Zopfiella erostrata strains were found to extensively colonize plant roots, forming a profuse melanized mycelium in the rhizosphere, which could be involved in improving water uptake and nutrient mineralization in plants. The aim of this study is to evaluate the effect of different strains of Z. erostrata on stress mitigation in wheat and tomato plants grown under water deficit conditions. General plant growth variables, as well as physiological and biochemical parameters, related to oxidative status were determined. Our data demonstrate that inoculation with both Zopfiella strains had a very significant effect on plant growth, even under water deficit conditions. However, we observed an even more pronounced impact, depending on the plant and strain involved, suggesting a certain degree of plant/strain compatibility. The biochemical aspects, the accumulation of proline, the oxidative damage to lipids, and the activity of antioxidant enzymes varied considerably depending on the endophyte and the plant evaluated.Drought is a major threat to plant growth in many parts of the world. During periods of drought, multiple aspects of plant physiology are negatively affected. For instance, water shortages induce osmotic imbalance, inhibit photosynthesis, decrease nutrient uptake, and increases the production of reactive oxygen species (ROS). In this context, it is necessary to develop sustainable strategies for crops that would help mitigate these conditions. In previous studies, endophytic  In agricultural environments, most crops are exposed to a diverse range of biotic and abiotic stresses, which profoundly affect their development and productivity, due to the inhibition of essential physiological and metabolic processes ,2,3. Dro55% of the total area of Argentina is made up of drylands , with temperature extremes and a variable moisture regime restricted mainly to the summer months . These rDry ecosystem plants harbor a wide variety of fungal endophytes, which grow in the root tissues of fungi without producing disease symptoms ,22,23. TInoculation with fungal endophytes helps increase the host plant’s tolerance to drought stress through various mechanisms ,31,32,33Zopfiella erostrata extensively colonized the roots of Eragrostis cilianensis (a desert grass), leading to the development of an abundant melanized mycelial network in the plant rhizosphere, which increases the water retention and nutrient mineralization of these plants [Z. erostrata strains on stress mitigation among wheat and tomato plants grown under water deficit conditions. Overall growth variables, as well as physiological and biochemical parameters related to oxidative status, were determined in inoculated and non-inoculated plants subjected to 14 days of water deficit conditions.In the Monte desert of La Rioja in Argentina, we observed that different strains of e plants ,51. In ae plants . HoweverZopfiella erostrata 1; (b) plants inoculated with the endophyte Zopfiella erostrata 2; and (c) non-inoculated control plants. Two watering regimens were applied to the plants: (1) well-watered conditions (100% water-holding capacity) throughout the experiment and (2) drought conditions (water application was reduced by 60–50%) during the last two weeks before harvest. Each treatment was replicated ten times with respect to a total of 60 plants for each plant species.The assay consisted of a randomized complete block design with three inoculation treatments for each plant species: (a) plants inoculated with the fungal endophyte v/v), sieved (2 mm) and sterilized by steaming (100 °C for 1 h for three consecutive days).The growth substrate consisted of a mixture of loamy soil (soil composition is shown in Zopfiella aff. erostrata endophytes (strains 1 and 2), which were isolated from arid environments in La Rioja, Argentina [Two fungal rgentina , were usTriticum aestivum L.) and tomato  were used as experimental plant hosts. Seeds were superficially sterilized by washing and shaking with 10% NaClO for 15 min and four times with sterile distilled water. The seeds were then placed on sterile vermiculite at 25 °C to germinate. Two-week-old seedlings were transferred to plastic pots containing 1 kg of sterilized substrate (one seedling per pot).Wheat  to allow adequate fungal establishment and colonization of roots. After this growth stage, half of the pots were subjected to water stress for two weeks. At this time, the plants were allowed to dry until soil water content reached between 60–55% field capacity (3 d required), which corresponded to 10% volumetric soil moisture . Plants were maintained under these conditions for an additional 12 d. To control the level of drought stress, the soil water content was measured daily using a ThetaProbe ML2 ) at the end of the afternoon and the amount of water lost was added to each pot to return soil water content to the desired 10% of volumetric soil moisture (60% of field capacity). However, during the 24 h period between each rewatering, the soil water content progressively decreased, reaching a minimum value of 50% of field capacity. Four replicates per treatment were performed for all evaluated parameters.Photosynthetic efficiency and stomatal conductance were measured in vivo (prior to the plant’s harvest) 14 days after drought stress imposition. After seven weeks, the plants were harvested and the shoot and root systems were separated in 0.5 g aliquots which were stored at −80 °C for future determination of antioxidant enzyme activities  and other biochemical parameters such as proline, lipid peroxidation and hydrogen peroxide concentrations. The dry weight (DW) of shoots and roots was measured after drying in a forced hot-air oven at 70 °C for 48 h. The percentage of root colonization was estimated by visual observation of the fungal presence (melanized hyphae and mycelium) in the plant roots. Roots subsamples were stained following the procedure described by Barrow , and werThe efficiency of photosystem II was measured using a FluorPen FP100 , which quantifies the yield of photosystem II as the ratio between the actual fluorescence yield in the light-adapted state (FV’) and the maximum fluorescence yield in the light-adapted state (FM’) . This deStomatal conductance was measured two hours after the light turned on by using a porometer system  according to the user manual instructions. Stomatal conductance measurements were taken in the third youngest leaf from six different plants of each treatment.g for 20 min at 4 °C. Total protein was determined according to the Bradford method [In order to determine enzymatic activities, 0.5 g of fresh leaves were ground in liquid nitrogen using a ceramic mortar until the tissue presented a homogeneous appearance. The material was then mixed with 1.5 mL of extraction buffer . After homogenization, the extracts were filtered through nylon pads and centrifuged at 20,000× d method  with bov−1 protein.SOD activity was measured according to the ferrocytochrome c method as described by MaCord and Fridovich . The rea2O2 (ꜫ = 23.5 mM−1 cm−1) at 240 nm for 1 min [2O2 and 25 µg protein extract in a volume of 1 mL.CAT activity was determined by following the consumption of Hor 1 min . The rea−1 cm−1) for 1 min in 1 mL of the reaction medium containing 0.1 M Tris–acetate buffer (pH 6.4), 350 µM ascorbic acid, 170 µM H2O2 and 25 ug protein extract [APX activity was estimated by following the oxidation of ascorbate (ASC) at 290 nm (ꜫ = 2.75 mM extract .−1 cm−1) for 1 min [GR activity was assayed by following the oxidation of NADPH at 340 nm  content as determined by the thiobarbituric acid reaction according to the method described by Buege and Aust . For the2O2) content was measured according to the method described by Patterson et al. [g for 10 min. The supernatant was filtered through a millipore filter (0.45 mm) and used for the assay. A 200 mL aliquot was brought to 2 mL with 100 mM K-phosphate buffer (pH 8.4) and 1 mL colorimetric reagent was added. This reagent was made on the same day by mixing 1:1 (v/v) 0.6 mM potassium titanium oxalate and 0.6 mM 4–2 (2-pyridylazo) resorcinol . The samples were incubated at 45 °C for 60 min and the absorbance at 508 nm was recorded. The blanks were made by replacing leaf extract with 5% TCA.Hydrogen peroxide , Duncan’s multiple range test for means comparison was applied [Z. erostrata 1 y Z. erostrata 2).The data were analyzed by one-way ANOVAs using INFOSTAT software . When si applied . NonlineZ. erostrata 1 increased their shoot and root dry weight. The shoot and root dry weights of wheat plants subjected to drought stress decreased by half as compared to the well-watered plants. Root colonization by Z. erostrata 1 significantly increased root growth under both watering conditions, and Z. erostrata 2 considerably increased root growth, particularly under well-watered conditions  significantly enhanced shoot and root biomass regardless of the water regime applied , whereas the plants inoculated with strain 2 of Z. erostrata showed lower levels of colonization (≈35%). However, in tomato plants, the endophyte Z. erostrata 2 exhibited the highest root colonization levels, regardless of the watering conditions applied  had a positive impact on the plant’s physical variables such as root and shoot dry weight, as well as photosynthetic efficiency and stomatal conductance, with respect to the non-inoculated controls (C_WW). In particular, inoculation with Z. erostrata 1_WW had a more pronounced effect on the growth parameters of wheat plants. Likewise, under drought conditions, inoculation with fungus Z. erostrata 1 produced a similar effect in the plants, as evidenced by the grouping of the samples in the same quadrant of the graph. Conversely, under drought conditions, inoculation with endophyte Z. erostrata 2 did not show any differences in SOD, MDA and proline levels with respect to control plants.With regard to wheat, the dispersion plot shows the distribution of samples (dark blue diamonds) with respect to the parameters determined in the plants a. As canZ. erostrata 1_WW and Z. erostrata 2_WW enhanced GR and APX enzymatic activity under well-watered conditions, whereas, under drought conditions, these fungi modified mainly MDA and proline levels, as well as SOD activity.In tomato plants, fungal inoculations improved plant development under both water regimes evaluated b. Thus, The adverse effects of drought stress on crop productivity have been widely reported ,67. One Zopfiella erostrata strain 1 and Zopfiella erostrata strain 2, on tomato and wheat plants grown under two different water regimes (well-watered conditions and drought stress). The results demonstrate that the growth responses of plants to Zopfiella erostrata inoculation were highly significant even under water deficit conditions. In general terms, both fungal endophytes improved the shoot and root development of the two plant species studied. However, the endophyte Z. erostrata 1 was found to perform better in terms of wheat plant growth, whereas fungus Z. erostrata 2 was found to have a more significant impact on both the shoot and root biomass of tomato plants regardless of the water conditions applied. These differences in growth responses between wheat and tomato plants have been detected in a diverse range of plant species [Nectria haematococca improved various growth parameters in tomato plants subjected to drought stress. These improvements in growth could be due to markedly higher nutrient