However, dermatologists achieved a slightly higher specificity (88.9% vs. 83.3%). (5) Conclusions: For the first time, a deep neural network was demonstrated to be capable of diagnosing PG, solely on the basis of photographs, and with a greater sensitivity compared to that of dermatologists.(1) Background: Pyoderma gangrenosum (PG) is often situated on the lower legs, and the differentiation from conventional leg ulcers (LU) is a challenging task due to the lack of clear clinical diagnostic criteria. Because of the different therapy concepts, misdiagnosis or delayed diagnosis bears a great risk for patients. (2) Objective: to develop a deep convolutional neural network (CNN) capable of analysing wound photographs to facilitate the PG diagnosis for health professionals. (3) Methods: A CNN was trained with 422 expert-selected pictures of PG and LU. In a man vs. machine contest, 33 pictures of PG and 36 pictures of LU were presented for diagnosis to 18 dermatologists at two maximum care hospitals and to the CNN. The results were statistically evaluated in terms of sensitivity, specificity and accuracy for the CNN and for dermatologists with different experience levels. (4) Results: The CNN achieved a sensitivity of 97%  84.2–99.9%) and outperformed dermatologists, with a sensitivity of 72.7% (CI 54.4–86.7%) significantly ( Pyoderma gangrenosum (PG) is a rare but serious autoinflammatory neutrophilic dermatosis , showingThe disease typically starts with a sterile pustule  that rapidly progresses, causing necrotising ulceration of variable depth and size with undermined violaceous wound borders. Most frequently, it occurs on the lower extremities, but other parts of the skin may also be affected. PG may be associated with either an inflammatory disease, such as inflammatory bowel disease or rheumatoid arthritis, or neoplastic diseases, such as haematological malignancy or solid tumours. Furthermore, retrospective cohort studies described a close association with paraproteinemia . AccordiTreatment relies on immunosuppressive drugs, with the aim of slowing down the activation of neutrophilic granulocytes. In fact, the only approved treatment is the oral administration of corticosteroids . Beyond In addition, efficient treatment with mycophenolate mofetil , azathioMore recently, the so-called PARACELSUS-Score was developed to standardize diagnosis ,19. The Nonetheless, failed or delayed diagnosis is common ,21 and rIn this paper, we present a deep neural network designed to support physicians and wound experts in diagnosing PG based on photographs of the wound. The application of deep learning techniques in healthcare has recently been a strong focus of computer vision researchers . It has Our dataset comprised 491 photographs of PG and conventional leg ulcers (most of which were due to proven venous insufficiency). The dataset was nearly balanced between the two diseases (PG:LU = 244:247). The patients were treated in the Klinikum Nuremberg and the Klinikum Stuttgart, both being large tertial referral centres for Dermatology between 2004 and 2021. The PG diagnosis was made in each case by the most experienced dermatologists (ES and PVDD), using the criteria of 1997, and all cases were additionally confirmed by the use of the recently described PARACELSUS-score [Out of this dataset, we randomly selected a balanced training dataset of 422 pictures (“dataset-422”) for the training of the convolutional neural network (CNN), and we put aside the remaining 69 pictures  for final CNN validation and comparison with the dermatologists’ classification performances.In order to measure classification performance, we applied the following performance measures  represent the numbers of correct/incorrect PG diagnoses, and TN/FN  represent the numbers of correct/incorrect ulcus cruris (UC) diagnoses):Furthermore, since the CNN yields probabilities for the conditions of PG and ulcus cruris (UC), and the decision threshold (cut-off) for one or the other diagnosis (50% throughout this work) can be manually varied, it is common to report the area under the curve (AUC) of the receiver operating characteristic  .Given the relatively small dataset, the implementation of a binary classification CNN based on transfer learning is the common approach, and has recently been successfully implemented in neural networks for melanoma classification ,30,31,32Prior to the input into the neural network, all original megapixel images were resampled to 300 × 300 pixel resolution (nearest neighbour interpolation), i.e., the image aspect ratio was changed to 1:1.In order to achieve optimal performance, we followed the deep learning good practice and performed an exhaustive hyperparameter grid search, including nested k-fold cross-validations. The tested hyperparameters comprised e.g., the visual network type , the classifier network size, dropout rates, learning rate, and optimizer. For the exhaustive search, each of the 972 possible configurations was evaluated in a 3fold stratified cross-validation procedure, i.e., the training set of 422 pictures was randomly split into three balanced subsets of 140 pictures; then, the configuration was trained on any combination of two subsets of 280 pictures and evaluated on the remaining third subset. The average performance score ACC and AUC of the three runs yield realistic, non-optimistic estimates of a configuration’s performance on unseen data. The best performing model from a grid of 972 configurations was finally trained on the full dataset-422 and evaluated on the validation set.To account for the relatively small number of training images, we applied the established technique of data augmentation, which multiplies the dataset by creating copies with arbitrary rotations, namely horizontal or vertical flips of images, without affecting the diagnosis. By doing so, the network learns that photographic perspective has no impact on the diagnosis and input information is spread across the entire visual input field. Throughout this work, we applied an augmentation factor of 4 .The optimal network’s performance was finally validated with the 69 unseen images from the validation set and the performance measures described above. In parallel, we compared the A.I.’s performance with the visual diagnoses made by the dermatologists on the same dataset, in an online survey.The optimal network performance was obtained with an architecture, which uses VGG16 visual processing, a 64-, 32-, and 2-neuron fully connected classification output see . When teFor the final assessment of the AI’s performance, we conducted an anonymous online survey, in which 18 dermatologists and dermatological trainees of the Klinikum Nuremberg and the Klinikum Stuttgart centres were asked to make purely visual judgements of the 69 images in the validation set, without getting any further clinical information on the cases given. The participants were initially informed about the number of PG and UC cases included. Images presented to the physicians were the original images  adjusted to the browser window size. Numerical results are summarized in p < 0.03), the experts’ sensitivity of 72.7% , and the juniors’ sensitivity of 45.5% . Differences in UC diagnosis (specificity) are not significant: with CNN: 83.3% (67.2–93.6%); all 88.9% (73.9–96.9%); experts: 88.9% (73.9–96.9%); and juniors: 83.3% (67.2–93.6%). For clarity, we have grouped the participants into groups of professional experience: (1) all dermatologists, (2) experts with >3 years of experience and (3) juniors with ≤3 years of experience. Within each group, the majority determined the group’s final diagnosis for the image under test. The CNN achieves a sensitivity of 97% (CI: 84.2–99.9%) and surpass-es all dermatologists’ sensitivity of 72.7% (CI: 54.4–86.7%) significantly . Graphical representation of the man–machine comparison on the receiver operating curve (ROC) are shown in p < 0.001).The CNN achieves an accuracy of 89.9% (80.2–95.8%), slightly better than all dermatologists and the experts with 81.2% (69.9–89.6%), and significantly better than juniors with 65.2%  , there aThe aim of the present study was to develop a CNN to facilitate the diagnosis of PG for primary care physicians and wound managers, who are not routinely treating patients with this autoinflammatory disease. We made the first step towards this goal by developing a convolutional neural network (CNN), trained with 422 carefully validated images. The network showed a significantly higher sensitivity to diagnosing PG than dermatologists did (sensitivity 97% versus 72.7%), whereas the specificity of dermatologists was slightly better than that of the CNN (88.9% versus 83.3%). However, it must be mentioned that the diagnosis of PG does not only rely on the clinical image, as this may vary during the course of the disease, but often needs additional information, such as a search for associated diseases, histopathology, and laboratory examinations.In an anonymous man vs. machine comparison, we demonstrated that sensitivity is clearly a matter of professional experience, as expected see . The autOur CNN tends to overestimate PG probability (expressed by the lower specificity); this is a welcome property, because the goal is to prevent the oversight of PG and to trigger further diagnostic procedures in dubious cases.Before initiating the inclusion of the A.I. in a routine clinical decision making process , furtherIn addition, tools for visual plausibility checks must be developed and evaluated, to provide physicians with sufficient comprehensive information to accept or reject the machine’s proposal. As shown in the previous section and in Even though the PARACELSUS score  in part The comparison of A.I. versus human experts’ visual diagnoses may be considered unfair, because in clinical practice, wound experts would consider additional non-visual information prior to making a diagnosis. Nonetheless, our results demonstrate that human experience greatly improves visual diagnosis and a visual AI tool may offer valuable, supplementary assistance for differentiating PG from leg ulcers.Due to the rarity of PG, we were limited to a relatively small number of training images and validation images, compared to melanoma/nevi CNN developments ,30,31,32


