assess morphological and physiological differences within the tumour mass. Other heterogeneity metrics analysed at the whole-tumour level are listed in SI Appendix, Fig. 2 and HbT heterogeneity in individual xy plane sections of all tumour types and depths  and the corresponding vascular patterns throughout entire tumours in vivo at resolutions never before demonstrated by another method. By doing so, we also demonstrated that by using optoacoustic imaging, we can observe different patterns between tumours, allowing study of their individualized morphology and functions.For the first time, we characterized the spatial heterogeneity of tumour hypoxia , provides a holistic view of tumours. In this way, the two techniques are highly complementary: MSOM can provide an overall view, within which specific regions can be analysed at higher resolution using intravital microscopy.2 on a tumour-to-tumour basis. The relatively high quality of the images provided by the MSOM system described here likely reflects critical improvements over our previously published design28. The linear transducer array with a central frequency of 15 MHz can capture acoustic signals at a high signal-to-noise ratio from light-absorbing anatomical structures in the tumour smaller than 100 μm (blood vessels) up to structures spanning several millimetres (tumour core). The tumour in our system was illuminated with broad-field illumination from four sides, helping provide homogeneous stimulation of light absorbers in the tumour, leading in turn to stronger and more uniform acoustic signals despite depth-dependent attenuation of light fluence. The custom-designed sample holder features a tumour hole with plastic snap rings of various inner diameters that can be optimized to the tumour size. In addition, a linear stage allows the sample holder to be raised or lowered to position the sample optimally within the field of view of the detector. Frequency equalization, in which the relative contributions of acoustic signals of different frequencies are balanced, improves image fidelity by recovering chromophores of various sizes in the tumour, such as smaller and larger vessels. This advanced performance was essential to provide a detailed analysis of and insights into tumour patterns.The results of this pilot study on technology feasibility clearly demonstrated differences in the heterogeneity present in different tumours. The study shows that the novel 15 MHz MSOM system can indeed interrogate the variability between tumour types and explore the relation between tumour type and heterogeneity in morphological and functional parameters such as vascularization and sO2 and less hypoxic than KPL4 or MDA-MB-231 tumours; nevertheless, as a future step, it would be interesting to investigate these findings in a larger tumour sample in the context of explaining the particularly aggressive growth and multi-site metastasis of 4T1 tumours48. Tumour physiological heterogeneity and, in particular, oxygenation parameters can only be resolved at resolution orders of magnitude lower using nuclear imaging techniques, which require radioactivity and thereby limit possibilities for longitudinal studies. The possibility of using high-resolution MSOM to analyse tumour heterogeneity may create new opportunities for studying tumour heterogeneity in a more detailed manner. Indeed, MSOM fulfils one set of proposed requirements49 for analysing three-dimensional variations in tumour properties: high spatial resolution (10–100 μm) and a unique contrast and feature set not available to other imaging modalities. The study suggests the potential of textural measures of spatial heterogeneity such as skewness and kurtosis for image-based analysis of breast cancer. Skewness describes tumour characteristics, and in our samples, more extensive angiogenesis (4T1) was associated with greater positive skew values than less extensive angiogenesis (MDA and KPL4). The kurtosis values in our samples were inversely proportional to noise. The contrast and variance in our samples correlated negatively with hypoxia but positively with angiogenesis, which can be seen in the sO2 difference between the tumour centre and rim and in the HbT values at the rim. Measures of heterogeneity such as kurtosis are employed in other imaging modalities, such as X-ray CT50, PET50 or MRI41, for assessing tumour malignancy or staging56, therapeutic response and efficacy57 and survival outcomes61. In this study, we employed these metrics for the first time in analysing optical contrast in high resolution, showcasing previously undisclosed optical and sO2 variability between the tumours studied. Compared to earlier work on tissue heterogeneity, such as spectrum analysis approaches64 and MSOT18, our work focused on providing a mesoscopic tool operating at a centre frequency of 15 MHz to offer in vivo three-dimensional functional images with resolutions of 38–92 μm throughout whole solid tumours. Therefore, MSOM allows us to observe fine details of cancer haemodynamic contrast that go beyond the intensity and frequency signals of optoacoustic images obtained at macroscopic resolutions.Our pilot study suggests that 4T1 may be less heterogeneous in sO2, lipids and melanin, as well as exogenous contrast based on nonspecific dyes and specific targeting agents65, at high resolution. Spectral unmixing allows the contributions of these various contrast agents to be separated, enabling the simultaneous imaging of different markers of health and disease. In the present work, we