Improved recovery of urinary small extracellular vesicles by differential ultracentrifugation.

Extracellular vesicles (EVs) are lipid-membrane enclosed structures that are associated with several diseases, including those of genitourinary tract. Urine contains EVs derived from urinary tract cells. Owing to its non-invasive collection, urine represents a promising source of biomarkers for genitourinary disorders, including cancer. The most used method for urinary EVs separation is differential ultracentrifugation (UC), but current protocols lead to a significant loss of EVs hampering its efficiency. Moreover, UC protocols are labor-intensive, further limiting clinical application. Herein, we sought to optimize an UC protocol, reducing the time spent and improving small EVs (SEVs) yield. By testing different ultracentrifugation times at 200,000g to pellet SEVs, we found that 48 min and 60 min enabled increased SEVs recovery compared to 25 min. A step for pelleting large EVs (LEVs) was also evaluated and compared with filtering of the urine supernatant. We found that urine supernatant filtering resulted in a 1.7-fold increase on SEVs recovery, whereas washing steps resulted in a 0.5 fold-decrease on SEVs yield. Globally, the optimized UC protocol was shown to be more time efficient, recovering higher numbers of SEVs than Exoquick-TC (EXO). Furthermore, the optimized UC protocol preserved RNA quality and quantity, while reducing SEVs separation time.

Biomarkers for Pre-Treatment Risk Stratification of Prostate Cancer Patients: A Systematic Review.

BACKGROUND: Prostate cancer (PCa) is one of the most frequently occurring malignancies. Although most cases are not life-threatening, approximately 20% endure an unfavorable outcome. PSA-based screening reduced mortality but at the cost of an increased overdiagnosis/overtreatment of low-risk (lrPCa) and favorable intermediate-risk (firPCa) PCa. PCa risk-groups are usually identified based on serum Prostate-Specific Antigen (PSA), the Gleason score, and clinical T stage, which have consistent although variable specificity or subjectivity. Thus, more effective and specific tools for risk assessment are needed, ideally making use of minimally invasive methods such as liquid biopsies. In this systematic review we assessed the clinical potential and analytical performance of liquid biopsy-based biomarkers for pre-treatment risk stratification of PCa patients. METHODS: Studies that assessed PCa pre-treatment risk were retrieved from PubMed, Scopus, and MedLine. PCa risk biomarkers were analyzed, and the studies' quality was assessed using the QUADAS-2 tool. RESULTS: The final analysis comprised 24 full-text articles, in which case-control studies predominated, mostly reporting urine-based biomarkers (54.2%) and biomarker quantification by qPCR (41.7%). Categorization into risk groups was heterogeneous, predominantly making use of the Gleason score. CONCLUSION: This systematic review unveils the substantial clinical promise of using circulating biomarkers in assessing the risk for prostate cancer patients. However, the standardization of groups, categories, and biomarker validation are mandatory before this technique can be implemented. Circulating biomarkers might represent a viable alternative to currently available tools, obviating the need for tissue biopsies, and allowing for faster and more cost-effective testing, with superior analytical performance, specificity, and reproducibility.

Aberrantly Glycosylated GLUT1 as a Poor Prognosis Marker in Aggressive Bladder Cancer.

Muscle-invasive bladder cancer (MIBC) remains a pressing health concern due to conventional treatment failure and significant molecular heterogeneity, hampering the development of novel targeted therapeutics. In our quest for novel targetable markers, recent glycoproteomics and bioinformatics data have pinpointed (glucose transporter 1) GLUT1 as a potential biomarker due to its increased expression in tumours compared to healthy tissues. This study explores this hypothesis in more detail, with emphasis on GLUT1 glycosylation patterns and cancer specificity. Immunohistochemistry analysis across a diverse set of human bladder tumours representing all disease stages revealed increasing GLUT1 expression with lesion severity, extending to metastasis, while remaining undetectable in healthy urothelium. In line with this, GLUT1 emerged as a marker of reduced overall survival. Revisiting nanoLC-EThcD-MS/MS data targeting immature O-glycosylation on muscle-invasive tumours identified GLUT1 as a carrier of short glycosylation associated with invasive disease. Precise glycosite mapping uncovered significant heterogeneity between patient samples, but also common glycopatterns that could provide the molecular basis for targeted solutions. Immature O-glycosylation conferred cancer specificity to GLUT1, laying the molecular groundwork for enhanced targeted therapeutics in bladder cancer. Future studies should focus on a comprehensive mapping of GLUT1 glycosites for highly specific cancer-targeted therapy development for bladder cancer.