and water uptake by the fungal hyphal network, as previously reported [Z. erostrata 1 showed higher levels of colonization in the wheat plants, whereas Z. erostrata 2, to a great extent, colonized the tomato roots. Thus, the effects on growth are dependent upon the host involved, which is one of the factors that deeply influences this symbiotic relationship [In the present study, we evaluated the effect of the fungal endophytes,  species . This ca species . Valli a species  have demreported ,39. In ationship . Therefotionship . In linetionship ,74. HoweDrought stress inhibits photosynthetic capacity and stomatal conductance (gs) in plants ,75. HoweRhizophagus irregularis under abiotic stress conditions. With regard to enzymatic activity in tomato plants, SOD and APX levels were lower following fungal treatments under stress conditions as compared to non-stressed conditions. Similarly, other studies have found that inoculation with strains of endophytes and AM decrease antioxidant enzyme activity in plants under diverse abiotic stress conditions [2O2 removal [2O2 values decreased significantly in the inoculated tomato plants. In addition, no CAT activity was detected following any treatment, and APX levels were minimal in the tomato plants, which may indicate that hydrogen peroxide is scavenged by other non-enzymatic mechanisms such as antioxidant compound production [Plants have developed various antioxidative strategies to flush out reactive oxygen species (ROS) produced during drought stress . Severalnditions ,87. Prevnditions . GR is a removal . In our oduction ,91. In foduction ,93. Anotoduction ,39.2O2 levels under drought stress conditions can be explained by enhanced CAT and APX enzyme activity. However, this correlation between the parameters mentioned above was not detected in our study. Although our results demonstrate the capacity of these endophytes to mitigate oxidative damage caused by drought, further research that provides a better understanding of the possible mechanisms involved is required.Fungal inoculation reduced hydrogen peroxide levels in all the plants subjected to stress, although the effect was more marked in tomato plants. This finding is in line with that observed in several AM-inoculated plants subjected to abiotic stresses such as drought ,95, and Z. erostrata 1, which showed an opposite behavior to that of wheat in tomato plants, increased peroxidation levels as compared to non-inoculated plants exposed to drought. Given that the plants can respond in different ways to similar stress conditions and to fungal inoculation, the results are quite likely to vary depending on these factors.Membrane lipid oxidation is a reliable measure of free-radical production and oxidative stress in plants . In thisZ. erostrata 1 decreased proline content, whereas Z. erostrata 2 significantly increased proline in these plants. Likewise, other studies have also reported a decrease in proline content in host plants in response to drought [Z. erostrata 1. As these plants appear to be less affected by drought and have better water status than the non-inoculated plants, they do not need to accumulate proline. In tomato plants subjected to drought, Z. erostrata 2 decreased proline content, whereas Z. erostrata 1 maintained these values similar to those for control plants. This suggests that the high colonization levels of Z. erostrata 2 could increase water absorption by the roots of tomato plants and also improve growth parameters and stomatal conductance. Thus, the inoculated plants exhibit lower proline content due to enhanced water uptake by the roots [Among the strategies more commonly used to cope with drought stress, many plants increase the osmotic potential of their cells through the accumulation of compatible osmolytes such as proline and mannitol, which are involved in osmotic adjustment ,100. The drought ,104,105.he roots ,107. StuZopfiella erostrata. In general, inoculated plants exhibited higher tolerance to drought than non-inoculated plants, as evidenced by their enhanced shoot and root biomass production, higher photosynthetic efficiency and stomatal conductance, as well as lower peroxide content under these conditions. However, the data demonstrate the differential effects of endophytic inoculation depending on the plant species evaluated, suggesting a certain degree of compatibility between endophytic strains and host plant. With regard to biochemical aspects, proline accumulation, oxidative damage to lipids, and the activity of the four antioxidant enzymes measured varied considerably depending on the endophyte considered and the host plant. Thus, future research should focus on conclusively elucidating the mechanisms by which each of these endophytic strains improve plant drought resistance according to the host plant involved.In the present study, the effects on water stress of two unknown and poorly studied fungal endophytes were evaluated. This is therefore the first report to address the physiological and biochemical aspects of drought tolerance in wheat and tomato plants inoculated with two strains of


Background: Scaphoid fracture is most often missed and mismanaged leading to scaphoid nonunion with or without avascular necrosis. When avascular necrosis of the proximal pole is confirmed with intraoperative evaluation, conventional bone graft is not enough. The treatment modalities are evolving day by day. The current trend is vascular bone grafting, which has shown good outcomes in terms of union and wrist function.Methods: Fifty patients with nonunion fracture of the scaphoid were treated with vascularized pedicle bone graft from the dorsum of the distal radius using the 1st and 2nd intercompartmental supraretinacular artery, from 2014 to 2022. Preoperative and postoperative clinical evaluation included pain, range of motion, grip strength, and satisfaction. The average follow-up period was 12 months.Results: Among 18 patients, 14 were clinically improved after a mean follow-up period of eight weeks. Thirteen patients reported the absence of any discomfort, three patients reported slight discomfort after hard work, and two patients reported pain with light work. The wrist range of motion improved significantly, and the hand grip strength also improved. According to the modified Mayo wrist scoring chart, clinical results were rated as excellent in 24 cases, good in 19 cases, and poor in four cases.Conclusion: 1,2 intercompartmental supraretinacular artery  is superficial to the extensor retinaculum and is a proper pedicle of vascularized bone graft due to the ease of visibility and dissection. The functional results and union rates were satisfactory in our study. Scaphoid fractures represent less than 2% of all fractures , but theAccording to Lichtman , scaphoiThe study was a prospective and multicenter study done at the National Institute of Traumatology and Orthopaedic Rehabilitation (NITOR) and one private hospital outside NITOR, Dhaka, Bangladesh from 2015 to 2022. Fifty participants who had scaphoid nonunion and met the selection criteria were selected. Lichtman type I and type II of scaphoid nonunion were included in the study. Patients with scaphoid nonunion with degenerative changes were excluded from the study. Preoperatively, all of them were evaluated both clinically and radiologically. Nonunion was defined as a fracture gap with a sclerotic zone on X-ray. Surgery was done following the technique described by Zaidemberg et al. in 1991 . PostopeClinical evaluationPatients with scaphoid nonunion usually present with a history of previous trauma, which may be a fall on the outstretched hand or hyperextension injury to the wrist with initial inadequate or no treatment. These patients complain of decreased wrist motion, weakness, inability to perform pushups, or radial site wrist pain. If the scaphoid nonunion remains untreated, it may lead to AVN of the scaphoid, which may result in carpal collapse, humpback deformity, or dorsal intercalated segmental instability in long-standing cases .ImagingUp to 20% of acute scaphoid fractures can be missed in initial X-rays . Union wMRI is very useful to diagnose nonunion and AVN. Low signal intensity on T1-weighted images and high signal intensity on T2-weighted images on MRI are suggestive of AVN. However, the absence of punctate bleeding intraoperatively confirms AVN . CT scanSurgical techniqueThis technique of vascularized bone graft was first described by Zaidemberg et al. in 1991 . With thPostoperative evaluationRadiologicalPosteroanterior and lateral views were taken postoperatively. The disappearance of the fracture line and trabecular realignment was considered a union of the fracture.FunctionalA modified Mayo wrist scoring system was used to evaluate functional outcomes postoperatively . The MayIn this study, we operated scaphoid fracture nonunion of 50 respondents. Of them, 45 were male, and most of them (38%) were between 40 and 49 years of age. The right hand was predominant (60%). The mean delay of surgery from the time of injury was 8.50 ± 9.37 (mean ± SD) months, where the majority of the respondents were operated on in less than six months (40%) following injury. Thirty (60%) respondents received no previous treatment, whereas 18 of them were managed in the form of cast application previously. All the respondents were evaluated with X-ray and MRI preoperatively. Waist fracture was found in 42 (84%) respondents, and the rest of them had proximal pole fracture (16%). The majority of them were classified as Lichtman type I (60%). Twenty (40%) of them had developed AVN preoperatively. All of the respondents were treated with 1,2 ICSRA as primary vascularized bone graft and fixed with Herbert screw. The mean time of union was 6.74 ± 1.71 (mean ± SD) weeks, where 40 (80%) were united in six weeks. The final range of movements  was compared with the opposite healthy site where the p-value was 0.002 (<0.05), which was statistically significant. Finally, the respondents were evaluated by the modified Mayo wrist score. According to these scores, 24 (48%) respondents had excellent results, 19 (38%) had good results, and four (8%) of them had poor results (Figure Table Table Figure Scaphoid is the most common carpal bone to be fractured. The fracture is often missed in the initial presentation, which may lead to nonunion and subsequently AVN. Conventional Russe  inlay boSeveral authors used different methods and different donor sites for vascularized bone grafts. Chacha  and KawaIn this study, the mean time of union was 6.74 weeks. According to the modified Mayo wrist score, 86% of the respondents had excellent and good results in the present study. Four of them had poor results in terms of pain, grip strength, and range of motion. This might be due to the location of the fracture in the proximal pole with late presentation and inadequate postoperative rehabilitation. This study used the 1,2 ICSRA as pedicle vascularized bone graft only, so a comparative study using free medial femoral condyle graft could be done for further study.Due to superficial location and ease of dissection, it may be concluded that vascularized bone graft using 1,2 ICSRA was useful in treating scaphoid fracture nonunion and AVN. Special surgical experience is required for this surgical technique. This study determined that the probability of a successful outcome is different and depends on the most effective surgical technique and careful patient and fracture selection.