demonstrate the simultaneous imaging of vasculature and gold nanoparticles at the level of entire tumours. This raises intriguing possibilities for tumour analysis that aims to correlate, for example, oxygenation and the activity of specific subsets of tumour and host cells, such as tumour-associated macrophages, which can be tracked in vivo using specifically designed nanoparticles66. Perfusion of gold nanoparticles into tumours could be accessed longitudinally in a non-invasive manner at a level of intra-tumoural detail not previously achieved with other tomographic set-ups67 and with a whole-tumour imaging volume inaccessible to intravital microscopy. This finding highlights the potential of MSOM for preclinical screening and assessment of anticancer therapeutics in vivo or in organoids. The technique’s combination of penetration depth, resolution, whole-tumour imaging, and non-invasiveness may give it advantages over intravital microscopy as a key tool in the drug discovery process69, such as for clarifying why nanoparticle-based cancer treatment often fails to improve therapeutic outcomes70.Uniquely among high-resolution imaging techniques, MSOM can visualize tissue features based on endogenous contrast from Hb, HbOFuture efforts to improve the current MSOM system should focus on reducing measurement time using lasers with high repetition rates and fast wavelength scanning. Future work will introduce more image analysis methods to improve the quantitative performance of MSOM in tumour heterogeneity analysis.Overall, MSOM is shown to image entire tumour morphology and physiology in vivo in a label-free, non-invasive manner at depths and resolutions never before demonstrated with an optical or optoacoustic method. By using a transducer array at a 15 MHz central frequency and applying frequency equalization, our method allowed unprecedented cross-sectional imaging and achieved resolutions never before achieved in whole-tumour imaging by an optical contrast imaging method. As a result, we were able to quantify patterns of heterogeneity at the entire-tumour level and differentially quantify patterns of heterogeneity within parts of the tumour, such as the core and the rim. With its demonstrated abilities and the potential for hardware and software improvement, MSOM may prove to be a uniquely powerful preclinical technology on the road towards precision oncology, which aims to characterize tumour heterogeneity in order to select the most appropriate therapies and optimize prognosis.Foxn1 nude mice  were injected into the mammary fat pad with 4T1 murine breast cancer cells  or KPL4 human breast cancer cells71 . NOD/SCIDSHrN nude mice  were injected into the mammary fat pad with MDA-MB-231 human breast cancer cells . Each type of cancer cell was injected into three animals under isoflurane anaesthesia. When tumours reached a diameter of ~8 mm, the animals were anaesthetized and imaged using MSOM. This tumour size was reached within 7–10 days in 4T1-injected animals, within approximately 40 days in KPL4-injected animals, and in 30–40 days in MDA-MB-231-injected animals. At this time, a mouse carrying a 4T1 tumour was injected intravenously with 50 μl of gold nanoparticles  suspended in 200 μl of phosphate-buffered saline (PBS) and then imaged in vivo.Animal procedures were approved by the Government of Upper Bavaria. Female athymic 73. Single motorized stages for rotation  and translation  allow scanning in discrete or continuous modes31. In the present study, in vivo imaging was performed in continuous scanning mode with an angle range of 255° and a translation range of 10 mm. Samples were excited optically using an optical parametric oscillator laser  tunable from 690 to 900 nm and capable of providing sub-10 ns pulses at a 10 Hz repetition rate and maximum pulse energy of 80 mJ at 760 nm. The laser beam was delivered to the sample using a custom-made bundle of 672 fibres  divided into four arms, each of which measured 6.5 × 2.5 mm and was positioned on a different side of the sample. This setup allowed homogeneous illumination of a volume of approximately 14 × 14 × 6 mm. Laser pulse energies were selected based on sample size and were always between 30 and 50 mJ to remain below the ANSI-defined limit for biological samples. The average laser power was measured before each experiment at each wavelength using a Vega power metre  to allow light fluence correction.Figure xy plane. The holder was attached to a linear stage  to allow adjustment of the holder position along the z axis. The anaesthetized mouse was placed on top of the holder such that the tumour protruded through a hole and into a water bath held at 33 °C. The fibre bundle heads and transducer array were also immersed in the water bath. This bath provided acoustic coupling between the tumour and the transducer array. Snap rings with the same outer diameter and different inner diameters were fitted onto the hole according to tumour size in order to optimize sample placement and minimize the generation of acoustic signals from neighbouring tissues.A sample holder was custom-designed to accommodate an entire mouse as well as the four heads of the fibre bundle, which were positioned at an angle of 8° from the The ultrasound response from the sample was detected using a custom-made linear transducer array  with 96 elements, a 15 MHz central frequency and an average −6 dB pulse-echo bandwidth of 45%. Each element has a pitch of 100 μm, an elevation of 1.5 mm and a cylindrically focused feature with a