Dear Editor,Paracoccidioidomycosis is the main systemic mycosis in Latin America.The clinical classification of the disease comprehends a chronic form that occurs in adults, responsible for 80% of cases, and the acute/subacute juvenile form, representing 20% of the cases.The authors report the case of an adult woman with a severe and atypical form of the disease, living in an urban area where several cases were described related to local environmental changes secondary to intense and prolonged soil movement during the construction of a metropolitan area highway that crosses urban centers in the state of Rio de Janeiro.A 61-year-old female patient, housewife, born and residing in the urban area of the municipality of Nova Iguaçu, state of Rio de Janeiro, Brazil, presented with jaundice associated with abdominal pain for five months. She developed ascites, with weight loss and an increased number of lesions. She denied recent travel, comorbidities, smoking and alcohol consumption.On physical examination, she had jaundice, a distended abdomen, hepatosplenomegaly with the right hepatic lobe located at three cm from the costal margin and a palpable spleen between the costal margin and the umbilicus. There was no palpable lymphadenopathy in the superficial chains. Dermatological examination showed multiple erythematous papules on the face, trunk, and limbs. In the cervical region, the papules were violaceous, with slight desquamation .Figure 13 µL; aspartate aminotransferase 90 U/L; alanine aminotransferase 52 U/L; alkaline phosphatase 2,563 U/L; gamma-glutamyl transferase 233 U/L; total bilirubin 12.76 mg/dL; direct bilirubin 9.22 mg/dL; indirect bilirubin 3.54 mg/dL; albumin 2.5 g/dL; globulin 3.5 g/dL; prothrombin index 49%; activated partial thromboplastin time 44.2 seconds. Serological tests for hepatitis B and C, syphilis, and HIV were negative.The laboratory tests showed anemia, jaundice with a cholestatic pattern and a negative immunodiffusion test for paracoccidioidomycosis. The results of the tests of interest were as follows: hemoglobin 7.8 g/dL; hematocrit 23%; leukocyte count 11,900 μL ; platelets 700 × 10Computed tomography of the abdomen and chest showed ectasia of the intrahepatic bile ducts, pancreas with heterogeneous volume and density, bilateral pleural effusion and hepatosplenomegaly, in addition to several enlarged intra-abdominal lymph nodes .Figure 3The main diagnostic hypotheses were hepatobiliary neoplasia, primary biliary cholangitis associated with lichen planus, and paracoccidioidomycosis.Biopsies of the skin, liver and an intra-abdominal lymph node were performed. Histopathological examination of the skin disclosed chronic granulomatous inflammation, with a large number of neutrophils and multinucleated giant cells containing rounded structures in the cytoplasm. The Grocott staining showed spherical structures stained in brown, with multiple budding cells with a “marine pilot’s wheel” and “Mickey Mouse ears” appearance .Figure 4Histopathological examination of the liver and lymph node biopsy revealed chronic granulomatous inflammation with necrosis and the presence of fungal structures.Histopathology confirmed the diagnosis of acute/subacute paracoccidioidomycosis and treatment with intravenous amphotericin B deoxycholate 1 mg/kg/day was started. However, the patient developed pancreatitis and acute renal failure, with the following laboratory tests: amylase 470 U/L; lipase 864 U/L; urea 118.8 mg/dL; creatinine 1.9 mg/dL. Therefore, the therapeutic regimen was modified to liposomal amphotericin B 5 mg/kg/day associated with meropenem 1 g IV 8/8 h and nutritional support measures, administration of intravenous fluids and amines. Blood cultures were performed and, although they did not show microorganisms, the patient developed a clinical picture compatible with refractory septic shock, due to pancreatic necrosis and died. An autopsy was not performed.Among the mycoses, paracoccidioidomycosis is the main cause of death in immunocompetent patients. In Brazil, from 1998 to 2006, the disease was responsible for 50% of hospitalizations due to mycoses, with in-hospital mortality of 5%.Although the disease is rare in urban areas, this study describes the increase in the disease incidence after deforestation and the massive removal of land for the construction of a highway in the metropolitan area that crosses urban centers in the state of Rio de Janeiro. Before the highway construction, there were 1.29 cases for every million inhabitants in the region. After that, there was an increase to 8.25 cases per million inhabitants.Regarding the diagnostic hypotheses, it should be emphasized that, initially, the possibility of hepatocarcinoma was investigated, but liver, skin and lymph node biopsies showed the presence of paracoccidioidomycosis, correlating with the skin lesions and the clinical picture. However, before that primary biliary cirrhosis associated with lichen planus was included among the hypotheses, due to the lichenoid aspect of the lesions and the reports of the association of liver disease with this dermatosis.7In this case, the presence of jaundice was noteworthy, due to the severe hepatobiliary involvement, which is one of the systems most affected by the disease.The main causes of jaundice related to paracoccidioidomycosis are biliary obstruction due to lymph node enlargement, pancreatitis, intraluminal granulomatous lesion, and hepatitis. It should be emphasized that the first two above-mentioned causes were identified in this patient. In general, obstructive jaundice occurs when there is extrinsic compression of the common biliary duct caused by lymph node enlargement near the hepatic hilum. Pancreatitis, on the other hand, results from pancreatic duct drainage obstruction by regional ganglion compression.In conclusion, in endemic areas, it is important to consider paracoccidioidomycosis among the diagnostic hypotheses for jaundice and other signs and symptoms of cholangitis.None declared.Fernanda Altoé Stringuini: Design and planning of the study; drafting and editing of the manuscript; collection, analysis, and interpretation of data; effective participation in research orientation; intellectual participation in the propaedeutic and/or therapeutic conduct of the studied cases; critical review of the literature.Priscila Oliveira Naback: Collection, analysis, and interpretation of data; intellectual participation in the propaedeutic and/or therapeutic conduct of the studied cases.Luciana Ferreira Araújo: Approval of the final version of the manuscript; collection, analysis, and interpretation of data; effective participation in research orientation; intellectual participation in the propaedeutic and/or therapeutic conduct of the studied cases; critical review of the manuscript.Ricardo Barbosa Lima: Approval of the final version of the manuscript; design and planning of the study; collection, analysis, and interpretation of data; effective participation in research orientation; critical review of the literature; critical review of the manuscript.Carlos José Martins: Approval of the final version of the manuscript; design and planning of the study; effective participation in research orientation; critical review of the manuscript.None declared.


Peyronie’s disease (PD) is a fibrotic disorder of the penile tunica albuginea. To date, only a few cases of recovery from PD following medical treatment have been reported in the literature. In this article, we describe three new cases of PD where patients achieved complete resorption of plaque following multimodal antioxidant treatment. In all three cases, treatment included the following antioxidants: bilberry, propolis, ginkgo biloba, silymarin, and vitamin E. Only in case nos. 1 and 2 did we also use the following antioxidant substances: L-carnitine, coenzyme Q10, and Boswellia. In all three cases, we also used a local therapy with diclofenac gel. Only in case no. 2 did we also use periodic perilesional injections with pentoxifylline. Although the sample of cases presented here was small, these patients incontrovertibly experienced complete plaque disappearance and recovery . Therefore, it is our conviction that urologists may find our experiences of considerable interest in their clinical practices. Peyronie’s disease (PD) affects adult men and is characterized by chronic inflammation of the tunica albuginea of the penile corpora cavernosa. Its hallmark is the formation of fibrous plaque, which may even undergo calcification and result in the deformation of the penis. PD symptoms are as follows: penile deformity , pain in the penis 20–70%), erectile dysfunction (approximately 30%), and anxiety/depression (48%) ,2,3.0–70%, erPD prevalence ranges from 3.2 to 13% ,5,6. AltDisease onset occurs as blood and fibrin accumulate at the site of the trauma, inducing the recruitment of inflammatory cells and excessive production of fibrogenic factors and reactive oxygen species ,12. OxidSurgical intervention involves the treatment of choice when PD has reached its stable phase and in cases presenting with severe erectile dysfunction and/or severe penile curvature preventing complete sexual intercourse, while medical therapy is indicated instead when PD is in its initial, inflammatory phase. Medical therapy comprises oral treatment with Potaba, colchicine, tamoxifen, vitamin E, phosphodiesterase-5 inhibitors, antioxidant substances, penile injections with anti-fibrotic and anti-inflammatory substances, antioxidants , as well as physical treatments  ,16,17.We have already published articles reporting on the full recovery of patients following antioxidant treatment.There was no photographic documentation in previous case reports/articles because the patients did not authorize the publication of photographs of their penis.We present here three new cases of PD where patients experienced complete plaque disappearance following multimodal antioxidant therapy at our PD Care Center.For two of these cases, photographs before and after treatment are included. Furthermore, unlike the previous articles, in case no. 3, we obtained complete regression of the disease in just four months.A 31-year-old non-smoking Caucasian man suffering from chronic prostatitis and an associated anxious–depressive state presented to our clinic in March 2019. He complained of painful erections and a penile bend that commenced approximately nine months before. The patient was asked to rate his pain using a visual analog score (VAS); his VAS was 8 (range 0–10). He was then asked to complete the International Index of Erectile Function (IIEF) questionnaire.We considered questions 1, 2, 3, 4, 5, and 15 regarding erectile function . The patient’s score was 26. The patient’s penis presented both a 10-degree ventral curvature and a lateral curvature with a 15-degree bend to the left  was used to take three-dimensional measurements of the plaque volume ,19.3)  , Table 1When gathering the patient’s informed consent, the patient was forewarned that the treatment would be lengthy, considering the disease’s chronicity. Fearing pain, the patient refused to receive penile infiltrations. The patient began treatment in March 2019 after granting his informed consent. Treatment comprised of the following: oral propolis 600 mg + bilberry 160 mg + silymarin 400 mg + ginkgo biloba 250 mg + L-carnitine 1000 mg + coenzyme Q10 100 mg + Boswellia 200 mg + vitamin E 30 mg/daily; diclofenac gel 4%/2× per day for 12 months.3)  had not varied. We then observed a reduction to 5 degrees in the lateral left penile curvature angle, and we no longer observed any ventral penile curvature (previously present with a 10-degree angle). Erections were no longer painful. Dimensions on the ultrasound were as follows: 9.0 × 6.97 × 2.6 mm (volume = 86 mm3) .The plaque volume was, therefore, 75.6% smaller than before treatment. In view of the good results achieved after the first cycle, a second identical 12-month treatment cycle was agreed on with the patient.3)  .The plaque’s volume had, therefore, decreased by 92.6%. In view of the excellent results after the second cycle, the decision was made to continue treatment, with no changes, for only six more months.At follow-up, after three full treatment cycles corresponding to an overall duration of therapy of two years and six months, the patient had the same IIEF score. Penile palpation did not detect any nodule and the two original curvatures were observed to have disappeared .The same physician performed the initial ultrasound scan as well as all follow-up scans, always employing the same Philips Affinity 70 G device .Antioxidant treatment was consequently suspended. Following treatment, the patient reported no side effects.A 52-year-old Caucasian man, non-smoker, with Dupuytren’s disease and Ledderhose disease, who had a 5-degree congenital dorsal penile curvature before PD onset, presented to our clinic in February 2015; the patient did not complain of any pain in the penis. At the time of our observation, the penis presented both a dorsal curvature, with a 20-degree angle, and a lateral left curvature of 20 degrees. The patient was, therefore, asked to fill in the IIEF questionnaire on erectile function, which yielded a score of 26. Upon physical examination, a fibrous nodule, approximately 25 mm long, was palpable at the middle and distal third of the penile shaft. After inducing erection with the injection of Alprostadil 10 mcg, a penile Doppler scan was performed.Cavernous artery flow and end-diastolic velocity were normal: peak systolic velocity = 70 cm/s on the right and 74 cm/s on the left; end-diastolic velocity = 0 cm/s on both sides.3); the second plaque, located at the distal third, measured 14.8 × 8.43 × 3.87 mm (volume = 252 mm3)   , Table 1When gathering the patient’s informed consent, the patient was forewarned that the treatment would be