Targeted and Self-Adjuvated Nanoglycovaccine Candidate for Cancer Immunotherapy.

Advanced-stage solid primary tumors and metastases often express mucin 16 (MUC16), carrying immature glycans such as the Tn antigen, resulting in specific glycoproteoforms not found in healthy human tissues. This presents a valuable approach for designing targeted therapeutics, including cancer glycovaccines, which could potentially promote antigen recognition and foster the immune response to control disease spread and prevent relapse. In this study, we describe an adjuvant-free poly(lactic-co-glycolic acid) (PLGA)-based nanoglycoantigen delivery approach that outperforms conventional methods by eliminating the need for protein carriers while exhibiting targeted and adjuvant properties. To achieve this, we synthesized a library of MUC16-Tn glycoepitopes through single-pot enzymatic glycosylation, which were then stably engrafted onto the surface of PLGA nanoparticles, generating multivalent constructs that better represent cancer molecular heterogeneity. These glycoconstructs demonstrated affinity for Macrophage Galactose-type Lectin (MGL) receptor, known to be highly expressed by immature antigen-presenting cells, enabling precise targeting of immune cells. Moreover, the glycopeptide-grafted nanovaccine candidate displayed minimal cytotoxicity and induced the activation of dendritic cells in vitro, even in the absence of an adjuvant. In vivo, the formulated nanovaccine candidate was also nontoxic and elicited the production of IgG specifically targeting MUC16 and MUC16-Tn glycoproteoforms in cancer cells and tumors, offering potential for precise cancer targeting, including targeted immunotherapies.

A multivalent CD44 glycoconjugate vaccine candidate for cancer immunotherapy.

Cancer presents a high mortality rate due to ineffective treatments and tumour relapse with progression. Cancer vaccines hold tremendous potential due to their capability to eradicate tumour and prevent relapse. In this study, we present a novel glycovaccine for precise targeting and immunotherapy of aggressive solid tumours that overexpress CD44 standard isoform (CD44s) carrying immature Tn and sialyl-Tn (sTn) O-glycans. We describe an enzymatic method and an enrichment strategy to generate libraries of well-characterized cancer-specific CD44s-Tn and/or sTn glycoproteoforms, which mimic the heterogeneity found in tumours. We conjugated CD44-Tn-derived glycopeptides with carrier proteins making them more immunogenic, with further demonstration of the importance of this conjugation to overcome the glycopeptides' intrinsic toxicity. We have optimized the glycopeptide-protein maleimide-thiol conjugation chemistry to avoid undesirable cross-linking between carrier proteins and CD44s glycopeptides. The resulting glycovaccines candidates were well-tolerated in vivo, inducing both humoral and cellular immunity, including immunological memory. The generated antibodies exhibited specific reactivity against synthetic CD44s-Tn glycopeptides, CD44s-Tn glycoengineered cells, and human tumours. In summary, we present a promising prototype of a cancer glycovaccine for future therapeutical pre-clinical efficacy validation.

Relationship of posterior peripheral corneal layers and the trabecular meshwork: an immunohistological and anatomical study.

BACKGROUND/AIM: With the popularity of endothelial keratoplasty (EK) procedures, Descemet membrane (DM) EK and pre-Descemet EK, considerable work has been done on understanding the posterior corneal anatomy. Most of the information available relates to the central cornea. We evaluated the peripheral cornea to explore the immunohistological and anatomical relationship between the pre-Descemet layer (PDL), DM and trabecular meshwork (TM). METHODS: Six donor human sclerocorneal discs were studied. PDL, DM and TM were examined by light microscopy, transmission electron microscopy (TEM) and immunohistology. The DM was peeled from the centre to the limit of its peripheral attachment, to reach the transition zone (TZ) between TM and peripheral cornea. Ten-micron sections were stained with antibodies against collagens 1, 2, 3, 4, 5, 6, 12, elastin, myocilin, wnt-1, aquaporin, tenascin C, laminin and integrin alpha 3. RESULTS: Collagens 2, 3, 4, laminin and myocilin were predominantly seen in the TZ between TM and peripheral cornea. Wnt-1, integrin alpha 3 and tenascin C were highly concentrated in TM. Collagen 1 was present predominantly in the corneal stroma. On TEM; DM was thinner with a denser banded structure spread throughout its thickness in the periphery compared with the central cornea where it presents as the distinct anterior banded layer. CONCLUSION: The TZ between DM, PDL and TM shows a unique histological structure at the periphery. The collagen and elastin fibres of the TM are continuous with the PDL. The structures are firmly attached to each other. These findings provide structural information that is relevant to the preparation of DMEK donor tissue.