The genetic mutations that contribute to the transformation of healthy cells into cancerous cells have been the subject of extensive research. The molecular aberrations that lead to cancer development are often characterised by gain-of-function or loss-of-function mutations in a variety of oncogenes and tumour suppressor genes. In this study, we investigate the genomic sequences of 20,331 primary tumours representing 41 distinct human cancer types to identify and catalogue the driver mutations present in 727 known cancer genes. Our findings reveal significant variations in the frequency of cancer gene mutations across different cancer types and highlight the frequent involvement of tumour suppressor genes (94%), oncogenes (93%), transcription factors (72%), kinases (64%), cell surface receptors (63%), and phosphatases (22%), in cancer. Additionally, our analysis reveals that cancer gene mutations are predominantly co-occurring rather than exclusive in all types of cancer. Notably, we discover that patients with tumours displaying different combinations of gene mutation patterns tend to exhibit variable survival outcomes. These findings provide new insights into the genetic landscape of cancer and bring us closer to a comprehensive understanding of the underlying mechanisms driving the development of various forms of cancer. Cancer genes include genes encoding cell surface receptors, oncogenes, tumour suppressor genes, kinases, phosphatases, and transcription factors6. Cancer genes of these classes transcribe mRNAs that encode proteins, which function in various oncogenic pathways that fuel oncogenesis by enabling various hallmarks of cancer7. The mutations in known cancer genes, unlike those in non-cancer driver genes10, qualitatively or quantitatively alter the function of genes and proteins and, consequently, alter the cellular processes in which these proteins participate. Moreover, oncogene mutations are linked with differences in patient survival, clinical outcomes, metastatic or recurrent tumours, and serve as predictors of tumour responsiveness to anti-cancer drugs12. There is, therefore, a need to understand the extent to which cancer genes are mutated in cancers of different tissues of origin.Cancer is a complex disease characterised by mutations in genes that control various hallmarks of the disease, including escaping programmed cell death, promoting genome instability and mutations, and proliferative signalling18. Furthermore, many genetic aberrations have been identified in human cancers, and several of the proteins encoded by these genes are well-established drug targets, while others are promising drug targets20. However, despite this impressive list of known gene mutations, it covers only a few cancer types. To comprehend the extent and consequences of gene alterations affecting function, it is crucial to study the alteration of cancer genes across all human cancers and within each cancer category. Such knowledge has been successfully applied in the design of therapies explicitly targeting proteins altered by somatic and germline mutations in cancer genes22. However, we still do not completely understand the extent to which cancer genes and the classes thereof are altered in all human cancers.Over the last few decades, our understanding of the genes, pathways, and their role in oncogenesis has grown significantly, leading to increased efforts to treat various cancer types24. Then, we comprehensively analyse known cancer gene mutations across different cancer types by integrating information on tumour genetic alterations with known gene annotations. Our analysis provides novel biological insights into the mutational landscape of these cancer genes and shows the extent to which they co-occur or are exclusive in tumours of various tissues and their association with patient outcomes.Here, we utilise publicly available datasets generated by various cancer sequencing projects to understand the extent of cancer gene alterations in human cancers. Furthermore, we obtain information on known cancer genes compiled by the Catalog of Somatic Mutations in Cancer (COSMIC), Cancer Gene Consensus (CGC) database24 and Cancer Gene Consensus (CGC)23 database. This list includes genes that encode oncogenes (383), tumour suppressor genes , transcription factors (150), kinases (75), phosphatases (9), and cell surface receptor proteins   factors 50, kinas factors 50, kinasVHL (and other genes) in acute myeloid leukaemia to 94% for the TP53 gene in small cell lung carcinomas  of pancreatic adenocarcinomas and PTEN (67%) of uterine corpus endometrial carcinomas, and JAK2 (75%) of myeloproliferative neoplasms , testicular germ cell tumours (KRAS and KIT), and thyroid carcinomas (BRAF and NRAS)  none of the samples, 2) less than 5 per cent of the samples, and (3) more than 5% of the samples. Our analysis revealed that most cancer genes were not mutated cancer types, and a limited number of genes were found to be mutated in over 5% of the samples. For instance, only two known cancer genes were found to be mutated in over 5% of thymomas , stomach adenocarcinoma (330 genes), and skin cutaneous melanoma (314 genes). This finding shows that the extent to which the cancer genes are mutated across cancer types varies and that some cancer types have few mutations within the coding sequences of known cancer genesMUC16 (mutated in 18.9% of tumours), PIK3CA (12.4%), and KRAS (11.1%) were the most frequently mutated across the profiled samples , CSMD3 (13.7%), and LRP1B (13.5%) were the most frequently mutated , BRAF (6.6%), and ATM (6.0%) were the top three mutated genes that encode protein kinases  and PTEN (6.4%) were the most frequently mutated among the genes that encode protein phosphatases  and KMT2C (8.6%) were the top-two frequently mutated among the genes that encode transcription factors  and LRP1B (13.5%) were the most frequently mutated , MUC16 (18.9%), CSMD3 (13.7%), LRP1B (13.5%), and PIK3CA (12.4%)  of cancer genes were mutated in at least one sample. Among the oncogenes, TP53  is the most frequently altered gene, followed by PIK3CA (12.4%)27. Furthermore, we found that the extent to which the cancer genes are mutated in different cancer types varies significantly, a pattern likely to impact the treatment strategies that could be applied to cancers of different tissues28.The mutation frequencies we report here are reasonably consistent with previous reports, which indicated that We were interested in evaluating the extent to which genes in particular categories of cancer genes  are mutated across human cancers. Here, we found mutations in the known cancer genes in all tumours. Of the six classes of cancer genes, the TSGs (91% of the tumours) and the oncogenes (89%) showed the highest frequency of mutations, followed by the transcription factors (72%), kinases (62%), receptors (60%), and the phosphatases (19%);  of all the tumours harboured mutations in genes involved in more than one of the six cancer gene classes , TP53 and BRAF (p = 8.0 × 10–13), and BRAF and KRAS (p = 9.5 × 10–8), see Fig. PTEN and PIK3CA (p = 2.1 × 10–70), BRAF and ERBB4 (p = 6.9 × 10–63), and EGFR and ERBB4 (p = 1.9 × 10–59), see Supplementary Data First, we assessed mutations in 127,765 gene pairs, both present in at least 1% of 20,331 samples across 41 human cancer types (see the “Methods” section). Here, collectively across all the cancer types, our analysis revealed 127,605 gene pairs with significantly co-occurring mutations, 15 pairs with mutually exclusive mutations, and 145 pairs with non-statistically significant mutations , KIT and PDGFRA in gastrointestinal stromal tumour (p = 3.3 × 10–37), and EGFR and KRAS in lung adenocarcinoma (p = 6.6 × 10–29) , TP53 and PTEN in uterine corpus endometrial carcinoma (p = 9.0 × 10–29), FGFR1OP and MAP3K1 in colorectal adenocarcinoma (p = 2.7 × 10–38) and uterine corpus endometrial carcinoma (p = 2.9 × 10–05). The complete list of mutually exclusive and co-occurring mutated gene pairs in specific cancer types can be found in Supplementary Data Second, we analysed the mutations in gene pairs that were found to be mutated in more than 5% of the tumours for each of the 41 cancer types. Our results indicate 284,709 gene pairs with significantly co-occurring mutations, 796 gene pairs with significantly mutually exclusive mutations, and 32,636 gene pairs that exhibit a non-statistically significant mutation pattern  than across all types   or CDKN1A and TP53 (p = 1.99 × 10–35) tended to exhibit worse survival outcomes than those with tumours in which one or none of these genes are mutated . The groups were: (1) no mutations, (2) and 3) only one of the genes in the pair is mutated, and (4) both genes are mutated. Here, we found that mutations in gene pairs are associated with varied overall survival durations of patients afflicted. For example, in the case of some of the most studied genes in cancer only one53 p = 1.9 × 10–3545. For example, mutations in the KRAS that co-occur with STK11, KEAP1, and TP53 genes in lung cancer patients have been associated with poorer prognosis and reduced survival44.Our findings highlight the importance of understanding the impact of different combinations of gene mutations on cancer development and progression. Specifically, some cancer patients may exhibit significantly different disease outcomes due to the specific combination of mutations present in their tumours, as has been demonstrated in multiple studiesTP53, KRAS, APC, SMAD4, and PIK3CA) that exhibit a co-occurring mutation pattern, while the second set of five genes  are exclusively mutated. Similarly, in acute myeloid leukaemia, we found two gene sets; the first set includes six genes  that exhibit a co-occurring mutation pattern, and the second set of four genes , exhibit an exclusive mutation pattern  that exhibit a co-occurring mutation pattern, and the second set of four genes   for the top 10 most frequently mutated cancer genes in various cancer types. Using a detailed analysis of gene pairs, we found intriguing variations in patterns of co-occurring and mutually exclusive mutations across different cancer types  Fig. c, exhibiHowever, our analysis revealed that driver gene mutations have a high co-occurrence in specific cancer types, including uterine corpus endometrial carcinoma, skin cutaneous melanoma, and basal cell carcinoma Fig. . We alsoHere, our findings suggest that different cancer types may exhibit distinct patterns of driver pathway mutations, and that further research is needed to fully understand the implications of these patterns for cancer subtypes and cancer treatment.Owing to the importance of cancer hallmarks in designing better treatment strategies, we sought to determine the extent to which genes associated with each hallmark of cancer are altered across different types of human cancer  is 378 times more likely than exclusive mutations (796). This demonstrates that genes involved