lengthy, considering the disease’s chronicity. The patient did not authorize the publication, however anonymous, of photographs of his penis. In March 2015, the patient provided his informed consent and began the following combined treatment: oral propolis 600 mg + bilberry 160 mg + silymarin 400 mg + ginkgo biloba 250 mg + L-carnitine 1000 mg + coenzyme Q10 100 mg + Boswellia 200 mg + vitamin E 30 mg/daily; topical diclofenac gel 4%/2× per day; pentoxifylline 100 mg periplaque injection (with a 30G needle) every two weeks for six months. In order to minimize trauma to the penile tissues, penile injections with pentoxifylline were performed in a perilesional fashion (and not inside the plaque). For the same reason, a very thin needle (30 G) was used.Having ended his first therapy cycle, the patient was followed up with the same examination process as previously. His IIEF score had risen to 27. A dorsal penile curvature could still be observed, but its angle was reduced to 15 degrees; we no longer observed any lateral penile curvature.3); the second plaque, located at the distal third, measured 12.7 × 7.73 × 2.93 mm (volume = 151 mm3)   .The total volume of the two plaques was 34.5% smaller compared to the initial total volume. Since the first cycle of the multimodal therapy had yielded a good response, the decision was made to continue the same oral and topical treatments, with the same agents and doses, for a further 12 months, while reducing pentoxifylline’s 100 mg injection frequency to one penile injection every month for 12 months.The patient then underwent follow-up with the IIEF questionnaire, physical exam, and penile Doppler ultrasound after the second cycle of treatment. His IIEF score was 27. We observed that the dorsal penile curvature was unchanged (15 degrees); we did not observe any lateral penile curvature.3); the second plaque, located at the distal third, measured 11.2 × 6.84 × 2.72 mm (volume = 109 mm3)   .The total size of the two plaques was 58.9% smaller compared to the original dimensions. In consideration of the good response achieved by the second treatment cycle, the decision was made to continue the same treatment for a third cycle, with the same agents and doses, while reducing pentoxifylline’s 100 mg injection frequency to one penile periplaque injection every other month.After completion of the third treatment cycle, the patient underwent a follow-up consisting of a physical exam and penile Doppler ultrasound. At the follow-up, the patient’s IIEF score was 27. We observed that the dorsal penile curvature reduced to 10 degrees.3) (The penile ultrasonography detected the presence of only one plaque (middle third), whereas the distal plaque was no longer detectable. Plaque dimensions on the ultrasound were 3.69 × 3.74 × 1.9 mm (volume = 14 mm3) .Since the previous examinations, the ultrasound device  in our clinic had been replaced with a new device , which was used in the latter and subsequent follow-ups. The same doctor performed all sonographic examinations.Upon completion of three treatment cycles, the total plaque volume was 96.7% smaller compared to the initial total volume of the two plaques. In view of the excellent response to the third multimodal treatment cycle, we elected to cease the periplaque injections with pentoxifylline and extend the oral and topical treatments .After four full treatment cycles, for an overall course of multimodal therapy lasting approximately four years and three months, the patient underwent follow-up with the IIEF questionnaire, physical exam, and penile Doppler ultrasound. No change was observed in the IIEF score, which was again 27. The angle of the dorsal penile curvature decreased to 5 degrees, a measure comparable to the condition of the patient before PD onset . No nodule could be detected on palpation. Plaque was no longer visible on the ultrasonography , Table 1Following treatment, the patient reported no side effects. The same physician performed all ultrasonography studies.A 30-year-old Caucasian man, non-smoker, with left varicocele, presented to our clinic in September 2014 complaining of pain in the penis with erections, with onset approximately one week earlier. The patient reported penile sexual trauma four weeks before. At the time of our observation, no penile deformity was present. The VAS score was 5. The IIEF score was 26. No nodules were found on penile palpation. Following the physical examination, a penile Doppler ultrasound was performed. After the erection was induced with 10 mcg of the intracavernous injection of Alprostadil, the erect penis showed no deformation ; peak systolic velocity = 84.9 cm/s on the left and 77.3 cm/s on the right.3)  , Table 1When gathering the patient’s informed consent, the patient was forewarned that the treatment would be lengthy, considering the chronicity of the disease.After receiving the patient’s informed consent, on 30 September 2014, we began the following combination therapy with oral antioxidants and topical NSAIDs: bilberry 160 mg + propolis 600 mg + silymarin 400 mg + vitamin E 800 IU + ginkgo biloba 250 mg/per day + diclofenac gel (4%) twice daily for six months.Respecting the patient’s decision (the patient was very anxious), follow-up was performed only four months after the beginning of treatment. No change was observed in the IIEF score, which was still 26. Erections were no longer painful. No penile deformation was observed .Although antioxidants are not included in current AUA and EAU guidelines for PD treatment, in the literature, there are three randomized studies describing the use of antioxidant substances in patients suffering from PD, as well as a number of controlled studies where antioxidants were used in association, with excellent benefits (plaque reduction and an improvement in penile deformity) ,25,26,27The European Association of Urology (EAU) guidelines and American Urological Association (AUA) guidelines strongly recommend infiltrative therapy with interferon alpha-2b or with collagenase clostridium histolyticum CCH ,27.We have never used interferon alpha-2b because of its high cost and the high possibility of side effects, such as diarrhea, dizziness, flu-like symptoms, nausea, fatigue, vomiting, etc. We have also never used CCH in treatment because its indications are Peyronie’s disease in the “stabilization phase”, and our PD patients are all in the active phase of the disease. It should be added that, in Italy, the drug Xiapex (CCH) (from 1 January 2020) was withdrawn from the market by the Italian Medicines Agency (AIFA).In recent years, we adopted the combination of antioxidants presented in this case report article. Our multimodal (or combined) treatment consisted of administering several therapeutic agents in various ways  and sometimes in association with iontophoresis. Multimodal therapy further improved outcomes compared with the results that may be achieved while employing a single active substance or therapy.Initially, many years ago, we only used propolis, bilberry, and then also pentoxifylline, but in this way, we only achieved a partial response. Therefore, we decided to progressively increase the number of substances, recently obtaining better results up to the complete regression of the plaque. We decided on the dosages for each substance while trying to respect the therapeutic dosage to avoid side effects.In this regard, we consulted other studies that had used some of the same substances. We have not used pentoxifylline orally for several years because side effects have previously occurred  (see our study) . We, theRecently, we published three case report articles, where we presented eight patients  with PD whose plaque disappeared completely after multimodal antioxidant treatment ,29,30. TPrior to our articles, the disappearance of PD plaque had already been described in an experimental study on rats .In the first two cases reported here, the period of time needed for our treatment was lengthy; the duration of treatment is due to the fact that, as PD is a “chronic” inflammatory disease, it takes time to achieve full plaque resorption. The necessary length of treatment is also linked to the size of the diseased area.It is likely that the excellent result achieved in the third case, with a markedly shorter treatment time compared with the other two cases, can be mainly ascribed to the fact that diagnosis was made extremely early in the disease (four weeks after trauma). In this case, it was possible to have the patient undergo a shorter cycle of treatment, which did not require periodic penile injections, as the plaque was limited in size, clearly because the disease was in an extremely early phase.In the last case presented, the ultrasound examination (performed with the latest generation machine) clearly detected a plaque with an ultrasound appearance typical of the first phase of the disease, i.e., hypoechoic. Furthermore, after four months of treatment, the plaque was no longer visible in the ultrasound examination. The deformation was absent because the plaque was newly formed; therefore, it was not yet fibrotic and was unable to cause a deformation in the form of curvature. We know from the literature that curvature is not always present during PD (present in 87–94% of cases) ,3,14.Although the sample of cases presented here is small, we believe all urologists may find our experience of great interest for their clinical practice.In the period 2014–2018, we treated 45 PD patients with similar multimodal treatment. Of these patients, 22 successfully completed the treatment until complete plaque regression (after a variable period between 28 and 53 months).In total, 18 of these patients also underwent periodic penile injections with pentoxifylline. The remaining 23 patients discontinued treatment after approximately 13–24 months despite the reduction in plaque volume and the improvement in the curve over time. These 23 patients felt that the treatment was too long. We believe that when the plaque volume is large, it takes some years to achieve complete regression of the disease (chronic inflammation).In our opinion, the success achieved by our therapy rests in great part on the properties of the antioxidants we employed ,13,14.The antioxidant substances we used also inhibit the NF-kB factor, a transcription factor for the production of pro-inflammatory and pro-fibrotic cytokines ,36,37,38When our treatment plan includes injection therapy, we gradually increase the time interval between each pentoxifylline injection over the course of treatment. The reason behind this is that even simple peri-lesional injections implicate trauma, be it ever so mild, and as trauma is beyond any doubt the trigger for the onset of PD, it should be avoided as much as possible ,8,9. TheWhereas ultrasonography in the literature is generally deemed inadequate for acceptable measurements of plaque, in our experience, plaque dimensions can be accurately obtained if an experienced physician performs the scans using a highly sensitive, cutting-edge ultrasound machine ,41,42. TThe limitations of this study are the limited number of patients and the relative absence of a control group.Although the sample of cases presented here is small, these patients incontrovertibly experienced the complete disappearance of plaque in the area affected by the disease, in one case after only a brief course of treatment.We attribute our success fundamentally to the following three factors: the choice of antioxidants used; the performance of ultrasonography with an extremely sensitive ultrasound machine by which we obtained precise plaque measurements; and our entrusting the ultrasound scan to a physician with extensive experience with PD cases.Therefore, it is our conviction that specialists may find our report, however limited, of great interest for their urology and andrology practice.Still, there is a need for randomized controlled studies enrolling a larger number of patients in order to prove the effectiveness of this multimodal antioxidant treatment for patients suffering from PD.