OncoUroMiR: Circulating miRNAs for Detection and Discrimination of the Main Urological Cancers Using a ddPCR-Based Approach.

The three most common genitourinary malignancies (prostate/kidney/bladder cancers) constitute a substantial proportion of all cancer cases, mainly in the elderly population. Early detection is key to maximizing the patients' survival, but the lack of highly accurate biomarkers that might be used through non-/minimally invasive methods has impaired progress in this domain. Herein, we sought to develop a minimally invasive test to detect and discriminate among those urological cancers based on miRNAs assessment through ddPCR. Plasma samples from 268 patients with renal cell (RCC; n = 119), bladder (BlCa; n = 73), and prostate (PCa; n = 76) carcinomas (UroCancer group), and 74 healthy donors were selected. Hsa-miR-126-3p, hsa-miR-141-3p, hsa-miR-153-5p, hsa-miR-155-5p, hsa-miR-182-5p, hsa-miR-205-5p, and hsa-miR-375-3p levels were assessed. UroCancer cases displayed significantly different circulating hsa-miR-182-5p/hsa-miR-375-3p levels compared to healthy donors. Importantly, the hsa-miR-155-5p/hsa-miR-375-3p panel detected RCC with a high specificity (80.54%) and accuracy (66.04%). Furthermore, the hsa-miR-126-3p/hsa-miR-375-3p panel identified BlCa with a 94.87% specificity and 76.45% NPV whereas higher hsa-miR-126-3p levels were found in PCa patients. We concluded that plasma-derived miRNAs can identify and discriminate among the main genitourinary cancers, with high analytical performance. Although validation in a larger cohort is mandatory, these findings demonstrate that circulating miRNA assessment by ddPCR might provide a new approach for early detection and risk stratification of the most common urological cancers.

An approach to reduce Descemet's membrane scrolling: Relevance to Descemet's membrane endothelial keratoplasty (DMEK).

Purpose: We aimed to determine whether Descemet's membrane (DM) scrolling occurs primarily along the vertical or horizontal axis and establish whether oval trephination along the axis of least scrolling can reduce the grade of the scroll. Methods: The longest limbus-to-limbus axis on 28 sclerocorneal discs was taken as the horizontal axis. The horizontal (n = 7) or (right angles to it) vertical (n = 6) axis was marked on DM before peeling it off. The direction and grade of scrolling was observed. Narrow strips (3-4 mm wide) were then cut along the two axes (n = 4 each) and the scrolling pattern was observed. Ellipses (7 × 9 mm) of DM were punched along the two axes (n = 6 each) and the scrolls graded. Immunofluorescent staining for elastin on horizontal and vertical tissue sections from three DM samples was performed. The intensity and thickness of elastin staining were measured. Results: Twenty-four (85.72%) DM samples showed scrolling along the horizontal axis, none showed scrolling along the vertical axis, and four (14.28%) samples showed a spiral scroll, regardless of which axis was marked (grade 3.7 and 3.6). Vertically oval discs showed significantly reduced scrolling (grade 1.2) compared to horizontally oval discs (grade 3.5). Narrow strips of DM showed a similar scrolling pattern. Immunohistology showed no difference in any of the parameters examined along the two axes or from the center to the periphery. Conclusion: DM scrolls primarily along the horizontal axis. Vertically oval DM samples show minimal scrolling, which can be an advantage in DMEK. Differential scrolling is not determined by the distribution of elastin.

Plastic pollution on the world's coral reefs.