in various cancer pathways have a greater tendency to co-mutate rather than mutate exclusively67. As a result, our findings suggest the presence of a limited number of context-dependent, co-occurring driver gene mutations, which could facilitate the identification of widely applicable drug targets and markers of aggressiveness across a multitude of patients' tumours.Despite the large amounts of genomic data this study analysed, we could not pinpoint commonly mutated cancer genes in samples of specific cancer types. This highlights the sparsity nature of gene mutations and the limited diversity of the presently available genome sequences, which impede the identification of commonly applicable drug targets and marker mutations within each cancer type70.Our findings also show that the co-occurrence and exclusive nature of cancer gene mutations significantly affect the disease outcome of patients with various forms of cancer. In addition, these results indicate that various gene alterations in specific gene pairs have a diverse impact on processes that drive disease aggressiveness23. Tier 1 genes have well-documented activities relevant to cancer, while Tier 2 genes have strong indications but with emerging evidence23. By not distinguishing between these tiers, we may have overlooked some nuanced differences in the role these genes play in oncogenesis. This could potentially affect the interpretation of the data, especially in the context of clinical relevance. In future studies, a separate analysis of Tier 1 and Tier 2 genes might provide more detailed insights into their distinct roles and contributions to the development and progression of cancer.However, a limitation of our study is that we did not account for the distinction between COSMIC Tier 1 and Tier 2 cancer-driving genes in our analysis42 and computational tools can be used to predict the potential impact of certain gene mutations or their combinations73, the limitations of available data and the complexity of cancer genetics make it challenging to comprehensively evaluate the impact of every possible combination on cancer development and drug response. As such, future experiments that allow the altering of cancer genes in normal cells in different combinations will ultimately help to unlock the impact of a combination of cancer gene mutations on oncogenesis, disease aggressiveness, and the chemosensitivity of tumours. By developing new molecular tools and continuing to expand our knowledge of cancer genetics, we can move closer to achieving precision medicine in cancer treatment, where patients receive tailored therapies based on the genetic profile of their cancer.In conclusion, to comprehensively evaluate the impact of different combinations of gene alterations on cancer development and treatment response, there is an urgent need for new molecular tools. While there is a vast amount of genomics data available, it is currently not feasible to study the impact of every possible combination of gene mutations due to the sheer size of the combinatorial space. It is important to note that the currently available genomics data is limited to a subset of patient tumours and cell lines, which only includes a fraction of the possible combinations of driver mutations that may exist. While databases74 version 5.2.5  of deeply sequenced tumours. To ensure a high level of specificity, we excluded studies that employed targeted sequencing, datasets of paediatric tumours, and cancer cell lines, resulting in a final dataset of 206 cancer studies. Further, we filtered the dataset again to return only those from 138 cancer studies that also had clinical information for the profiled afflicted patients. The final datasets encompassed 20,331 patient-derived tumour samples representing 41 distinct human cancer types sequenced from the year 2012 to 2021 , specifically from the Consensus Cancer Gene Database23 , which includes a total of 727 cancer-driving genes that have been rigorously vetted for evidence and manually curated, with 576 in Tier 1 and 151 in Tier 2. To further classify these known cancer genes, we divided them into five classes: oncogenes, tumour suppressor genes, kinases, phosphatases, cell surface proteins, and transcription factors. To achieve this, we obtained information on gene annotations from various databases, including the Sanger Consensus Cancer Gene Database23 (699 oncogenes and TSGs), the UniProt Knowledgebase75 (304 oncogenes and 741 TSGs), the TSGene database76 , the ChEA transcription factor database77 (645 transcription factors), the TF2DNA database78 , the Kinase Enrichment Analysis database79 (428 kinases), the ONGene database80 (725 oncogenes), and the Surfaceome database81 . We then collated the cancer genes from all the above databases to obtain a list of 727 cancer genes (after removing overlapping genes), including 383 oncogenes, 370 tumour suppressor genes, 9 phosphatases, 75 kinases, 63 cell surface receptors, and 150 transcription factors  for each gene across the 20,331 samples across each cancer type  for each of the 41 human cancer types represented among the 20,331 samples  of all tumours across all the samples. Next, we applied the Fisher test to each pair of the selected genes and utilised a cut-off p-value of 0.05 to identify statistically significant gene pair correlations. Furthermore, we used the magnitude of the odds ratio to identify gene pairs with co-occurring mutations (odds > 1 and p < 0.05) and gene pairs with mutually exclusive mutations (odds < 1 and p < 0.05). Additionally, we used the approach to identify cancer gene pairs within co-occurring or mutually exclusive mutation patterns within each of the 43 human cancer types  only one gene pair mutated, (2) the other gene pair mutated, (3) none of the gene pair mutated, and (4) both gene pairs mutated.We used the Kaplan–Meier83. This algorithm employs a mathematical programming method to identify de novo driver pathways in cancer from mutation profiles. Specifically, we aimed to identify pathways with mutated cancer genes that exhibit both high coverage  and high exclusivity, and show a statistically significant co-occurrence pattern.To identify the patterns of mutations associated with each cancer type and the corresponding cancer gene combinations, we applied the CoMDP algorithm84. In cases where MutSigCV2 identified fewer than 10 genes as significantly mutated, we included additional genes to bring the gene set size to 10. If a gene with very long coding regions or very long introns  were not identified as being significantly mutated using MutSigCV2 in a specific cancer type, it was excluded from the analysis for that cancer type84.To begin, we selected the top 10 mutated cancer genes for each cancer type as input for the CoMDP algorithm. These genes were selected from the significantly mutated genes identified using the MutSigCV2 algorithm29.Next, we ran the CoMDP test for each cancer type with K = 10, where K equals the gene set size. The CoMDP analysis returned mutated driver pathways associated with the genes in each cancer type (Supplementary Data 85 and the mutated gene sets, we accessed information on genes and proteins associated with various Hallmarks of Cancer from the COSMIC database24. We then calculated the number of mutated genes at a frequency of more than 1% in at least one cancer type within each class based on the various cancer hallmark gene sets (Supplementary Data To investigate the relationship between the Hallmarks of Cancer86 for all statistical analyses. We used two-sided statistical tests with a p-value < 0.05 to indicate statistical significance. To correct for multiple statistical testing, we applied the Benjamini & Hochberg procedure87, resulting in a q-value < 0.05 for each comparison.We used MATLAB 2022aThe study protocol was approved by The University of Cape Town; Health Sciences Research Ethics Committee IRB00001938. The publicly available datasets were collected by the cBioPortal.Supplementary Legends.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Figures.


HSP60 is a heat shock proteins (HSPs) family member and help mitochondrial protein to fold correctly. Survivin is one of the inhibitors of apoptosis protein family member, which plays a significant part in cancer progression. They were capable of forming HSP60-survivin complexes and involved in the development of various tumors.The Cancer Genome Atlas (TCGA) database demonstrated that HSP60 and survivin and their correlation on mRNA expression level with OSCC patients. Besides, expression of HSP60 and survivin proteins was studied utilizing immunohistochemistry in tissue microarrays (TMA) in OSCC and in adjacent non-cancerous squamous epithelium (Non-CCSE) tissues.P < 0.05). Additionally, elevated HSP60 displayed a positive correlation with survivin in terms of mRNA and protein expression levels . Patients with OSCC who had advanced clinical stage or lymph node metastasis (LNM) showed higher HSP60 expression . Higher levels of the proteins HSP60 and survivin were significantly inversely correlated relationship with OSCC patients’ overall survival rates in multivariate survival analysis . From the above results, overexpression of HSP60 and survivin protein may serve as independent biomarkers predicting poor prognosis in OSCC.Significantly increased levels of HSP60 and survivin in most cancers compared to normal tissue by pan-cancer analysis. HSP60 and survivin proved a significantly increased expression in OSCC samples compared to Non-CCSE both on mRNA and protein (both Elevated HSP60 and survivin might be served as novel poor prognosis biomarkers for surgically resected OSCC patients. Oral cancer is a prevalent malignant tumor worldwide, with a high incidence and mortality rate, bringing a significant negative impact on global public health , 2. Due Heat shock proteins (HSPs) known as a family of conserved mitochondrial chaperone with critical roles in maintaining cellular homeostasis in response to hyperthermia, chemical agents, and other stress conditions . Heat shAs an anti-apoptosis protein belongs to apoptosis protein (IAP) family, survivin contains a baculovirus IAP repeats (BIR) domain specific to the formation of dimer and the inhibition of apoptosis , to playCurrently, inhibition of tumor cell apoptosis is a novel biomarker of the occurrence and development of most or all multiple types of tumors. Nevertheless, the precise relationship between HSP60 and survivin in OSCC is currently unknown. Therefore, in our current investigation, 79 patients of OSCC and 22 cases of Non-CCSE had HSP60 and survivin protein detected by IHC in tissue microarrays (TMA). Thus, this study aimed to detect the expression of HSP60 and survivin and thereby explore the association between the expression of those two proteins and clinicopathological features in OSCC.We collected the TCGA database to conduct a pan-cancer bioinformatics analysis of HSP60 and BIRC5. And we used 32  and 