White rice is a staple food for Japanese, a population at high risk for colorectal cancer (CRC). We investigated the association between white rice intake and CRC among Japanese Americans in the Multiethnic Cohort (MEC) study.The MEC study is a prospective study established in Hawaii and California in 1993–1996. Usual dietary intake was assessed using a validated quantitative food frequency questionnaire at baseline. Cox proportional hazards models were used to compute hazard ratios (HRs) and 95% confidence intervals (CIs) for quartiles of intake and to perform trend tests across sex-specific quartiles with adjustment for relevant confounders.trendP = 0.11) or women (trendP = 0.56). After excluding participants with a history of diabetes, the inverse associations were significant for CRC  and tumors of the distal colon  among men but not women.We identified 1,553 invasive CRC cases among 49,136 Japanese Americans  during a mean follow-up of 19 years. White rice consumption was not associated with overall CRC incidence in men (White rice consumption was not associated with an increased risk of overall CRC among Japanese Americans. An inverse association was observed with risk of CRC and distal colon cancer in men without a history of diabetes. Rice  is a major energy source for Japanese Americans.6 Lifestyle factors, such as obesity,7,8 physical inactivity,9,10 smoking,11,12 alcohol consumption,13,14 and dietary patterns low in dietary fiber,15,16 calcium and vitamin D,17,18 and folate19,20 and rich in red meat21,22 may contribute to the rising risk for CRC across populations. In particular, Japanese Americans have experienced a very high CRC incidence and are at increased CRC risk compared to Caucasians as demonstrated in the Multiethnic Cohort (MEC) Study .23 However, the causes for this increased risk are not fully understood.Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer death in men and women in the United States.24 In contrast, a case-control study in Fukuoka, Japan, revealed a reduced risk of CRC with rice intake.25 Previous studies on rice consumption and CRC have not distinguished white rice from other rice, such as brown rice. Therefore, the current analysis aimed to specifically investigate the association of white rice consumption with CRC risk among the Japanese American population of the MEC. We hypothesized that white rice consumption may significantly increase the risk of incident CRC in men and women because white rice increases risk for T2DM, which is a known risk factor for CRC.The evidence for an association of white rice consumption with CRC has been limited. For instance, no significant relationship between rice consumption and CRC was observed in a Japanese cohort study with 11 years of follow-up.26 and was approved by the Institutional Review Boards of the University of Hawaii and the University of Southern California.The MEC is a prospective cohort study established from 1993–1996 to examine lifestyle factors, in particular diet, related to cancer and other chronic diseases. In brief, a multiethnic population was recruited consisting of more than 215,000 adults aged 45–75 years residing in Hawaii and California. The participants completed a mailed baseline questionnaire on demographics and lifestyle, including diet. The study design has been described previouslyn = 56,806). Participants who had CRC prior to cohort entry reported in the baseline questionnaire or by linkage to tumor registries (see below) (n = 823), implausible diets based on total energy intake  or its components  , or missing information for continuous covariates  were excluded from the current analysis. Missing information for categorical covariates was kept as a dummy variable. As a result, a total of 49,136 participants  were analyzed in the present report.The current study consisted of the Japanese Americans enrolled in the MEC (https://www.uhcancercenter.org/for-researchers/mec-questionnaires), which was developed based on 3-day food records and validated against 24-hour recalls collected from random samples of each ethnic group.27 In the validation study, the average correlation coefficients between nutrient densities from the QFFQ and the 24-hour recalls among Japanese were 0.66 for men and 0.61 for women.Dietary intake was assessed at baseline using a self-administered quantitative food frequency questionnaire (QFFQ) with more than 180 food items . The serving size was measured in three portion sizes, which were small (1/2 cup or 1 scoop or less), medium (1 rice bowl or 1 cup or 1 musubi), and large (2 rice bowls or 2 musubi or more). Daily white rice intake (g/day) was transformed into energy density . Intake of other foods, such as brown rice and meat, was estimated using the same method. Micronutrients were computed by converting grams per day of each food to amounts of the dietary component by applying a study-specific food composition table and summing across items.Incident cases of invasive adenocarcinoma of the colon and rectum were identified through linkage with the Surveillance, Epidemiology, and End Results Program (SEER) state tumor registries in Hawaii and California. Case ascertainment was complete through December 31, 2013. Cases were limited to invasive adenocarcinoma with other types of CRC cases censored at the date of diagnosis. According to the International Classification of Disease (ICD) codes, the anatomical subsites were categorized as follows: C18.0–C18.9, C19, and C20 for CRC, C18.0–C18.5 for proximal colon tumors, C18.6–C18.7 for distal colon tumors, and C19 and C20 for rectal cancer. Tumors overlapping subsites (C18.8–C18.9) were excluded in the subsite-specific analyses. Deaths and causes of death were identified through linkage to the death certificate files in Hawaii and California and the National Death Index. The end of follow-up was determined as the earliest of these events: a diagnosis of CRC, death, or closure date of December 31, 2013.2), cigarette smoking (pack-years), alcohol consumption (g/day), moderate to vigorous physical activity (hours/day), and intakes of total energy , red meat , processed red meat , dietary fiber , calcium from food and supplement , folate from food and supplement , and vitamin D from food and supplement . Moderate to vigorous physical activity (hours/day) was derived from self-reported time spent in these activities per week and was found to provide reasonable agreement with estimates of energy expenditure from doubly labelled water.28 Intakes of magnesium , vitamin B6 , vitamin B12 , n-3 PUFA , and sodium  were considered as additional covariates; however, the results were unchanged by their adjustment and are not shown.The models included covariates from the baseline questionnaire: indicator variables for history of diabetes (yes or no), family history of CRC (yes or no), history of polyps of the intestines (yes or no), education , nonsteroidal anti-inflammatory drugs (NSAID) use , multivitamin use (yes or no), and hormone replacement therapy in women ; and continuous variables for age (years), body mass index , with the cases at other subsites considered as censored at time of diagnosis. The analysis was repeated with participants limited to those without a history of T2DM at baseline since participants with T2DM may have modified their diet as the results of their T2DM diagnosis; in particular, they may have been asked by their physicians to avoid eating white rice.29 All statistical tests were two-sided, and the significance level was 0.05. SAS software, version 9.4 was used .White rice consumption was measured as energy density  and divided into sex-specific quartiles. Characteristics of participants are shown according to quartiles of white rice intake. Cox proportional hazards models of CRC were performed to compute adjusted hazard ratios (HRs) and 95% CIs. Separate models for men and women were fit, and age was used as the time metric. In adjusted models, covariates were selected a priori based on previous studies.We identified 1,553 incident cases of CRC after a mean of 19 years of follow-up: 867 cases among 23,595 men and 666 cases among 25,541 women  of 50.9, 109.5, 166.9, and 261.2 in men, respectively. The corresponding values in women were 36.2, 89.3, 133.5, and 219.0 g/1,000 kcal/day. The Japanese men with a high intake of white rice were more likely to be younger at cohort entry and less educated, to report more pack-years of cigarette smoking, a lower alcohol consumption, and to be less likely to use NSAID and multivitamins. As to diet, men in higher quartiles of white rice intake tended to have a lower intake of total energy, energy from protein and fat, brown rice, red meat, dietary fiber, sodium, calcium, folate, and vitamin D. Similarly, women with higher white rice consumption had less education, a greater pack-years of cigarette smoking, lower alcohol consumption, and less NSAID and multivitamin use. Hormone replacement therapy was not significantly different by group. Also, lower intakes of total energy, energy from protein and fat, brown rice, dietary fiber, calcium, folate, and vitamin D were observed in women who consumed more white rice. The prevalence of T2DM at baseline among the participants was 12% in men and 8.9% in women. Among men, those in the lowest quartile of rice intake had the highest prevalence of T2DM (14%), while in women, those in the highest quartile had the highest prevalence (9.7%).trendP = 0.11) and women (trendP = 0.56). In terms of specific anatomical subsites of CRC in men, an inverse association in quartile 3 for distal colon cancer was significant  but no evidence for trend (trendP = 0.057). Similarly, no significant trends were observed in men for proximal colon cancer (trendP = 0.28), and rectal cancer (trendP = 0.74), and in women for proximal colon cancer (trendP = 0.54), distal colon cancer (trendP = 0.87) and rectal cancer (trendP = 0.44).Table n = 44,064, 90% of the total population; 20,792 men and 23,272 women). The age-truncated incidence of CRC was 217 per 100,000 person-years in men and 143 per 100,000 person-years in women. A trend for CRC in men was significant  but not in women . In subsite analysis, a significant reduction in risk was observed for distal colon carcinomas in men: quartile 2 , quartile 3 , and quartile 4  with a significant trend (trendP = 0.006). No significant HRs or trends were observed in women.As shown in Table This study is the first population-based study investigating the association of white rice consumption with CRC among Japanese Americans. This analysis in a large cohort with a long follow-up revealed that white rice consumption was not associated with an increased risk for overall CRC in men and women. However, after excluding participants who had a history of T2DM at baseline, an inverse association was observed for white rice with risk of overall CRC and distal colon cancer among men only.24,25 A large cohort study in Japan reported no association between rice consumption, predominantly white rice, and CRC.24 In that study, the trend tests for overall CRC were not significant in men  or women . In contrast, a case-control study in Fukuoka25 observed a significant inverse association of rice consumption across quintiles with CRC  among men and women who were analyzed in the same model. In a subsite analysis, the P for trend was 0.61 for proximal colon, 0.08 for distal colon, and 0.06 for rectum. The studies didn’t show the results stratified by diabetes. Overall, these results suggest that white rice is not associated with CRC risk in Japanese population.The overall null association of white rice consumption with risk for CRC in MEC appears to be consistent with previous studies in Japanese.30 After excluding participants with a history of T2DM, white rice showed a significant inverse trend for distal colon cancer in men. A study in Japan showed that a Western dietary pattern was associated with distal CRC risk only in men.31 There was also a strong positive association between the ratio of fat/carbohydrate consumption as an energy source and risk of distal colon cancer. The distal colon may be more susceptible to the carcinogenic effect of diet than the proximal colon and rectum. In our study, the participants without a history of T2DM in quartile 1 for rice had the highest median for a ratio of fat/carbohydrate consumption (0.62 in men and 0.54 in women), in contrast to those in quartile 4 (0.39 in men and 0.41 in women). This ratio was much lower in the Japanese study, with a range of 0.10–0.20. Taken together, the participants who consume less white rice were more likely to consume protein and fat, such as red meat and processed meat, which could increase CRC risk. Furthermore, a study investigating diet quality in relation to CRC incidence in the MEC study has shown that all four diet quality scores were inversely associated with cancer risk for the distal colon and rectum but not for the proximal colon, even in Japanese men.32 These results support the idea that tumors of the distal colon