Coral reefs are losing the capacity to sustain their biological functions1. In addition to other well-known stressors, such as climatic change and overfishing1, plastic pollution is an emerging threat to coral reefs, spreading throughout reef food webs2, and increasing disease transmission and structural damage to reef organisms3. Although recognized as a global concern4, the distribution and quantity of plastics trapped in the world's coral reefs remains uncertain3. Here we survey 84 shallow and deep coral ecosystems at 25 locations across the Pacific, Atlantic and Indian ocean basins for anthropogenic macrodebris (pollution by human-generated objects larger than 5 centimetres, including plastics), performing 1,231 transects. Our results show anthropogenic debris in 77 out of the 84 reefs surveyed, including in some of Earth's most remote and near-pristine reefs, such as in uninhabited central Pacific atolls. Macroplastics represent 88% of the anthropogenic debris, and, like other debris types, peak in deeper reefs (mesophotic zones at 30-150 metres depth), with fishing activities as the main source of plastics in most areas. These findings contrast with the global pattern observed in other nearshore marine ecosystems, where macroplastic densities decrease with depth and are dominated by consumer items5. As the world moves towards a global treaty to tackle plastic pollution6, understanding its distribution and drivers provides key information to help to design the strategies needed to address this ubiquitous threat.

Immunomodulatory glycomedicine: Introducing next generation cancer glycovaccines.

Cancer remains a leading cause of death worldwide due to the lack of safer and more effective therapies. Cancer vaccines developed from neoantigens are an emerging strategy to promote protective and therapeutic anti-cancer immune responses. Advances in glycomics and glycoproteomics have unveiled several cancer-specific glycosignatures, holding tremendous potential to foster effective cancer glycovaccines. However, the immunosuppressive nature of tumours poses a major obstacle to vaccine-based immunotherapy. Chemical modification of tumour associated glycans, conjugation with immunogenic carriers and administration in combination with potent immune adjuvants constitute emerging strategies to address this bottleneck. Moreover, novel vaccine vehicles have been optimized to enhance immune responses against otherwise poorly immunogenic cancer epitopes. Nanovehicles have shown increased affinity for antigen presenting cells (APCs) in lymph nodes and tumours, while reducing treatment toxicity. Designs exploiting glycans recognized by APCs have further enhanced the delivery of antigenic payloads, improving glycovaccine's capacity to elicit innate and acquired immune responses. These solutions show potential to reduce tumour burden, while generating immunological memory. Building on this rationale, we provide a comprehensive overview on emerging cancer glycovaccines, emphasizing the potential of nanotechnology in this context. A roadmap towards clinical implementation is also delivered foreseeing advances in glycan-based immunomodulatory cancer medicine.

The pre-Descemet's layer (Dua's layer, also known as the Dua-Fine layer and the pre-posterior limiting lamina layer): Discovery, characterisation, clinical and surgical applications, and the controversy.

The pre-Descemet's layer/Dua's layer, also termed the Dua-Fine layer and the pre-posterior limiting lamina layer, lies anterior to the Descemet's membrane in the cornea, is 10 µm (range 6-16) thick, made predominantly of type I and some type VI collagen with abundant elastin, more than any other layer of the cornea. It has high tensile strength (bursting pressure up to 700 mm of Hg), is impervious to air and almost acellular. At the periphery it demonstrates fenestrations and ramifies to become the core of the trabecular meshwork, with implications for intraocular pressure and glaucoma. It has been demonstrated in some species of animals. The layer has assumed considerable importance in anterior and posterior lamellar corneal transplant surgery by improving our understanding of the behaviour of corneal tissue during these procedures, improved techniques and made the surgery safer with better outcomes. It has led to the innovation of new surgical procedures namely, pre-Descemet's endothelial keratoplasty, suture management of acute hydrops, DALK-triple and Fogla's mini DALK. The discovery and knowledge of the layer has introduced paradigm shifts in our age old concepts of Descemet's membrane detachment, acute corneal hydrops in keratoconus and Descemetoceles, with impact on management approaches. It has been shown to contribute to the pathology and clinical signs observed in corneal infections and some corneal dystrophies. Early evidence suggests that it may have a role in the pathogenesis of keratoconus in relation to its elastin content. Its contribution to corneal biomechanics and glaucoma are subjects of current investigations.

Does the placement of a collagen-fibrin sealant reduce complications of radical inguinal lymph node dissection? - Comparative study in patients with penile cancer.