329 OSCC samples from TCGA database to make tumor/normal differential expression of HSP60 and survivin and analyze their correlation.The Ethics Committee of the Second Xiangya Hospital of Central South University approved this study. All experimental procedures were in accordance with the 1964 Helsinki declaration and its later amendments of comparable ethical standards. Written informed consent was obtained from every research sample. For the minors, however, the guardian must sign a written consent on their behalf.In this study, all OSCC patient with OSCC were submitted to surgical treatment at the Department of Oral and Maxillofacial Surgery, the Second Xiangya Hospital of Central South University  from 2007 to 2011. All OSCC samples and non-cancerous squamous epithelium were obtained from Department of Pathology, the Second Xiangya Hospital of Central South University. These patients had undergone standard staging, a decisive surgical removal of a portion of the tongue, and a methodical neck lymph node dissection. According to the WHO’s histological categorization of oral cancer, OSCC was confirmed histologically in all patients. In accordance with the 8th edition of the AJCC/UICC TNM staging system of OSCC, the current analysis’ staging classification was completed. No patients had been previously treated with chemotherapy and radiotherapy at the time of original operation. All patients’ full clinical histories and follow-up information were accessible. Informed consent was obtained from all patients before surgery. Follow-up time for analyses of survival for the 79 OSCC cases was calculated from the date of surgery to the date of death, loss-to-follow-up, or 2011, whichever came first. The characteristics of patients were detailed in Table The following primary antibodies were used for staining: HSP60 ; survivin  at 4 °C overnight.IHC staining of HSP60 and survivin in OSCC and Non-CCSE TMAs was performed using the previous protocol . As a neHSP60 and survivin staining in TMAs were evaluated at 200x magnification using light microscopy by two pathologists (SF and QW) blinded to the clinicopathologic data of the patients. A formula to calculate the overall expression marks based on the intensity and distribution of IHC staining was created: total score = intensity mark + percentage mark. Simply put, we visually regard as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong) to rate the intensity of staining for HSP60 and survivin. Meanwhile, positive tumor cell percentages were assigned as 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). All samples had a total score between 0 and 7 for HSP60 and survivin protein levels. Also, on the basis of overall survival (OS) of OSCC using the log-rank test, the optimal critical levels of the two proteins were 5 and 4, respectively. A score above 5 and 4 indicated high expression of the two proteins, whereas lower scores indicated poor expression in final analysis. Two pathologists reached consensus on all IHC scoring disagreements, and their level of agreement was 95%.SPSS version 24.0 software was utilized to conduct statistical analysis. The correlation between HSP60 and survivin, as well as analysis of survival rate curve and survival rate, were evaluated through the use of the log-rank test, Kaplan-Meier analysis, the Spearman’s rank correlation coefficient and regression analysis, respectively. The diagnosis to date of death was utilized to compute overall survival rates. Cox proportional hazard regression model was carried out to evaluate if HSP60 and survivin are independent prognostic markers. Every P value was derived from a statistical analysis with two branches, and a value of p < 0.05 was used as the cutoff for determining whether or not the results were statistically significant.Significantly increased levels of HSP60 Fig. A and surP < 0.05). In line with this finding, the expression of HSP60 was found to have a positive correlation with survivin mRNA levels in patient tumors , although expression of survivin was not significantly correlated with clinical stage (P > 0.05). Higher levels of HSP60 expression were found in patients with LNM status in OSCC (P = 0.004). The data presented in Table P = 0.047). Age, gender, smoking, drinking, and chewing areca nuts were not found to have any prognostic significance in OSCC .We then used the univariate chi-square test to examine the relationships between HSP60 and survivin expression and a variety of clinicopathological parameters of OSCC patients, including gender, age, clinical stages, lymph node metastasis (LNM) status, pathological differentiation, and living habits like tobacco use, alcohol consumption, and areca nut chewing. As observed in Table P < 0.001; R2 = 0.2119, P < 0.0001, respectively).We examined the relationship between HSP60 and survivin expression in OSCC using these data. Table We utilized Kaplan-Meier survival curves of all 79 OSCC patients, and the statistical significance was evaluated by the log-rank test when examining the effect of HSP60 and survivin protein expression on OSCC patient survival. There were 23 (29.1%) total deaths among OSCC patients at the time of the study.P = 0.009, Fig. P = 0.025, Fig. Figure P = 0.018, P = 0.040, respectively), as well as tobacco using (P = 0.009). Besides, no impact was detected with alcohol, pathological differentiation, clinical stages, LNM status of OSCC .Multivariate Cox proportional hazard regression analysis was used to further verify whether the expression of HSP60 and the survivin protein were the independent prognostic factors in OSCC patients, which was show in Table We came to the conclusion that the levels of HSP60 and survivin expression in OSCC were significantly higher in mRNA as well as protein when compared to adjacent Non-CCSE tissues in current research. HSP60 expression was found to be higher in patients with advanced clinical stages and LNM and demonstrated a strong connection between high survivin expression and poor pathological differentiation of OSCC patients. Additionally, those with OSCC who had increased HSP60 expression exhibited a poorer survival rate than those with decreased HSP60 expression. These statistics indicated that overexpression of HSP60 and survivin has potential prognostic value and could be new prognostic marker for OSCC. HSP60 as one of the most common molecular chaperones is able to support cancer cell survival under adverse physiological and stress circumstances like hypoxia, viral agents, low pH as well as exposure to UV light and chemical . HSP60 eHSP60 staining was observed in the cytoplasm in our present study, which is consistent with the previously founding in patients with colorectal cancer . FurtherBoth the mRNA and protein levels of HSP60 and survivin showed a positive correlation in the data, and the presence of elevated HSP60 and survivin expression revealed that these parameters were poor independent prognostic factors for OSCC patients’ survival. It has been proved that HSP60 helps to stabilize mitochondrial survivin and knockdowns of mitochondrial HSP60 can promote apoptosis though breaking the interaction of HSP60-survivin in tumor cells , 29. FurThe use of tobacco and heavy alcohol drinking are the two most important risk factors for OSCC , and othHowever, the specific mechanisms and other players involved in the relationship between HSP60 and survivin have not been characterized. Our work on the creation of HSP60 and survivin inhibitors may be appealing, offering a promising line of inquiry for targeted cancer therapy and reduce both the adverse effects and drug resistance while also enhancing the efficiency of targeting cancerous cells.Summing up our study, OSCC patients had clearly elevated HSP60 and survivin protein expression. Overexpression of HSP60 and survivin could be viewed as novel biomarkers for poor prognosis, who possess potential application value for OSCC targeted therapy.


Tetraselmis genus, reflects the genetic background.Climate changes and anthropogenic pressures are causing a biodiversity decline in terms of species number and genetic diversity, reducing the adaptability and evolvability of natural communities. Transitional water ecosystems are more sensitive to habitat reduction and degradation and, thus, are more exposed to biodiversity declines requiring biodiversity monitoring programs for their conservation. Environmental DNA (eDNA) metabarcoding represents a high-throughput tool for biodiversity assessment that is facilitating data collection for biodiversity monitoring. In this study, we applied, for the first time, eDNA metabarcoding in a Mediterranean coastal lagoon to assess the ecological features of eukaryotic phytoplankton communities. We sampled water in seven different lagoon sites and amplified the extracted DNA with primers targeting the variable region 4 (V4) of the 18S rRNA gene marker. The results demonstrated the validity of eDNA studies to provide insights into lagoon phytoplankton composition, establish the structure and spatial variation of phytoplankton communities, and evaluate its correlation to abiotic factors. Finally, the genetic distances analysis suggests that the different spatial distribution of OTUs, at least for the  In addition to a decrease in species composition, habitat reduction and degradation are also causing a loss of species’ genetic diversity4. The genetic diversity of natural populations and communities ensures adaptive potential and evolvability5, reducing the risk of extinction6. To address these problems, monitoring actions and conservation strategies are essential. The inter-governmental group on Earth Observations and Biodiversity Observation Network (GEO BON) has developed the Essential Biodiversity Variables (EBVs), which are a set of variables aiming to capture different levels of biodiversity and are divided into 6 classes: genetic composition, species populations, species traits, community composition, ecosystem function, ecosystem structure. Specifically, the class of genetic composition includes genetic diversity and genetic differentiation or genetic distance; the class of species populations includes species distribution and abundances; the class of community composition includes community abundance and taxonomic/phylogenetic diversity7.Climate changes and anthropogenic pressures are the main drivers of a biodiversity decline characterized by an unprecedented rate of species extinctionThe advent of environmental DNA (eDNA) metabarcoding as a high-throughput tool for biodiversity assessment is certainly facilitating data collection for biodiversity monitoring.9. The first method involves DNA to be extracted from a bulk sample of organisms. The bulk sampling method has been applied to small planktonic organisms, but also to larger and multicellular species10. The second method uses traces of DNA collected by filtering water or using soil or sediment samples11. The sources of this DNA include