may be more strongly related to diet among Japanese American men without a history of T2DM.In the MEC, the prevalence of T2DM was high (12% in men and 9.8% in women), reflecting the prevalence of this disease in the Japanese American population in the United States.1 Homemade Japanese foods consist of white rice, miso soup, grilled fish, and pickles, which may improve general health among the Japanese population.33 Japanese Americans have generally adopted a more American diet, but older individuals, as represented in the current study, still consume a significant amount of traditional foods.34 Inflammation is considered a hallmark of CRC risk, as evidenced by NSAIDs reducing CRC risk, including among Japanese American men, by lowering inflammation in the large bowels.35 White rice and fish could minimize inflammatory reactions by replacing Western foods, such as processed foods and red meat, which potentially increases inflammation in the distal colon.36–38 In our study, the participants consumed fewer percent of energy from white rice (19% in men and 16% in women) than in Japan (29%).5 Taken together, the Japanese Americans who consumed white rice may be more likely to consume homemade Japanese foods and fewer pro-inflammatory western foods.The biological plausibility for an association between white rice and a reduced risk of distal colon cancer remains unclear. There are two possible explanations related to either an indirect or direct mechanism of protection against CRC. First, white rice intake could be a proxy for the traditional Japanese diet, as consumed in Japan before the introduction of western foods. Japanese immigrants who consume more white rice are more likely to consume a more traditional Japanese diet.39 Starch is a main nutritional component of white rice and could have biological functions similar to dietary fiber after being converted into resistant starch. Although risk reduction from starch remains controversial,40 some studies report an inverse relation with CRC.41 In vivo, starch may improve the microbiome and total stool composition and stool volume to promote shifts in microbial diversity that may reduce inflammation by controlling COX-2, NF-kB, TNF-a, and IL-1b.42Second, starch may directly ameliorate the gut microbiome in favor of a profile that may be protective against CRC.The present study had several strengths, such as a population-based prospective design with a large number of participants and identification of cancer cases through high-quality tumor registries. Refined rice was distinguished from brown rice, and results were adjusted for a set of comprehensive covariates of CRC risk. The long follow-up allowed for the identification of a substantial number of cases. The limitations of the study include the exclusion of participants (13.5%) who had missing data, which may have introduced selection bias. White rice consumption was measured using a self-reported questionnaire, a possible source of information bias. However, misclassification due to measurement error is more likely to be non-differential, biasing the HRs toward the null.In conclusion, this prospective analysis in the MEC suggests that, overall, white rice consumption is not associated with an increased incidence of CRC among Japanese Americans men and women. After excluding the participants with a history of T2DM at baseline, a significant inverse association was identified for overall CRC and distal colon cancer in men but not in women. This analysis may support the hypothesis that a diet with a large proportion of energy from white rice may be protective against CRC in men without a history of T2DM. However, it is not clear which component of a white rice-based diet, such as a high intake of resistant starch or a low intake of red meat, or other related lifestyle factors are responsible for this observation and why this benefit does not extend to women.


Nymphaea L. (water lily) is the most diverse genus in the family Nymphaeaceae, with more than 50 species worldwide, including 11 species distributed in Africa. The complex and variable morphology of Nymphaea makes it extremely difficult to accurately identify species based on morphological characteristics alone. DNA barcoding has the potential to identify species accurately. In this study, 158 Nymphaea populations from seven African countries were collected for species identification by ITS, trnT-trnF and rpl16. Additionally, the three candidate DNA barcodes were evaluated for genetic distance and barcoding gap. Based on the comprehensive analysis of sequence similarity, genetic distance method and phylogenetic tree, a total of 137 populations of seven Nymphaea species from African were well-identified, including N. lotus, N. petersiana, N. zenkeri, N. nouchali var. caerulea, N. micrantha and N. guineensis. ITS has more obvious advantages over trnT-trnF, rpl16 and trnT-trnF+rpl16 in the intraspecific and interspecific variation differences and barcoding gap and can identify most species. trnT-trnF and rpl16 can identify some species that cannot be identified by ITS. The results showed that it is more appropriate to apply the combination of ITS and trnT-trnF (or rpl16) as the DNA barcoding of Nymphaea. Additionally, this study further enriches the DNA barcoding database of Nymphaea and provides a reference basis for studying taxonomy, phylogenetics and evolutionary origin of Nymphaea.The genus  Nymphaea L.) is the most species-rich, phenotypically diverse and geographically widespread genus in the order Nymphaeaceae, with about 50 species worldwide [Nymphaea into five subgenera in his monograph, including subg. Anecphya, Brachyceras, Hydrocallis, Lotos and Nymphaea. These five subgenera were also supported by molecular evidence and are now generally accepted [Nymphaea were well-resolved, interspecific relationships within these taxa remain uncertain [Nymphaea was published by Conard [Nymphaea are still ongoing. A quarter of the currently accepted Nymphaea species were completed more than 40 years ago, and the status of many species is unknown, especially subg. Brachyceras and Lotos [Nymphaea species makes their correct identification problematic, and even the morphological characteristics of the same species are diverse [Nymphaea increased the difficulty of classical classification [Nymphaea species using morphology alone is difficult, and additional evidence is needed to address the taxonomic complexity of the genus.The water lily , a total of 11 Nymphaea species are distributed in Africa. However, this is only based on morphological records and lacks the support of molecular data. Therefore, it is necessary to carry out the DNA barcoding study to help the taxonomic identification of African Nymphaea species. Currently, studies have applied the ribosomal DNA (nrDNA) ITS and the chloroplast DNA (cpDNA) trnT-trnF to the phylogenetic analysis of the genus Nymphaea and suggested that ITS and trnT-trnF could be used as potential barcodes for the identification of Nymphaea species [trnT-trnF and rpl16) for a DNA barcoding analysis of African Nymphaea species. The present study aimed to identify African Nymphaea species using the three DNA barcodes and assess the ability of the three candidate DNA barcodes to identify African Nymphaea species.Two subgenera,  species ,21. In tNymphaea. The sequences obtained were submitted to the GenBank database, and the accession numbers are shown in trnT-trnF and rpl16 after sequence alignment were 731 bp, 1507 bp and 766 bp, respectively, and the number of parsimony information sites were 196, 70 and 15, respectively. The GC content of the ITS region was 51.8%, which was much higher than that of the two cpDNA sequences. Moreover, the number of variation sites (212) and variation rate (29.73%) of ITS was the highest, followed by trnT-trnF . Therefore, ITS sequences were considered ideal DNA barcoding for Nymphaea plants. The sequence characteristics of three DNA barcodes of the above Nymphaea are shown in The PCR amplification and sequencing success rates of the three candidate barcodes were 98.1–100% and 96.2–100%, respectively, indicating that these primers had good versatility in trnT-trnF with the sequences of the GenBank database by BLAST. The similarity of ITS was above 98.7%, except for the similarity (97.7%) of No. 733, 743 and 761, and the species identified by ITS and trnT-trnF belonged to the subg. Brachyceras and Lotos . No. 714, DID, HOH and KES were inconsistent in the subgenera identified by ITS and trnT-trnF, and it is possible that the samples were contaminated. Based on a BLAST search, 64 samples identified by ITS, and 50 samples identified by trnT-trnF were consistent with morphological classification. Additionally, 109 samples were identified based on ITS and trnT-trnF together, of which 63 unknown species were identified.We found that some species may have morphological identification errors through homology matching and analysis. For example, No. KISW, MSLK and MTR were identified as subg. Nymphaea species were divided into two groups (asap-score = 2.0) by the ASAP analysis of ITS, corresponding to Brachyceras and Lotos, and the species could not be successfully identified  and three groups, respectively, which also could not distinguish species well  did not show a great advantage over the single sequence  in trnT-trnF provided sufficient information for species identification [Nymphaea species based on trnT-trnF, with an identification efficiency of 83%. However, the efficiency of identifying Nymphaea species using trnT-trnF based on the BLAST search or constructing a phylogenetic tree was far less than 83% in this study. In contrast, we found that, in Borsch’s study, most of the samples were one to two per species, and the inadequate sampling resulted in an apparently high resolution. Additionally, the phylogenetic tree based on trnT-trnF in this study could only identify N. petersiana and N. nouchali but could not identify most species of the subg. Brachyceras, which was consistent with the results of previous studies [rpl16 and the combined sequence (trnT-trnF+rpl16) were basically consistent with that of trnT-trnF in this study.Since the advent of universal primers for the taxonomy . HoweverF region ,25. The fication . Borsch  studies . In additrnT-trnF, but the number of variation sites was close to twice that of trnT-trnF. Additionally, the variation rate of ITS was as high as 29.73%. Additionally, compared with cpDNA sequences, ITS had the largest intraspecific and interspecific genetic distances, with less overlap between the intraspecific and interspecific variants and an obvious barcoding gap. These characteristics indicated that ITS could be used as a DNA barcode for Nymphaea. However, ITS could identify some species of N. zenkeri, N. guineensis, N. micrantha and N. nouchali var. caerulea by the phylogenetic tree, and a considerable number of species could not be identified, while trnT-trnF and rpl16 could identify some species that could not be identified by ITS. Therefore, it is more appropriate to consider the joint application of ITS as the main DNA barcode of Nymphaea, complemented by two chloroplast sequences (trnT-trnF and rpl16).Previous studies have found that the evolutionary rate of ITS is three to four times higher than that of the cpDNA regions . Thus, tNymphaea was based on the ASAP analysis and cannot accurately identify species. It is possible that the interspecific genetic variants of the subgenera of Nymphaea were too low. Therefore, this method is not applicable to the identification of species with small interspecific genetic variants. In this study, we also tried a new method (ASAP) for species identification, which divides species partitions based on the hierarchical clustering of DNA sequence pairwise genetic distances . HowevertrnT-trnF, rpl16 and trnT-trnF+rpl16 cannot completely distinguish the African Nymphaea species. Therefore, there is an urgent need to develop new DNA barcoding or methods to apply to the species identification of African Nymphaea species. Hollingsworth et al. [Panax [Taxus [Rhododendron [Nymphaea and applying next-generation sequencing technologies to the identification of Nymphaea species are the next research directions.According to the above analysis, ITS, h et al.  proposed. [Panax , Taxus [x [Taxus  and Rhododendron , and theNymphaea populations from seven African countries were sampled in 2018 to 2019  according to the manufacturer’s protocol. DNA concentration was checked using the Nanodrop spectrophotometer , after which it was stored at −20 °C. Then, ITS, L spacer ,36, and  spacers . The seqrps4-5R-trnL110R and primers c–f) for 1 min and 72 °C for 90 s, a final extension step at 72 °C for 10 min and termination by a final hold at 4 °C. The final PCR products were sequenced using an ABI 3730XL automated sequencer .PCR amplification was carried out in a 25-μL reaction mixture containing 20 ng of template DNA, 2.5 μL of 10 × Buffer (pH 8.3), 0.5 μL dNTPs, 0.5 μL of each primer, 1 unit of Taq polymerase and DNA-free water. The PCR protocols involved initial denaturation at 94 °C for 5 min, followed by 35 cycles at 94 °C for 40 s, primer-specific annealing temperature  Each edited sequence was submitted to the GenBank database . Homology searches were performed using BLAST  to confirm the identity of the sequences. Due to the few rpl16 sequences of Nymphaea species in the GenBank database, we only performed a homology search of the obtained ITS and trnT-trnF sequences with the GenBank database. When the highest similarity between query sequences and matched sequences was over 98%, and ITS and trnT-trnF were jointly matched to the same species, the identification of the species was considered successful. (ii) In addition, a total of 51 sequences were downloaded from the GenBank database, including 45 sequences of Nymphaea species, 3 sequences of Euryale ferox and 3 sequences of Victoria cruziana  with default parameters, except for the model set to the Kimura 2 parameter (K2P). (iii) Phylogenetic analyses of the three barcodes were performed using neighbor-joining (NJ) and Bayesian Inference (BI) phylogeny. The NJ tree was constructed using MEGA7 software based on the K2P model with the bootstrap set to 1000. The best-fit model of the nucleotide substitution was determined by ModelFinder [The raw sequences were spliced, cut and edited using DNAMAN v.9.0 . In this study, African cruziana . All seqelFinder  with AkaelFinder , runningThe intraspecific and interspecific genetic distances of the species were calculated based on the K2P model using MEGA7. To estimate the barcoding gap, the minimum interspecific and maximum intraspecific distances were compared for each barcode . Then, w