OBJECTIVE: Management of patients with penile cancer (PeC) with palpable inguinal lymph nodes (ILNs) relies on radical ILN dissection (RILND). Low burden of nodal metastatic disease may lead to long-lasting survival with surgical management. Nevertheless, RILND involves significant postoperative morbidity. We compared the complications of patients undergoing RILND with (RILND-T) and without (RILND-0T) placement of a collagen-fibrin sealant patch on the resection bed. MATERIALS AND METHODS: We conducted an observational retrospective study. Data from men submitted to RILND-T and RILND-0T from Jan/2001 to Feb/2022, in a tertiary care centre were compared. The primary endpoint was the overall incidence of complications until 1 month after the procedure and their respective severity in both cohorts (Clavien-Dindo classification system). Secondarily, length of hospital stay (LOHS) was analysed. The placement of a collagen-fibrin sealant patch was left at the surgeon's discretion. RESULTS: Seven patients underwent RILND-T and 20 underwent RILND-0T, respectively. There were no differences in pathologic TNM stage nor in the total number of ILNs removed (17 ± 4 vs. 20 ± 8, p = 0.37). Overall, 23 (85.2%) patients had complications. The complication rate was similar in both cohorts (85.7% vs 85%, p = 0.73). Surgical wound infection (3/7 vs. 11/20) and lymphocele (4/7 vs. 11/20) were the most reported complications. Patients undergoing RILND-T were discharged faster (mean length of hospital stay 9 ± 3 vs 19 ± 20 days, p = 0.22). CONCLUSIONS: The application of a collagen-fibrin sealant patch on the resection bed does not seem to reduce the postoperative complication rate in patients undergoing RILND. Nevertheless, a trend towards a shorter LOHS in patients with RILND-T cannot be excluded and should be validated by further studies with a higher number of patients.

Neoadjuvant chemotherapy for muscle-invasive bladder cancer: does variant histology matter?

PURPOSE: The most frequent histology of bladder tumors is urothelial carcinoma. Most are pure urothelial carcinomas (PUC) but up to one-third of the cases present variant histological (VH) features. The aim of this study was to evaluate the role of variant histology in neoadjuvant chemotherapy (NAC) response in patients with urothelial muscle-invasive bladder cancer. METHODS: We retrospectively analyzed data from 77 patients with bladder cancer who performed neoadjuvant chemotherapy at two institutions. RESULTS: Complete pathological response (ypT0) was higher in patients with PUC (38.5%), comparing with VH (12%). Logistic regression analysis demonstrated that variant histology is associated with an 89% lesser likelihood of tumor downstaging, with advanced clinical T stages and positive smoking history as independent predictors. The estimated mean cancer-specific survival was 68.91 months for PUC patients and 50.23 months for VH patients (log rank test, P = 0.024). Multivariate Cox regression analysis demonstrated that VH and clinical T stage were independent predictors of cancer-specific survival, indicating a worse outcome for patients with VH and advanced clinical T stages. CONCLUSIONS: There are only a few retrospective studies evaluating the clinical impact of variant histology tumors, which are mainly managed as PUC. Our results demonstrate that VH is associated with a worse likelihood of tumor downstaging after NAC and a worse cancer-specific survival in bladder cancer patients. There is a need for further studies and genetic analysis to identify the patients most likely to achieve ypT0 status and downstaging after NAC.

Variant histologies of urothelial carcinoma: Does it change the survival outcomes in patients managed with radical cystectomy?

OBJECTIVE: To investigate the impact of variant histologies (VH) of urothelial carcinoma (UC) on survival outcomes after radical cystectomy (RC). MATERIALS AND METHODS: Data from 181 patients with UC treated with RC between January 2013 and December 2019 at a single tertiary care referral center were retrospectively accessed. All RC specimens were assigned by genitourinary dedicated pathologists. Overall survival (OS), disease-specific survival (DSS) and recurrence-free survival (RFS) were evaluated using the Kaplan-Meier methodology and the Cox proportional hazards regression. RESULTS: Of 181 patients, 43.1% (n = 78) had VH, with the most common being squamous differentiation (n = 29), followed by mixed variants (n = 18), micropapillary variant (n = 10) and other subtypes (n = 21). The median (range) follow-up was 35 (18-59) months. Kaplan-Meier survival analysis shows that median OS and DS were significantly worse for VH patients (78 vs 31 months, p = 0.038; not reached vs 42 months; p = 0.016). At 5 years, VH was associated with a 12% and 14% decrease in OS and DSS, respectively. No significant statistical difference between the two groups was reached regarding RFS. However, after adjusting for confounders, such as, demographics characteristics, comorbidities and pathological features, VH were not associated with any survival outcomes. CONCLUSIONS: Our study evidenced the high incidence of bladder cancers with VH. Although clearly associated with features of more aggressive behavior, VH had not any significant impact in survival expectancies when all confounders are adjusted in multivariate analyses.