cells, faeces, mucous, gametes, degrading cells or tissues and fragments resulting from predation9.Environmental DNA (eDNA) refers to DNA from an environmental sample and it is possible to identify two main ways to obtain this genetic material14. Among ecological indicators, phytoplankton has been subjected to monitoring studies using eDNA metabarcoding in lakes and rivers18.eDNA metabarcoding as a biodiversity assessment tool has already been applied in a variety of environments indicating its capability to unveil the macro-guilds used as ecological indicators as well as ecological communities structure22. Therefore, phytoplankton surveys are particularly important in the current context of critical environmental changes. However, despite the importance of phytoplankton in aquatic communities, accurate classification of phytoplankton species is notoriously difficult due to the occurrence of cryptic sibling species and cell dimensions, and eDNA metabarcoding can be considered an asset for rapid surveying of phytoplanktonic communities, including taxa hard to identify with traditional methods.Phytoplankton is a valuable ecological indicator due to its adaptability to environmental variations, the effect of its carbon sequestration ability on climate regulation, and its role in primary productivityIn this study, we applied eDNA metabarcoding to assess, for the first time, the biodiversity of eukaryotic phytoplankton communities in a Mediterranean coastal lagoon protected under the European Natura 2000 Network. This work suggests the validity of eDNA studies in establishing the structure and spatial variation of phytoplankton communities in transitional water ecosystems, as well as to correlate the phytoplankton communities structure to abiotic drivers, including also evidence of genetic diversity for some phytoplankton genera.The Aquatina Lagoon  3 outer sampling stations adjacent to the coastal dunes named A, B, and C; (ii) 3 inner sampling stations named D, E, and F; and (iii) 1 sampling station (named AF) at the end of the channel for the communication with the Adriatic Sea  are routinely monitored in the Aquatina lagoon in correspondence with the 7 sampling sites using the multiparametric probe YSI ProQuatro® .23. The obtained amplicon is about 390 bp in length. The reaction was performed in a volume of 50 μL composed of 5 μL of 10× reaction buffer, 1 μL of MgCl2 (50 mM), 1 μL of dNTP mix (10 mM), 1 μL of each primer (10 mM), 10 ng DNA, 0.2 μL of Platinum Taq (5 U/μL)  and sterile water to reach a volume of 50 μL. The amplification program included the following phases: initial denaturation at 95 °C for 5 min, 30 cycles of denaturation (95 °C for 30″), annealing (50 °C for 30″), extension (72 °C for 30″) and a final extension at 72 °C for 10′. All PCR products were purified with a PureLink PCR purification kit . Celero™ DNA-Seq Library Preparation Kit cod. 0360A  was used for library preparation following the manufacturer’s instructions, without fragmenting the amplicons. Both input and final libraries were quantified by Qubit 2.0 Fluorometer  and quality tested by Agilent 2100 Bioanalyzer High Sensitivity DNA assay . Libraries were sequenced through the MiSeq instrument  using 300 bp paired-end mode by IGATech, Udine, Italy. The generated FastQ files are deposited in GenBank (SRA) of NCBI with identification number PRJNA880489.The V4 region of 18S rRNA gene amplification was performed using the primers pair TAReuk454FWD1 and TAReukREV3QIIME pipelines and the USEARCH algorithm  were used for (i) chimaera filtering; (ii) grouping of replicate sequences, and (iv) OTU identification. The RDP classifier and the reference database SILVA 138 were used for taxonomic annotation. The resulting OTU list was filtered to contain only phytoplankton annotated OTUs.OTU richness has been calculated as the total number of OTUs per sampling site. OTU occurrence has been calculated as the percentage of OTUs occurring in the same number of sampling sites. All calculations have been done through the software Microsoft Excel. Bray–Curtis distance-based Redundancy Analysis (db-RDA) was performed by using the R Package Vegan. Response variables are represented by the occurrence of OTUs in each sampling site, while explanatory variables are represented by the mean abiotic parameter values registered in the Aquatina lagoon during routine surveys, specifically during the sampling period . Two-way ANOVA was performed for significance values. The results are visualised as an RDA plot obtained using the R Package ggplot2.OTUs consensus sequences were multiple aligned using Clustal Omega 1.2.2 through the Geneious Prime alignment options, with settings automatically adjusted based on the number of sequences. The obtained multiple sequence alignment was used to construct the Neighbour-Joining tree. For the tree construction, Resampling Method was set on Bootstrap with 674,476 random seeds and 1000 replicates. The genetic distance model was set on Jukes-Cantor, with no outgroup. Tree Build Method was set on Neighbour-Joining.To analyse the biodiversity of phytoplankton communities characterizing the study area, we identified seven sampling sites along the entire lagoon. DNA was extracted from water samples of the selected sites, and a region of the 18S rRNA gene marker was amplified with primers spanning the V4 region of the gene. Illumina sequencing generated a total of about 3,000,000 raw 18S rRNA reads with an average of about 420,000 sequences per sample, of which 41% were retained after bioinformatic processing Table . The GooOstreococcus, Micromonas and Bolidomonas.Following taxonomic assignment, only the OTUs annotated as phytoplankton taxa and classified beyond the D2 level of the SILVA 138 database were retained, resulting in a total of 2293 phytoplankton OTUs. This eDNA metabarcoding experiment detected a large fraction of phytoplankton groups Table , of whicInterestingly, eDNA from water samples, in our experimental conditions, was able to detect a wide fraction of phytoplankton taxa ranging over different phytoplankton size classes. In addition, both the total and relative numbers of OTUs belonging to the identified phyla present a specific and different distribution in the seven selected sites of the lagoon Fig. .Figure 3To quantify the differences in phytoplankton OTU richness and composition, we compiled the OTU presence/absence matrix Table . Based oOTU richness represents the number of phytoplankton OTUs observed per sampling site, revealing that sites A (n. of OTUs 1771), E (n. of OTUs 1599), and C (n. of OTUs 1417) host most of the OTU richness, site AF (n. of OTUs 496) appears to be the poorest, and sites B (n. of OTUs 899), D (n. of OTUs 909), and F (n. of OTUs 959) present intermediate values.The OTU occurrence has been calculated as the percentage of phytoplankton OTUs occurring in the same number of sampling sites and visualised as a bar plot Fig. . The majp-values calculated for each of the abiotic parameters, it results that salinity (p < 0.01) and temperature (p < 0.01) are the main explanatory variables for the phytoplankton OTUs distribution across the sampling sites, while pH (p < 0.05) and especially DO (p < 0.1) do not seem to affect it.To infer the dissimilarity between sampling sites and correlate it to the main abiotic drivers that characterize the environment under study through a constrained ordination, we performed a Bray–Curtis distance-based Redundancy Analysis (db-RDA). All loading and score values are summarised in Table The biplot of this analysis displays sites A, C, and D grouped, while sites AF, B, E, and F do not show any grouping pattern. This result reflects the similar environmental conditions occurring in sites A, C, and D, where comparable levels of the considered environmental variables have been registered. The proximity of these three sites in the biplot also indicates similarities in the phytoplankton OTUs composition across these sites. At the same time, the four sites far apart in the biplot represent areas with distinct environmental differences, influencing the phytoplankton OTUs distribution in a way that separates them apart and from the other three sites.Tetraselmis genus because the relative and different OTUs presented a clearly different distribution among sampling sites. The bioinformatics analysis recovered 8 OTUs assigned to the Tetraselmis genus highly differently occurring across the sampling sites  clustered and separated from OTUs widely distributed across the sampling sites, especially Tetraselmis 1, 2, 3, but significantly also from Tetraselmis 5.Interestingly, the OTUs present only in the marine site AF , displaying a high difference between site AF, located at the interface between the sea and the lagoon, characterized by high salinity and low temperature, site F, located in proximity to the freshwater input, characterized by low salinity and higher temperature, and the other sites, characterized by a greater intermediate homogeneity. The specific pattern observed in the db-RDA biplot shows that similar environmental conditions correspond to similarities in the phytoplankton community composition, while distinct environmental differences reflect in separated communities. These results confirm that environmental heterogeneity can represent the spatial factor playing a key role in influencing the phytoplankton community structure in transitional water ecosystems30.In this study, eDNA also detected significant correlations between phytoplankton OTUs occurrence and lagoon abiotic components. Specifically, the significant environmental explanatory variables were salinity and temperature (Tetraselmis genus. The absence of species assignment is probably related to the absence of 18S DNA barcodes for Mediterranean Tetraselmis species in the reference databases. OTUs belonging to the Tetraselmis genus and highly differently distributed across the sampling sites were analysed for the genetic distances. The results outlined that genetic backgrounds correlate with specific spatial distribution. Although the analysis is limited to a short fragment of a conserved gene, the results suggest that sequencing data from eDNA amplicons could be useful to inspect directional genetic variations.Different OTUs were taxonomically assigned to the A high genetic variation is important for the ability of phytoplanktonic assemblages to be extremely viable in highly heterogeneous environments such as coastal lagoons, which are subjected to strong variations driven by extrinsic factors such as climate and tides, other than by intrinsic variability given by abiotic heterogeneity.Overall, this work demonstrates that eDNA metabarcoding is a valuable tool for rapidly surveying the phytoplanktonic communities and describing their spatial distribution in transitional water ecosystems.Supplementary Information.Supplementary Table S2.Supplementary Table S3.Supplementary Table S4.Supplementary Table S5.