One’s working environment can be a risk factor for suicide. In this study, we examined whether suicidal ideation can be predicted using individual characteristics, emotional states, and working environments. (2) Methods: Nine years of data from the Korean National Health and Nutrition Survey were used. A total of 12,816 data points were analyzed, and 23 variables were selected. The random forest technique was used to predict suicidal thoughts. (3) Results: When suicidal ideation cases were predicted using all of the independent variables, 98.9% of cases were predicted, and 97.4% could be predicted using only work-related conditions. (4) Conclusions: It was confirmed that suicide risk could be predicted efficiently when machine learning techniques were applied using variables such as working environments. Adult workers spend most of their days working. Work is good for one’s mental health, but a poor work environment can cause physical and mental health problems . When a The working environment refers to all factors that affect work activities, including various managerial factors, human relationships, and physical conditions related to job performance, as well as the physical environment in terms of job performance. In other words, the working environment can be divided into the physical environment, including the type of employment and type of business, the labor structure, which includes the number of employees, working hours, and shift work, and the socio-psychological environment, which is affected by the characteristics of the work being performed .It was found that the longer the working hours, the higher was the risk of depression and suicidal ideation ,4,5. In In addition, irregular working environment factors, such as overtime and irregular working hours, can cause changes in an individual’s physiological rhythm and threaten their health . In the Job characteristics include job control and autonomy, and it is known that exposure to excessive noise, sunlight, and specific substances  can affect the work environment . It has Although these characteristics can be used to evaluate the risk of suicide in a worker group, there have been no studies on the overall risk of several factors. Suicide does not occur due to a single cause; rather it occurs as a result of the complex contribution of several factors such as biological vulnerability, psychological state, and clinical characteristics .Although the early identification and treatment of high-risk patients is a key suicide prevention strategy , high-riTo date, no studies have applied machine learning techniques to predict suicide risk in workers. Therefore, in this study, the demographic characteristics, medical history, and personal lifestyle differences of a group with suicidal thoughts were first examined. Their emotional status, stress levels, subjective health status, and depression factors were then examined to determine whether there were differences. In addition, we aimed to determine how work characteristics such as occupational classification, working hours, and work type affect suicidal ideation. This study aimed to investigate whether the suicide risk of workers could be efficiently predicted using machine learning techniques based on working environments and emotional factors.This study used data from the Korean National Health and Nutrition Examination Survey (KHANES). Data from a total of 9 years  were used . In 2014Occupation type: (1) managers, experts, and related workers; (2) office workers; (3) service and sales workers; (4) skilled workers in agriculture, forestry, and fisheries; (5) technicians, equipment operators, machine assembly and operation workers; (6) simple labor workers.Occupational status: (1) full-time; (2) temporary; (3) day worker.Working hour system: (1) full-time worker; (2) part-time worker.Type of shiftwork: (1) day work; (2) evening shift (14:00~24:00); (3) night shift (21:00–08:00); (4) regular day and night shift work; (5) 24 h shift work, split work (more than two working hours per day), irregular shift work, and other forms of shift work.Working hours: average working hours per week.Classification of regular and nonregular workers, which are thought to have an impact on suicidal ideation, were excluded as the survey was conducted only at 13, 15, and 17 years.Occupations were classified according to the major classification codes of Korea’s standard occupational classification system. Among the KHANES data, the work-related variables commonly investigated in the 9 years of data were as follows:This study evaluated the suitability of the suicidal ideation prediction model for wage workers by selecting 23 analyzable variables, presented in Among machine learning algorithms, random forest (RF) is a method for finding optimal results through randomly combining results and constructing several decision trees. Random forest is widely used due to its good predictive performance, especially in mental health-related studies ,22. TherThe subjects were analyzed by dividing the training and test data by 70% and 30%, respectively. Repeated 10-fold cross-validation was used to develop the analysis model. In the 10-fold cross validation, the data is divided into 10 folds of the same size, and the remaining 9 folds are used for training while each fold is used as a validation set. This is repeated five times to avoid overfitting and to generalize the prediction model. To find the optimal parameter of the RF, the analysis was conducted with the grid search method and the optimized hyperparameter of the RF was applied to the final model. The fitted model was used to predict the suicidal ideation of wage workers and it was compared with actual suicidal ideation using a confusion matrix with the training data. The performance of the predictive model was evaluated by calculating the accuracy, sensitivity, specificity, positive predictive value, negative predictive value, and area under the curve. Area under the curve (AUC) means the area under the roc curve drawn with a false positive rate as the x-axis and true positive rate as the y-axis. An AUC value of 0.5 indicated no discriminative value. An AUC value ≥0.75 is considered clinically useful . R versip < 0.001). The lower the household income (p < 0.001) and the lower the education level, the higher the rate of suicidal ideation (p < 0.001). Subjects in the suicidal ideation group were divorced (7.3%), separated, or widowed (9.1%), and the ratios of these factors were higher than those in the nonsuicidal group. Marital status was also significantly related to suicidal ideation (p < 0.001). Hypertension (p = 0.165), stroke (p = 0.469), cancer (p = 0.626), and diabetes (p = 0.994) were not associated with suicidal ideation, but hyperlipidemia , myocardial infarction, and angina pectoris  were, and the rates of these in the suicidal ideation group was significantly higher than that in the group without suicidal ideation. In the suicidal ideation group, the proportion (8.4%) of the group who drank more than four times a week was higher than that of the group without suicidal thoughts , and smoking (p = 0.049) was significantly associated with suicidal ideation. In the suicidal ideation group, the suicidal ideation rate was significantly higher in the group who felt extreme stress in their daily life . Bad (26.5%) or very bad (5.1%) subjective health status reports were also significantly higher in the suicidal group (p < 0.001). The rate of experiencing depressive symptoms lasting more than two weeks was overwhelmingly higher in the suicidal group (51.3%) compared to in the nonsuicidal group (7.1%) (p < 0.001), and the lower the EQ-5D score, the higher the rate of suicidal thoughts. This ratio was also significantly high (p < 0.001). BMI was not significantly associated with suicidal ideation (p = 0.395).A suicide-related questionnaire was administered to 12,816 subjects, and it included the question: “During the past year, have you ever felt that you were willing to die?” A total of 915 people answered yes (7.14% of the subjects). The proportion of women who had suicidal thoughts in the past year was 66.7%, which was higher than that of women who had not (47.3%) (p < 0.001). In the group with suicidal thoughts, the ratio of regular workers was lower and the ratio of temporary and daily workers was higher than in the group without suicidal thoughts (p < 0.001). The proportion of part-time workers was significantly higher in the suicidal ideation group (26.0%) than in the nonsuicidal group . In the suicidal ideation group, the proportion of shift workers was higher, as was the ratio of evening shift workers (9.8%) to night shift workers . Working hours per week were shorter in the group with suicidal thoughts (38.97 ± 17.65) than in the group without suicidal ideations . In the nonsuicidal group, the proportion of officials, experts, and related workers was highest at 25.7% , and for women, it was the highest among temporary workers (5.6%) .Work-related difficulties, such as excessive job demands, relationship conflicts, and emotional labor, can increase job stress, job satisfaction, emotional exhaustion, and negative health symptoms, such as depression ,36. HoweSuicide prevention programs in the workplace should be structured to prevent each of these stressors and enable the early detection and treatment of suicidal ideations . This stThe limitations of this study are as follows: First, only cross-sectional factors were used, and an overall evaluation of work environments was not performed. In situations where it is impossible to evaluate various working environments with a single evaluation index, it will be helpful to index the work environment by evaluating these environmental aspects through indicators that can be evaluated in the future. Second, only the cross-sectional area evaluation results were used. In the future, it will be necessary to confirm these findings via a cohort study of workers. Third, only suicidal ideation was evaluated. Suicidal behavior can be divided into suicidal thoughts, plans, and attempts. In the end, suicidal thoughts can lead to suicide plans and even suicide attempts, but it should be taken into account that not all suicidal thoughts lead to suicide attempts. Meanwhile, studies using machine learning or artificial intelligence to predict suicide have been conducted. However, these methods may be limited by the quality of the input data, and their clinical utility has not yet been established .The prediction of suicide using machine learning is mainly based on the results of suicide-related questionnaires or emotional evaluations ,38. HoweThis study attempted to predict suicide accidents among wage workers using variables from the National Health and Nutrition Examination Survey. When suicidal ideation cases were predicted using 23 work-, life-, and emotion-related variables with the random forest method, 98.9% were predicted more accurately (F1 score was 0.915), and 97.4% could be predicted using only work-related variables. It was confirmed that when the machine learning technique was applied to workers using working environment factors, suicide could be efficiently predicted. In the future, it will be necessary to identify risk factors using various variables and to make efforts to protect the emotional health of workers.