focal length of 7.8 mm. The array is mounted at 45° on a custom-made tilting module connected to the translation-rotation scanner, allowing conical scanning around the tumour sample. The radius of the central plane traced by the central (48th) element in the array was 5.8 mm. The linear array rotated and translated continuously at speeds chosen to provide optimal sample coverage at the 10 Hz repetition rate of the laser. Between two successive laser pulses, the array rotated by ~0.035° and translated ~200 nm.The MSOM system was used to visualize a phantom consisting of black polyethylene microspheres with a diameter of 20 μm  uniformly dispersed in agar gel cylinders with a diameter of 12 mm. These absorbers generate broadband optoacoustic signals with a frequency band wider than the maximum bandwidth of the linear array for the given laser pulse duration. The data collected with this phantom were used to calibrate the MSOM set-up, to measure the average speed of sound and to determine the spatial resolution of the system.Approximately 7000 × 96 optoacoustic signals were acquired at each wavelength and digitized using a custom-built data acquisition card  operating at 10 Hz and sampling at 125 MS/s with 12-bit resolution over a 16 mV range. The total scanning time per wavelength was 12 min.74 to compensate for frequency-dependent acoustic attenuation. Three-dimensional images were reconstructed using a backprojection algorithm75 with a voxel size of 24 × 24 × 24 μm. Reconstructed images were corrected for light fluence as previously described76. Frequency equalization was performed by reconstructing optoacoustic images in two frequency bands of 1–7.5 MHz and 4–28 MHz, and the image features from each band were coloured differently and overlaid using ImageJ77. Finally, linear spectral unmixing78 was used to generate separate reconstructions of the distribution of endogenous or exogenous contrast agents. The unmixing algorithm assumed a constant background spectrum and absorption spectra of Hb, HbO2, and gold nanoparticles  that are in arbitrary units and relative across different structures in the reconstructed images. The sO2 values are relative rather than absolute, as they are calculated as ratios from the Hb and HbO2 values.Ultrasound data collected after excitation at five wavelengths were used for tumour image reconstruction Fig. . Raw datles Fig. . Unmixed37. Coarseness, contrast and directionality were calculated using Tamura texture analysis36. The centre and rim regions of tumours were partitioned using cut-off points identified from the lines connecting the centre point and boundary points. Relative differences were calculated as the ratio between a metric’s absolute difference between the centre and rim to the metric’s sum for the centre and rim.The presented metrics were calculated by averaging the metric values in MIPs of 400 μm sections at different tumour depths throughout the entire tumour. Variance, skewness, kurtosis, energy and entropy were calculated as previously described2 or gold nanoparticles. Then, the norm of the residuals relative to the norm of the signal was displayed as an image to identify the relative uncertainty associated with different parts of the tumour image  were prepared using standard procedures . For haematoxylin-eosin staining, sections were thawed, dried on a heating plate, fixed in paraformaldehyde and stained with haematoxylin (30 s) and eosin (1 s) using standard procedures. For immunohistochemistry, sections were dried on a heating plate, fixed in cold acetone for 10 min, and then rinsed in Tris-buffered saline containing 0.3% Triton X-100 (TBST). Slices were blocked for 1 h at room temperature with TBST containing 10% goat serum. The blocking solution was removed, and sections were immunostained overnight at 4 °C with a rat primary antibody against mouse CD31  to show the blood vessel distribution. Sections were washed three times (10 min each) with TBST, incubated for 1 h at room temperature with an Alexa Fluor 594-conjugated goat anti-rat secondary antibody , and then washed again three times (10 min each) with TBST. Alternatively, sections were immunostained against HIF-1α as described above except using a rabbit primary antibody against mouse HIF-1α  and an Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody . All antibodies were diluted in TBST containing 1% goat serum. Sections were mounted using ProLong Gold Antifade Reagent with DAPI  and analysed using a Zeiss Axio Imager M2 microscope and Zeiss ZEN 2 Pro (Blue edition) software.81. Tumour sections were prepared and immunostained as described above using the mouse primary monoclonal antibody supplied in the Hypoxyprobe Kit and an Alexa Fluor 594-conjugated goat anti-mouse secondary antibody .In some experiments, animals were injected intravenously with pimonidazole-HCl (80 mg/kg) from the Hypoxyprobe Kit  and sacrificed 1 h later. Pimonidazole distributes to all tissues and forms adducts with thiol-containing proteins in cells where the oxygen partial pressure is below approximately 10 mmHg at 37 °CProfiles in Fig. 35. Hamming distance can be used to measure the differences between two hash values within the range from 0 to 64 and then identify whether two images are perceptually different or similar. The maximum Hamming distance of 32 in Fig. Based on an image’s visual appearance, a discrete cosine transform (DCT)-based image hashing algorithm can produce a hash value for each image stored in an unsigned 64-bit integerSupplementary Information