Glycoproteogenomics characterizes the CD44 splicing code associated with bladder cancer invasion.

Rationale: Bladder cancer (BC) management demands the introduction of novel molecular targets for precision medicine. Cell surface glycoprotein CD44 has been widely studied as a potential biomarker of BC aggressiveness and cancer stem cells. However, significant alternative splicing and multiple glycosylation generate a myriad of glycoproteoforms with potentially distinct functional roles. The lack of tools for precise molecular characterization has led to conflicting results, delaying clinical applications. Addressing these limitations, we have interrogated the transcriptome and glycoproteome of a large BC patient cohort for splicing signatures. Methods:CD44 gene and its splicing variants were assessed by Real Time-Polymerase Chain Reaction (RT-PCR) and RNAseq in tumor tissues. The co-localization of CD44 and short O-glycans was evaluated by proximity ligation assay (PLA), immunohistochemistry and double-immunofluorescence. An innovative glycoproteogenomics approach, integrating transcriptomics-customized datasets and glycomics for protein annotation from nanoLC-ESI-MS/MS experiments, was developed and implemented to identify CD44 variants and associated glycosignatures. The impact of CD44 silencing on proliferation and invasion of BC cell lines and glycoengineered cells was determined by BrdU ELISA and Matrigel invasion assays, respectively. Antibody phosphoarrays were used to investigate the role of CD44 and its glycoforms in the activation of relevant oncogenic signaling pathways. Results: Transcriptomics analysis revealed remarkable CD44 isoforms heterogeneity in bladder cancer tissues, as well as associations between short CD44 standard splicing isoform (CD44s), invasion and poor prognosis. We further demonstrated that targeting short O-glycoforms such as the Tn and sialyl-Tn antigens was key to overcome the lack of cancer specificity presented by CD44. Glycoproteogenomics allowed, for the first time, the comprehensive characterization of CD44 splicing code at the protein level. The concept was applied to invasive human BC cell lines, glycoengineered cells, and tumor tissues, enabling unequivocal CD44s identification as well as associated glycoforms. Finally, we confirmed the link between CD44 and invasion in CD44s-enriched cells in vitro by small interfering RNA (siRNA) knockdown, supporting findings from BC tissues. The key role played by short-chain O-glycans in CD44-mediated invasion was also demonstrated through glycoengineered cell models. Conclusions: Overall, CD44s emerged as biomarker of poor prognosis and CD44-Tn/ Sialyl-Tn (STn) as promising molecular signatures for targeted interventions. This study materializes the concept of glycoproteogenomics and provides a key vision to address the cancer splicing code at the protein level, which may now be expanded to better understand CD44 functional role in health and disease.

Pelagic observations of the midwater scorpionfish Ectreposebastes imus (Setarchidae) suggests a role in trophic coupling between deep-sea habitats.

During pelagic video transects off Santo Antão, Cabo Verde, we encountered the midwater scorpionfish Ectreposebastes imus in midwater between 300 and 800 m over a bottom depth of about 1000 m. The fish were typically positioned vertically with their heads pointing upwards. These first midwater observations of E. imus suggest migratory (potentially feeding) behaviour into the pelagic realm and hence a possible role of this species in the trophic coupling between the pelagic and benthic habitats in the deep seas of Cabo Verde and elsewhere in its global distribution.

Contributions to the taxonomy of the mugilid genus Chelon Artedi (Teleostei: Mugilidae), with a major review of the status of C. persicus Senou, Randall & Okiyama, 1995.

New data based on multigene phylogenetic analyses using the COI, 16S, and cytb genes and subsequent molecular species delimitation revealed that the mugilid genus Chelon contains 11 species. Of these, two species, Chelon sp. A and Chelon sp. B, remained unidentified in previous studies. While Chelon sp. B seemingly is a close relative to C. dumerili (Steindachner, 1870), no more detailed information were provided for Chelon sp. A which is present along the east coast of South Africa. Genetic analyses performed in this study, revealed that specimens from the Arabian/Persian Gulf and the Red Sea are nested within one genetic lineage alongside Chelon sp. A. Morphological, morphometric, and meristic analyses of specimens from all three areas demonstrated that they belong to Chelon persicus Senou, Randall & Okyiama, 1995. Chelon persicus was originally described in the genus Chelon but was later placed either in the genus Liza or Planiliza. However, results presented herein confirm its placement in Chelon and its distribution range is extended in the Western Indian Ocean from the type locality (Arabian/Persian Gulf) west to the Red Sea and south to South Africa. Further, the uncertain validity of the recently described Chelon caeruleus Deef, 2018 from the Mediterranean Sea and a previously proposed generic character distinguishing Chelon from Planiliza and Parachelon, the shape of the paired postzygapophyses of the second vertebra, are briefly discussed.