Mycoplasma periprosthetic joint infection (PJI) is rather difficult due to its rarity and difficult in isolation, there are not standardized diagnostic procedure for Mycoplasma PJI presently. This study aimed to reported a metagenomic next-generation sequencing (mNGS)-based diagnostic strategy for Mycoplasma PJI.The diagnosis of Mycoplasma PJI that were precisely diagnosed by mNGS and verified by optimized microbial culture methods and (or) 16S PCR polymerase chain reaction (PCR).In the present study, we have reported the largest number of Mycoplasma PJI was 57.14% and 71.43%, respectively. The infections were well controlled by targeted treatment in all cases.The positive rate of optimized microbial culture methods and 16S PCR in the detection of Mycoplasma PJI, which might also be provided as a novel diagnostic strategy for rare bacterial PJI. The standardized and optimized procedure based on mNGS presented in this study is useful for the diagnosis of  Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Pseudomonas aeruginosa through surgical incision infection or hematogenous infection (Periprosthetic joint infection (PJI) is one of the serious complications after joint arthroplasty, multiple debridement operations and extended period antibiotic treatment are need . PJI is nfection . With thnfection . Rare baMycoplasma is the smallest prokaryotic organisms, widely exists in people and animals, and most of them are not pathogenic. Mycoplasma is a common causative agent in pulmonary and genitourinary tract infections, so clinicians usually use microbial culture and polymerase chain reaction (PCR) for pathogen detection , a new molecular diagnostic tool, in the diagnosis of osteoarticular infection for long time . It is aJI cases . In ordeMycoplasma PJI according to microbial culture and mNGS results; 2) patients with complete medical data. Exclusion criteria were as follows: 1) insufficient clinical and laboratory data to diagnose PJI; 2) contamination or suspected contamination of samples; 3) infections at other sites or in the body.This study included the suspected PJI patients who were admitted in our hospital from 2015 to 2021. The diagnosis of PJI was based on Musculoskeletal Infection Association (MSIS) criteria: at least one of following three criteria should be met: 1) sinus tract was communicating with the prostate; 2) pathogen was isolated by culture from two separate tissue or fluid samples obtained from the affected prosthetic joint; and 3) patient met four of the following six criteria: (a) elevated ESR and CRP; (b) elevated synovial fluid WBC count; (c) elevated synovial fluid neutrophil percentage; (d) presence of purulence in the affected joint; (e) isolation of a microorganism in one periprosthetic tissue or fluid culture; and (f) > 5 neutrophils per high-powered field in 5 high-power fields observed from histologic analysis of periprosthetic tissue at ×400 magnification. Inclusion criteria were as follows: 1) patients who were suspected of Specimens were collected and transported to the microbiology laboratory within 30 minutes. For the synovial fluid, 100μL was inoculated on the blood agar, 1000μL was used for mNGS, and the rest was injected into the blood culture bottles for microbial culture, when the culture results were positive, they were transferred to the blood agar. For the tissue specimens, they were ground in sterile tryptic soy broth Ep tubes and then the homogenate was inoculated on the blood agar. For the removed prosthesis, they were immersed with sterile saline, sonicated, supernatant was removed after centrifugation, resuspended with sterile saline, and then injected into blood culture bottles for microbial culture.http://biobwa.sourceforge.net). The remaining sequence alignment to the microbial reference database, obtained from National Center for Biotechnology Information (NCBI) and classified as viruses, fungi, bacteria, parasites, etc. 5) Interpretation of mNGS results: the interpretation was conducted by the PJI panel, including at least one senior microbiologist, infectious disease expert and orthopedic expert dedicated to PJI research.The mNGS was performed according to previous describe methods . In brieMycoplasma reported by mNGS preoperatively, liquid combined with solid culture method (optimized culture method) was used for validation after sufficient samples were collected intraoperatively . In the liquid medium, micropores were closed with paraffin, after which samples were immersed and cultured at 37°C with 5% CO2, and the results were recorded at 24, 48 and 72 hour (h). When the bacteria concentration ≥ 104 cfu/ml, the micropores became red and were confirmed to be positive; for solid medium, samples were directly incubated in elective solid medium and cultured at 37°C in a 5% CO2 incubator for 24, 48, and 72 h, observed under low magnification (10×10) to confirm that the colonies were Mycoplasma if they were small and fried egg-like.For PJI cases with ratively . Liquid Mycoplasma reported by mNGS preoperative, sufficient samples were collected intraoperatively and verified by 16S PCR. Briefly, 200 μL specimen was placed in a disposable 2 mL sterile centrifuge tube, and the total DNA was extracted by DNeasyBlood & Tissue kit  as per manufacturer’s instructions. Digested with protease K for 4 h, and the extracted DNA was dissolved in 100 μL double distilled deionized water. The bacterial/fungal genomic DNA Mix was used as the positive control. The PCR conditions are: pre-denaturation at 94°C for 2 min, denaturation at 94°C for 20 s, annealing at 55°C for 30 s and extension at 72°C, with 30 cycles, extension at 72°C for 10 min and preservation at 4°C. The product was subjected to agarose gel electrophoresis and judged as negative amplification if no band was shown or if the band was not recognized by two PCR lab technicians. If positive, the PCR products were sent for sequencing and analysis , and the sequencing results alignment to Gen Bank to determine the species.For PJI cases with Mycoplasma based on the results of mNGS and other verification methods, combined with the clinical efficacies, and the accuracy of mNGS and other verification methods were also confirmed according to the clinical efficacies of Mycoplasma targeted treatment. All these cases were followed up regularly and the clinical data like CRP, ESR, infection recurrence and the incidence of antibiotic-related complications were recorded.For acute PJI, surgical treatment was performed with debridement, antibiotic irrigation and implant retention (DAIR), while for chronic PJI, two-stage revision was selected. After surgical treatment, PJI cases received intravenous antibiotic treatment for 2 weeks and oral antibiotic therapy for 1-3 months after discharge. PJI panel re-formulated the antibiotics regimen for Mycoplasma PJI cases were included in this study, which was the largest sample of Mycoplasma PJI cases so far ,3,4,6) could be successfully isolated with a positive rate of about 57.14% (4/7), while the positive rate further increased to 71.43% (5/7) by 16S PCR (Case 2 (left), 3, 4,6,7), but was still lower than that of mNGS. However, the relatively small number of Mycoplasma PJI cases and the lack of controls prevented comparison of positivity rate between mNGS and other validation methods.In this study, all cases were precisely diagnosed by mNGS. Although some case reports demonstrated that Mycoplasma was the true pathogenic bacteria combined with the patient’s medical history, symptoms, and other examination results. In this study, although Mycoplasma was only detected from 5 patients , the panel still diagnosed the other 2 patients as Mycoplasma PJI (case 5) or Mycoplasma complicated with other bacterial infections PJI (case 1) according to the poor clinical efficacies of routine microbial culture results oriented or empirical antibiotics treatment. Therefore, after surgical treatment (DAIR or two-stage revision), all patients received doxycycline or tetracycline for anti-infection treatment. All patients responded well to the targeted treatment and eventually controlled the infection. At the last follow-up, all patients had good joint mobility and functional scores, no patients need surgical debridement or long-term antibacterial therapy due to recurrence of infection, only one patient (case 1) developed antibiotic-related complications , which was relieved after symptomatic treatment. Thus, based on the results of mNGS and various validation methods, Mycoplasma PJI can be successfully diagnosed and cured with targeted treatment.Based on mNGS, routine and optimized microbial culture and 16S PCR results, PJI panel judged whether 6/L and the synovial fluid polymorphonuclear neutrophils (SF-PMN) percentage was 89.9%. To better identify the pathogenic organisms, routine microbial culture and mNGS were both performed preoperatively. About 48 hours later, the mNGS results indicated that it was a multiple infection caused by Staphylococcus epidermidis (S. epidermidis) and Mycoplasma hyorhinis, while microbial culture and 16S PCR only showed the potential infection by S. epidermidis. Unfortunately, CRP and ESR remained at high levels and the infection could not be controlled after microbial culture results oriented antibiotics treatment. Based on symptoms, physical examination and medical history, the PJI panel reconsidered that the Mycoplasma hyorhinis was also the true pathogen rather than background bacteria. Therefore, a two-stage revision was performed and a modified antibiotic regimen (levofloxacin + doxycycline) was adopted, and finally the inflammatory indexes decreased to normal and the infection was well controlled.Case 1 was a 77-year-old woman who underwent total hip arthroplasty (THA) for femoral neck fracture 5 years ago. Her hip pain had worsened for about 2 weeks, and she was admitted with a C-reactive protein (CRP) of 7. 38 mg/L and an erythrocyte sedimentation rate (ESR) of 36 mm/h. A synovial fluid aspiration and test was performed preoperatively, the synovial fluid white blood cell (SF-WBC) was 56367×106/L) and SF-PMN% (90.8%), a diagnosis of acute PJI was made. In order to timely identify the pathogen, routine microbial culture and mNGS were both performed preoperatively. mNGS showed Staphylococcus aureus (S. aureus) and Mycoplasma hominis infection, while the culture only indicated S. aureus. The diagnosis of acute PJI was made and DAIR was performed. Samples were also collected intraoperatively for optimized culture and 16S PCR, the 16S PCR results was negative while optimized microbial culture indicated the infection by Mycoplasma hominis. Therefore, this case was treated as mixed infection with a combination antibiotic regimen (levofloxacin+ doxycycline) determined by the PJI panel for about six weeks and the infection was controlled well. One year later, the patient underwent a total knee arthroplasty (TKA) on another knee for severe osteoarthritis. Unfortunately, his left knee was swollen and painful again after surgery, with elevated inflammatory indexes on admission. At this time, mNGS showed that this might be a Mycoplasma hominis PJI case, while the microbial culture results were negative. Interestingly, 16S PCR also indicated that the pathogenic bacteria might be Mycoplasma hominis, validating the accuracy of mNGS. Therefore, the patient was treated with DAIR and doxycycline, and the clinical outcome was satisfactory.Case 2 was an 88-year-old male who received total knee arthroplasty (TKA) on the right knee due to osteoarthritis. Five days post-operation, his right knee had been swollen and painful, and a small amount of pus was exuded from the incision on admission. Laboratory tests showed elevated CRP (>90 mg/L), ESR (120 mm/h), SF-WBC . The pathogens were identified by mNGS and routine microbial culture results were all negative, but optimized microbial culture results were positive in cases 3,4, 6, and 16S PCR results were positive in case 3,4,6,7. Among these cases, case 5 was a patient who underwent UKA on the left knee 4 years ago  optimized microbial culture method; 2) after DNA extraction from samples, 16s PCR amplification and sequencing could be performed. Finally, the PJI panel comprehensively judged the results of these methods, and decided whether to modify the antibiotic regimens according to Mycoplasma reported by mNGS based on the therapeutic effect of the primary antibiotics. The final diagnosis of Mycoplasma PJI was made by the clinical efficacy of targeted treatment , 3, 4, 6, 7 by 16s PCR, and failed in case 1 (mixed infection), case 2  and case 5. This suggested the limitation of 16S PCR in mixed infection.Although this method provided the reference for physicians, we should be cautious to interpret the results, as g et al. , Snellerg et al.  and Murag et al.  reportedg et al.  and Snelg et al.  isolatedu et al. , on the r et al.  reportedr et al.  successfr et al. . This want years . HoweverMycoplasma PJI, and all cases in this study were treated successfully after following this procedure. This procedure can also be used as a novel strategy for the diagnosis of PJI caused by other rare pathogenic bacteria.Based on previous reports and the results of this study, we proposed a standardized and optimized diagnostic procedure for Mycoplasma PJI cases were included in this study so far, the sample size was still far from adequate, so it was necessary to further expand the sample size by combining multiple centers to verify the true clinical value of this procedure; 2) Because Mycoplasma was difficult to culture and isolate, there might be other cases of Mycoplasma PJI that existed during the study, but were missed diagnosis due to the absence of mNGS test or negative mNGS results; 3) It was important to state that mNGS could only detected the nucleic acid of the microorganism, so it was impossible to determine whether the microorganism was alive or not. Therefore, it was necessary to consider and modify the antibiotic regimen according to the clinical reality.This study had some limitations: 1) Although the largest number of Mycoplasma PJI and then summarize a mNGS based standardized and optimized procedure for the diagnosis of Mycoplasma PJI, which might also be offered as a new diagnostic strategy for rare pathogenic bacteria PJI.In conclusion, we reported the largest number of cases of The data presented in the study are deposited in the China National GeneBank Database (CNGBdb) repository, accession number CNP0001047; more details are available from the corresponding author on a reasonable request.The studies involving human participants were reviewed and approved by The First Affiliated Hospital, Fujian Medical University. The patients/participants provided their written informed consent to participate in this study.WZ and XF designed the study; YQC, HD and YC performed the experiments and wrote the manuscript; CH, XC and CZ collected clinical samples and patient clinical information; ZH performed genotyping analysis; YH and WL analyzed data. All authors contributed to the article and approved the submitted version.