Background: Volatilomics is a powerful tool capable of providing novel biomarkers for medical diagnosis and therapy monitoring. The objective of this study is to identify potential volatile biomarkers of gastric cancer. Methods: The volatilomic signatures of gastric tissues obtained from two distinct populations were investigated using gas chromatography with mass spectrometric detection. Results: Amongst the volatiles emitted, nineteen showed differences in their headspace concentrations above the normal and cancer tissues in at least one population of patients. Headspace levels of seven compounds  were significantly higher above the cancer tissue, whereas eleven volatiles  were higher above the non-cancerous tissue. One compound, isoprene, exhibited contradictory alterations in both cohorts. Five compounds, pyridine, ethyl acetate, acetoin, 2,3-butanedione, and 3-methyl-1-butanol, showed consistent cancer-related changes in both populations. Conclusions: Pyridine is found to be the most promising biomarker candidate for detecting gastric cancer. The difference in the volatilomic signatures can be explained by cancer-related changes in the activity of certain enzymes, or pathways. The results of this study confirm that the chemical fingerprint formed by volatiles in gastric tissue is altered by gastric cancer. As a whole, they provide specific biochemical signatures that contain information on various normal and abnormal metabolic processes occurring in the body. Pathological processes such as oxidative stress, changes in enzyme activity, carbohydrate metabolism, lipid metabolism, modifications of proteins, or activation of genes alter these signatures [A number of recent studies have provided robust evidence that volatilomics is effective in identifying health issues and diagnosing diseases ,2. The tgnatures ,4,5. Volgnatures ,6,7 or bgnatures . TherefoGastric cancer is the fifth most diagnosed malignancy worldwide with over 1 million estimated new cases per year, and is the third most common cause of cancer death globally . Mortalibreathprints’) rather than identifying the volatiles and apply them in searching for a particular disease. Instruments based on these technologies have been demonstrated to discriminate gastric cancer patients from other patients and controls with a sensitivity and specificity ranging from 0.67 to 1.0 and from 0.71 to 0.98, respectively [Over the last decade, volatilomics has been employed to detect and classify gastric cancer using volatiles contained in human exhaled breath, yielding promising results ,11. Prinectively . A partiectively  reportinectively ,14,15.The ambition of this study is to identify the potential volatile biomarkers of gastric cancer. Specifically, this study centres upon the volatilomic signatures of gastric tissues to capture alterations in their metabolism induced by cancer. This embraces the identification of VOCs released by cancer and non-cancerous tissue and the identification of the volatile species being expressed at different levels in order to provide an alternative source of information on gastric cancer-related biomarkers. To improve the reliability of information, two distinct geographic populations of gastric cancer patients, namely, in Latvia and Ukraine, have been included in this study. The volatilomic signatures of tissue samples were investigated using the headspace solid phase microextraction (HS-SPME), followed by GC-MS.All standard mixtures were produced using high-purity liquid substances. Reference chemicals with stated purities of 95–99.9% were purchased from Merck (Austria) and Fluka (Switzerland). Standards were prepared by injecting and evaporating several microlitres of the liquid compound into evacuated and heated 1 L glass bulbs . Different calibration concentrations were obtained by transferring appropriate volumes from the bulb mixtures into Tedlar bags  that were pre-filled with purified and humidified air (relative humidity 100% at 34 °C). Ultimately, gas mixtures with VOC volume fractions ranging from 0.07 to 160 parts per billion by volume (ppb) were used for calibration. The calibration curves relied on using seven distinct and independent concentration levels.A cohort of 94 patients diagnosed with gastric cancer was recruited, all of whom were scheduled for elective gastric surgery. These patients were recruited in two clinical centres: the National Cancer Institute of Ukraine (UNCI) and the Riga East University Hospital, Latvian Oncology Centre (LOC). Details on the patient numbers, sex, and age range and median from these two centres are summarised in The tissue sampling, sample storage, and transport followed the standardized protocols outlined in detail elsewhere . Therefo−1) and crimped with 1.3 mm butyl/PTFE septa.Volatiles released by tissue samples were extracted using headspace solid phase microextraction (HS-SPME). Shortly before analysis, samples were thawed at 40 °C for 5 min and 100 mg (±10%) of a frozen tissue sample were transferred into a headspace vial . Next, the vial was rinsed with high purity nitrogen 99.9999% . Extraction was achieved by inserting a 75 μm Carboxen-PDMS SPME fibre  into the vials and exposing it to the headspace gas for 50 min. Subsequently, the fibre was introduced into the inlet of the gas chromatograph where the volatiles of interest were thermally desorbed at 290 °C in a splitless mode (1 min). The fibre was conditioned at 290 °C for 5 min prior to each analysis. In parallel with each pair of tissue samples, one blank sample containing nitrogen was analysed using the same protocol to identify possible contaminants stemming from sources other than the tissues. The resulting concentration levels were subtracted (where applicable) from the respective values in the associated tissue samples. Samples were analysed in batches on a daily randomized regime.−1. The column temperature programme was as follows: 37 °C for 12 min, followed by 5 °C min−1 up to 150 °C, then 10 °C min−1 up to 290 °C, and finally remaining at 290 °C for 8 min. The untargeted VOC analysis relied on the mass spectrometer working in a SCAN mode with the associated m/z ranging from 20 up to 250. The peak integration was based on extracted m/z ratio chromatograms, and such an approach allowed for a separation of the majority of peaks of interest from their neighbours. The quadrupole, ion source, and transfer line were kept at 150 °C, 230 °C and 280 °C, respectively.GC-MS analysis relied on an Agilent 8890/7079B GC-MS . The GC injector was equipped with an inert SPME liner  and operated in the splitless mode (0.75 min), followed by split mode with a ratio 1:50. Extracted compounds were separated using an Rxi-624Sil MS column  operated in constant helium flow at 1.4 mL minVOC identification was performed using a two-step process. First, the spectrum of a peak was checked against the NIST mass spectral library database. Next, the NIST identification was confirmed by comparing the retention times of peaks of interest with retention times obtained for reference standards prepared as outlined above.The validation parameters are provided in Collectively, a total of 235 compounds were found in the headspace of the tissue samples provided by both centres. Amongst these, 198 VOCs were identified in gastric cancer tissues and 199 in non-cancerous samples. A total of 165 volatiles were found in both types of samples. The majority of these species exhibited very low incidence. Only 32% of VOCs exhibited incidence higher than 20%. Excluding some obvious hospital environment-related species and their metabolites , ten volatiles  occurred in at least 80% of both types of samples and thereby can be considered as omnipresent. Moreover, pyridine and 1-propanol exhibited an incidence higher than 80% in tumour tissues, whereas in non-cancerous samples, this threshold was exceeded by acetoin and 2,3 butanedione. The distribution of VOCs with incidence above 20% according to their chemical classes is presented in It is not surprising that the distribution of the VOCs according to their chemical classes in both types of samples is similar. Each pair of tissue samples stems from the same individual and shares the same exposure to environmental, dietary, disease, or treatment-related VOCs, for examples. The predominant chemical classes in both tissues are hydrocarbons (17%) and alcohols (16%). These are followed by aldehydes , ketones (both 11%) and aromatics (both 10%). The signal of several compounds  exceeded the dynamic range of the MS detector. Consequently, it was not possible to compare the emission of these metabolites between respective tissue samples.Both populations under study exhibited some differences in the volatilomic signatures. For instance, in samples obtained from the Ukrainian cohort, 76 VOCs exhibited occurrence exceeding 20%, whereas in the Latvian group of patients, this number amounted to 58. Interestingly, there were differences in the distribution of compounds according to their chemical classes between both involved populations. Considering the cancer tissue, they were manifested mainly by the higher number of detected alcohols (15 vs. 7) and hydrocarbons (16 vs. 10), as depicted in p < 0.05 was taken as being significant. For this purpose, only compounds with incidence above 50% were taken into consideration. The list of compounds exhibiting a difference in emission between normal and cancerous tissues is presented in A Wilcoxon signed rank test was used to compare the emissions of VOCs from cancer and non-cancerous tissues, and a Amongst the volatiles detected, 19 showed consistent differences in their headspace concentrations above samples under study. Of these, 11 compounds were found to have reduced emissions from the cancer tissue, and hence the other eight showed reduced release from the healthy one. Four compounds, ethyl acetate, acetoin, 2,3-butanedione, and 3-methyl-1-butanol, exhibited a downregulated production in tumour tissue in both populations. Furthermore, three other volatiles, 2-pentanone, γ-butyrolactone, and DL-limonene, were found to be released in lower amounts only from cancer tissue obtained from Latvian patients. In comparison, four VOCs, benzaldehyde, 2-methyl-1-propanol, benzonitrile, and 3-methyl-butanal, showed analogous alteration