Glycoproteomics identifies HOMER3 as a potentially targetable biomarker triggered by hypoxia and glucose deprivation in bladder cancer.

BACKGROUND: Muscle invasive bladder cancer (MIBC) remains amongst the deadliest genitourinary malignancies due to treatment failure and extensive molecular heterogeneity, delaying effective targeted therapeutics. Hypoxia and nutrient deprivation, oversialylation and O-glycans shortening are salient features of aggressive tumours, creating cell surface glycoproteome fingerprints with theranostics potential. METHODS: A glycomics guided glycoproteomics workflow was employed to identify potentially targetable biomarkers using invasive bladder cancer cell models. The 5637 and T24 cells O-glycome was characterized by mass spectrometry (MS), and the obtained information was used to guide glycoproteomics experiments, combining sialidase, lectin affinity and bottom-up protein identification by nanoLC-ESI-MS/MS. Data was curated by a bioinformatics approach developed in-house, sorting clinically relevant molecular signatures based on Human Protein Atlas insights. Top-ranked targets and glycoforms were validated in cell models, bladder tumours and metastases by MS and immunoassays. Cells grown under hypoxia and glucose deprivation disclosed the contribution of tumour microenvironment to the expression of relevant biomarkers. Cancer-specificity was validated in healthy tissues by immunohistochemistry and MS in 20 types of tissues/cells of different individuals. RESULTS: Sialylated T (ST) antigens were found to be the most abundant glycans in cell lines and over 900 glycoproteins were identified potentially carrying these glycans. HOMER3, typically a cytosolic protein, emerged as a top-ranked targetable glycoprotein at the cell surface carrying short-chain O-glycans. Plasma membrane HOMER3 was observed in more aggressive primary tumours and distant metastases, being an independent predictor of worst prognosis. This phenotype was triggered by nutrient deprivation and concomitant to increased cellular invasion. T24 HOMER3 knockdown significantly decreased proliferation and, to some extent, invasion in normoxia and hypoxia; whereas HOMER3 knock-in increased its membrane expression, which was more pronounced under glucose deprivation. HOMER3 overexpression was associated with increased cell proliferation in normoxia and potentiated invasion under hypoxia. Finally, the mapping of HOMER3-glycosites by EThcD-MS/MS in bladder tumours revealed potentially targetable domains not detected in healthy tissues. CONCLUSION: HOMER3-glycoforms allow the identification of patients' subsets facing worst prognosis, holding potential to address more aggressive hypoxic cells with limited off-target effects. The molecular rationale for identifying novel bladder cancer molecular targets has been established.

Distribution, associations and role in the biological carbon pump of Pyrosoma atlanticum (Tunicata, Thaliacea) off Cabo Verde, NE Atlantic.

Gelatinous zooplankton are increasingly acknowledged to contribute significantly to the carbon cycle worldwide, yet many taxa within this diverse group remain poorly studied. Here, we investigate the pelagic tunicate Pyrosoma atlanticum in the waters surrounding the Cabo Verde Archipelago. By using a combination of pelagic and benthic in situ observations, sampling, and molecular genetic analyses (barcoding, eDNA), we reveal that: P. atlanticum abundance is most likely driven by local island-induced productivity, that it substantially contributes to the organic carbon export flux and is part of a diverse range of biological interactions. Downward migrating pyrosomes actively transported an estimated 13% of their fecal pellets below the mixed layer, equaling a carbon flux of 1.96-64.55 mg C m-2 day-1. We show that analysis of eDNA can detect pyrosome material beyond their migration range, suggesting that pyrosomes have ecological impacts below the upper water column. Moribund P. atlanticum colonies contributed an average of 15.09 ± 17.89 (s.d.) mg C m-2 to the carbon flux reaching the island benthic slopes. Our pelagic in situ observations further show that P. atlanticum formed an abundant substrate in the water column (reaching up to 0.28 m2 substrate area per m2), with animals using pyrosomes for settlement, as a shelter and/or a food source. In total, twelve taxa from four phyla were observed to interact with pyrosomes in the midwater and on the benthos.