Positive correlations were observed between the amount of urinary leakage according to the 1-h pad test 1, 3, 6, 9, and 12 months after RARP and the change in the cross-sectional area of PPM by RARP . A positive correlation was also noted between the amount of urinary leakage 6 and 12 months after RARP and the preoperative RUM diameter (p < 0.05). The amount of urinary leakage 1, 3, 6, 9, and 12 months after RARP negatively correlated with the change in the antero-posterior diameter of the membranous urethra (MU diameter) from the static to dynamic phases during the Valsalva maneuver by cine MRI. Furthermore, the change in the MU diameter negatively correlated with the change in the cross-sectional area of PPM (p < 0.05). PPM and RUM play significant roles as a supportive mechanism to maintain urinary continence by functioning as a urethral support.The present study investigated the role of a urethral support system to maintain urinary continence after robot-assisted radical prostatectomy (RARP), with a focus on pelvic floor muscles, such as the puboperinealis muscle (PPM) and rectourethralis muscle (RUM). Finally, 323 patients who underwent RARP were analyzed in this study. All patients performed a one-hour pad test 1, 3, 6, 9, and 12 months after RARP to assess urinary incontinence and MRI before and 9 months after RARP to evaluate the pelvic anatomical structure. The preoperative cross-sectional area of PPM (2.21 ± 0.69 cm Preoperative risk factors for urinary incontinence after prostatectomy include an advanced age, higher BMI, larger prostate volume, severe preoperative lower urinary tract symptoms, and a history of prostatectomy for benign prostatic hyperplasia2. Preoperative anatomical variables measured on magnetic resonance imaging (MRI), such as the length of the membranous urethra (MU) and periurethral supporting structures, have also been associated with urinary incontinence after RARP3. However, the mechanisms underlying the development of urinary incontinence have not yet been examined in detail.Although robot-assisted radical prostatectomy (RARP) has recently become the gold standard treatment for localized prostate cancer, postoperative urinary incontinence is still a complication that decreases the quality of life (QOL) of patients7. It comprises the puborectalis, pubococcygeus, and iliococcygeus muscles. The puboperinialis muscle (PPM) is the medial part of the puborectalis, arising from the posterior surface of the pubis on each side and passing posteriorly beside the urethra8. The rectourethralis muscle (RUM) is located at the interface between PPM and the rectum. Right and left PPM muscle edges are located close together in an area between the urethra and rectum, immediately lateral to RUM. Since these structures may form a strong sling and influence the stabilization of the MU, we considered the preoperative status of and surgical damage to these structure to potentially be important factors contributing to urinary incontinence after RARP. In the present study, we conducted an anatomical analysis of the structures around the urethra using MRI before and after RARP, and discussed the mechanisms underlying postoperative urinary incontinence.The levator ani muscle is an important anatomical and functional structure for maintaining urinary continence, and its preservation improves urinary continenceWe conducted a prospective observational cohort study. Patients who underwent RARP in our hospital between 2014 and 2019 were included in the present study. Patients with organ-confined prostate cancer were considered to be candidates for RARP and were included in the study. Inclusion criteria were (1) clinically localized prostate cancer, (2) age < 75 years, and (3) an Eastern Cooperative Oncology Group performance status of 0. During this period, 533 patients who met the inclusion criteria underwent RARP. The Ethics Committee of Fukushima Medical University Hospital approved this study  and all patients provided written informed consent. This study was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.9. This approach is initiated with transverse peritoneotomy between the bladder and rectum, followed by retrovesical dissection. After dissection of the bladder neck, the prostatic vascular pedicles are ligated and the prostate is removed. Preservation of the neurovascular bundles is recommended to patients with low or favorable intermediate risk prostate cancer. Posterior reconstruction was performed on all cases in the present study. Vesicourethral anastomosis was conducted using the Van Velthoven procedure. RARP was performed or supervised by a single surgeon (Y.K.).RARP was performed using a four-arm da Vinci Si™ surgical system  by the transperitoneal posterior approach reported by Guillonneau et al. in conventional laparoscopic procedures10. We considered it important to perform the assessment of urinary incontinence objectively because the anatomical analysis was objectively conducted by MRI. Patients performed the one-hour pad test 1, 3, 6, 9, and 12 months after RARP to evaluate the amount of urinary leakage. Pad test information was collected by urology nurses.Urinary incontinence was evaluated by the one-hour pad test recommended by the International Continence SocietyMRI was performed before and 9 months after RARP in all cases because the effects of postoperative inflammation in muscles needed to be excluded. Static and dynamic MRI were performed using a 1.5 T MR scanner . Dynamic MRI consisted of a T2-weighted, single slice True Fast Imaging with Steady-state Procession (TrueFISP) sequence in the sagittal, coronal, and transverse planes. The cross-sectional area of PPM and left–right (transverse) diameter of RUM (RUM diameter) were measured on T2-weighted fast spin echo (FSE) sequences. The left and right cross-sectional areas of PPM at the height of RUM on the dorsal side of the urethra were calculated using coronal sections, and the sum of the left and right cross-sectional areas was defined as the cross-sectional area of PPM . The significance of differences was considered when p < 0.05. Parametric comparisons between before and after RARP were performed using the paired 10, continence rates were 85.1, 44.6, 75.5, 75.5, 76.5, and 78.0% before and 1, 3, 6, 9, and 12 months after RARP, respectively  were enrolled in the present study  was significantly reduced by 19% after RARP . No significant change was observed in the RUM diameter between before and after RARP . There was no significant correlation between the change in the cross-sectional area of PPM and preoperative anti-androgen administration in the simple regression model (p = 0.49). There was also no significant correlation between the change in the cross-sectional area of PPM and neurovascular bundle (NVB) preservation in the simple regression model (p = 0.17). In addition, this correlation was not found regardless of unilateral or bilateral neurovascular bundle (NVB) preservation .We compared the cross-sectional area of PPM and RUM diameter using MRI before and after RARP Table . The pre11. In the simple regression model, the amount of urinary leakage 1 or 3 months after RARP positively correlated with BMI . The amount of urinary leakage 1, 6, or 9 months after RARP negatively correlated with NVB preservation during RARP . The amount of urinary leakage 6 months after RARP positively correlated with prostate volume  preservation during RARP or prostate volume, which are considered to be factors which may affect urinary continenceWe performed a correlation analysis between the amount of urinary leakage according to the one-hour pad test after RARP and MRI parameters, such as the cross-sectional area of PPM or RUM diameter Table . In the The multiple regression model revealed a negative correlation between the amount of urinary leakage 3, 9, or 12 months after RARP and the postoperative cross-sectional area of PPM  enhances the postoperative function of pelvic muscles, including the levator ani muscle, reduces urinary incontinence after prostatectomy, and improves QOL outcomes related to urinary continenceThere are several limitations that need to be addressed. First, this was a single-center analysis. Furthermore, postoperative MRI was only performed 9 months after RARP in all cases because the effects of postoperative inflammation in muscles need to be excluded. Second, we conducted pre- and postoperative anatomical analyses of the structure around the urethra using MRI, but not a functional analysis, such as the urethral pressure profile. Third, Valsalva effort is variable and subjective. The degree of dynamic movement is influenced by this effort. Therefore, further detailed studies are warranted.PPM and RUM play significant roles in the supportive mechanism to maintain urinary continence as a urethral support. Surgical interventions during RARP lead to the atrophy of PPM, the loss of its function as a urethral sling, and, ultimately, urinary incontinence after RARP. The preoperative measurement of PPM by MRI may predict urinary incontinence after RARP. The further development of surgical procedures to prevent or minimize damage to and reinforce this urethral support system may contribute to the mitigation of urinary incontinence after RARP.