only in Ukrainian subjects. The largest drop in the emission by tumour tissue was found for alcohols. Thus, for example, the production of 2-methyl-1-propanol in cancer tissue samples was reduced by a factor of 3.4, whereas 3-methyl-1-butanol exhibited 3 to 7.7 fold downregulated production in these type of samples.Both populations of interest also showed significant differences with respect to the cancer-related changes leading to the upregulation of VOC production. The species emitted in higher amounts by cancerous tissue comprised two aldehydes , two ketones (cyclohexanone and 2-nonanone), two heterocyclic VOCs (pyrrole and pyridine), and phenol. One compound, isoprene, exhibited contradictory alterations in emission in both cohorts. In the Latvian cohort, isoprene showed lowered emission from cancer tissue samples, whereas the opposite alteration took place in the Ukrainian population. Only one species, pyridine, exhibited elevated emission from cancer tissues as compared to the normal ones in both populations of patients. Four VOCs, pyrrole, phenol, cyclohexanol, and hexanal, were released in higher amounts by cancer tissues in the Latvian population only, whereas nonanal and 2-nonanol were found to be elevated in the headspace of the tumour samples obtained from the Ukrainian patients. The highest cancer-related change in the emission was noted for pyridine. The cancerous tissue exhibited 2.2 (Latvian patients) and 6.3 (Ukrainian patients) fold upregulated production of this VOC.An effort was made to quantify all compounds showing the differences in emission. Sixteen compounds were quantified using the aforementioned procedures. Three VOCs, benzaldehyde, acetoin, and phenol, were not quantified due to problems related to the preparation of reliable reference mixtures. Instead, their levels were assessed only based on peak areas. The median headspace concentrations ranged from 0.2 to 163 ppb. However, the majority of quantified species had emissions below 10 ppb. The highest median levels were noted for pyridine . The observed concentrations of volatiles of interest over the tissue samples are provided in The comparison of levels of VOCs showing consistent alterations in both populations of interest is shown in Although the underlying metabolic routes leading to the production of VOCs of interest are in many cases uncertain, a number of pathways could be responsible for their emission. Primary alcohols such as 2-methyl-1-butanol or 3-methyl-1-butanol may result from the activity of alcohol dehydrogenases (ADHs) that can reversibly reduce aldehydes to alcohols . Thus, 2Three ketones exhibited differences in the emission between the normal and cancer tissues. However, two of them (2-nonanone and cyclohexanone) showed upregulated production in cancer tissue, whereas the emission of 2-pentanone was found to be downregulated in this tissue. In principle, two metabolic pathways may be responsible for the production of ketones in humans: (i) oxidation of secondary or cyclic alcohols catalyzed by ADHs, or cytochrome p450 CYP2E1, and (ii) β-oxidation of fatty acids . The forWhen it comes to aldehydes, several mechanisms could be responsible for their generation. These include (i) metabolism of alcohols by ADHs, (ii) lipid peroxidation, or (iii) diet. Production of C3–C10 aldehydes, such as n-hexanal or n-nonanal, was demonstrated to be associated with the reduction of hydroperoxides by cytochrome P450 (CYP450) through the lipid oxidation of omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) . Thus, nInterestingly, both populations under study exhibited contradictory alterations in isoprene emission from cancer tissues. Isoprene is a terpenoid emitted by humans in large quantities . Accordiγ-Butyrolactone occurs naturally in humans. However, it is also the metabolite of fluoropyrimidine (UFT), which is used in the treatment of different cancers, including gastric cancer .+), nicotinamide adenine dinucleotide phosphate (NADP+), and their respective reduced forms (NAD(P)H). However, at this stage it is not clear what mechanisms underlie the upregulated emission of pyridine and pyrrole from cancer tissue. Perhaps these volatiles are the breakdown products of the aforementioned compounds. Further studies are required to elucidate the role of these VOCs in gastric cancer and reveal the underlying metabolic processes leading to their overproduction.Two heterocyclic compounds, pyrrole and pyridine, were found to be emitted in higher amounts by cancer tissue. Both VOCs are the components of numerous biologically active compounds. For instance, the pyrrole ring is a component of hemoglobin, myoglobin, and vitamin B12, and is produced during their biosynthesis . The pyrIn the context of this study, the most interesting are metabolites exhibiting (i) high emission rates from the tissue samples, and at the same time (ii) the most notable difference in the emission between normal and cancer tissue samples in both populations. Only five compounds showed consistent alterations in both populations of patients. Amongst them, four volatiles, ethyl acetate, acetoin, 2,3-butanedione, and 3-methyl-1-butanol, were characterized by a reduced production in cancer tissue. A further compound, pyridine, was found to be overproduced by cancer tissue. From these observations, pyridine appears to have the highest potential as a volatile biomarker for gastric cancer. High emission rates and the significant change in its production induced by cancer render this species a very interesting candidate for a gastric cancer probe. Furthermore, pyridine overproduction by cancer tissue was also reported in our pilot study which also focused on gastric tissues . Thus, tThe aim of this study was to characterize the volatile chemical patterns associated with cancer and normal tissues obtained from gastric cancer patients and identify potential volatile biomarkers of gastric cancer. The ex vivo GC-MS analysis resulted in the identification of 235 compounds, for which only 32% exhibited an incidence higher than 20%. Fourteen volatiles were found to be omnipresent (occurrence > 80%) in at least one type of tissue sample.Nineteen volatiles showed consistent differences in their headspace concentrations above the samples under study. Headspace levels of eight species were significantly higher above the cancer tissue, whereas eleven VOCs were found to be elevated in the head space above the non-cancerous tissue. The difference in the emissions of the aforementioned species may be explained by cancer-related changes in the activity of certain enzymes or pathways.Both populations investigated in this study also showed significant differences with respect to the cancer-related changes. Only one species, pyridine, exhibited elevated emission from cancer tissues in both populations of patients. Four species, pyrrole, phenol, cyclohexanol, and hexanal, were observed to be released in higher amounts by the cancer tissues only in the Latvian population, whereas nonanal and 2-nonanol showed analogous alteration only in the gastric tissues from the Ukrainian patients. When it comes to downregulated VOCs, four compounds, ethyl acetate, acetoin, 2,3-butanedione, and 3-methyl-1-butanol, exhibited this hallmark in both populations under study. Furthermore, seven volatiles, 2-pentanone, γ-butyrolactone, DL-limonene, benzaldehyde, 2-methyl-1-propanol, benzonitrile, and 3-methyl-butanal were found in only one group of subjects.The results obtained from this study imply that the human volatilome exhibits high interpopulation variability. This can be the reason for the disparities found between the volatile biomarkers of gastric cancer identified in different studies. In other words, clinical studies involving only one local population may provide only “local” biomarkers. It is not simple to explain the reason for this variability. However, local factors such as patients’ exposure to environmental VOCs, diet, or local microbiota may play a significant role. From this, we can state that the identification of “global” bioindicators is one of the major challenges in the use of volatilomics. However, the involvements of various cohorts of patients from different geographic locations could help to identify and overcome this problem.In the context of this study, volatiles showing similar cancer-related changes in both populations might be considered as candidate biomarkers for gastric cancer. From our results, this applies to five compounds, namely, pyridine, ethyl acetate, acetoin, 2,3-butanedione, and 3-methyl-1-butanol. Owing to the levels produced, pyridine appears to be the most promising of these compounds. Although the metabolic pathways leading to the production of pyridine in humans are unclear, the high emission rates and its overproduction by cancerous tissue render this species a very interesting candidate for further investigations in terms of its use as a probe for gastric cancer. These further studies are required to elucidate specifically the role of pyridine in gastric cancer and to reveal the underlying metabolic processes leading to its overproduction.The results obtained from this study confirm that the chemical fingerprint formed by volatiles in gastric tissue is definitely altered by gastric cancer. The volatile components of this fingerprint secreted from humans through breath could serve as biomarkers or probes of gastric cancer. They could lead the way to the development of non-invasive breath tests for the diagnosis of this disease. More specifically, the identification of volatiles associated with gastric cancer is an important step for guiding the design and optimisation of novel sensor technologies toward the robust and sensitive detection of disease-specific volatile markers.There are several limitations of this study. First, the available tissue samples were relatively small (∼100 mg), which must have affected the sensitivity of the applied analytical method, and thereby the recognition of species emitted in trace quantities. Secondly, the healthy tissue samples could be contaminated by the VOCs released by the malignant tissues, as the paired samples stemmed from the same stomach. Thus, the observed differences in emission may be diminished, affecting the identification of potential biomarkers.