Cells prepare for fluctuations in nutrient availability by storing energy in the form of neutral lipids in organelles called Lipid Droplets (LDs). Upon starvation, fatty acids (FAs) released from LDs are trafficked to different cellular compartments to be utilized for membrane biogenesis or as a source of energy. Despite the biochemical pathways being known in detail, the spatio-temporal regulation of FA synthesis, storage, release, and breakdown is not completely understood. Recent studies suggest that FA trafficking and metabolism are facilitated by inter-organelle contact sites that form between LDs and other cellular compartments such as the Endoplasmic Reticulum (ER), mitochondria, peroxisomes, and lysosomes. LD-LD contact sites are also sites where FAs are transferred in a directional manner to support LD growth and expansion. As the storage site of neutral lipids, LDs play a central role in FA homeostasis. In this mini review, we highlight the role of LD contact sites with other organelles in FA trafficking, channeling, and metabolism and discuss the implications for these pathways on cellular lipid and energy homeostasis. Maintaining energy homeostasis is obligatory for cellular survival and fitness. In general, cells store energy in the form of fatty acids (FAs) when nutrients are abundant or in excess. Stored FAs can be released under stress conditions or when nutrients are limited, in order to be used for the synthesis of membrane lipids or to fuel energy production. FAs are stored in the form of neutral lipids, with triacylglycerol (TAG) and sterol esters (SE) being the most abundant in most cells. These neutral lipids are incapable of forming membranes; therefore, they are sequestered in specific organelles known as Lipid Droplets (LDs) that form at the ER . LDs havVice versa, under carbon restriction, stored FAs are utilized, leading to reduction in LD size and number. Notably, it was also reported that during prolonged periods of nutrient deprivation when autophagy is activated, the amount of LDs increases sequestering free FAs released during autophagic degradation of membranous organelles. This provide a buffering system to reduce lipotoxic accumulation of free FAs  complex as acyl-CoA, and this process takes place in the cytosol. FA synthesis starts from acetyl-CoA (C2-CoA), which is elongated in a cyclic reaction using malonyl-CoA (a C3-CoA) as the carbon chain donor. Malonyl-CoA is synthesized from acetyl-CoA by the acetyl-CoA carboxylase (ACC) enzymes, and production of this substrate is a rate-limiting step for FA synthesis.Bulk FAs are synthesized n) acyl chain is coupled to the malonyl-ACP in a condensation reaction, yielding 3-ketoacyl-ACP. The 3-keto group is reduced to an alcohol, yielding 3-hydroxyacyl-ACP, which is subsequently dehydrated to yield 2-enoyl-acyl-ACP. Finally, the C2–C3 double bond is reduced, yielding the elongated acyl chain (Cn+2). After several reaction cycles, the end product acyl chain is transferred from ACP to acyl-CoA.FA synthesis starts with ligation of the acetyl- and malonyl- to an acyl carrier protein (ACP), by a trans-acylase enzyme. Acetyl-ACP is elongated with the addition of two carbon atoms in a cyclic cascade of four subsequent reactions. First, the . There are 13 different ACS isoforms in mammals that function in different tissues and subcellular locations . Acyl-CoA and FFAs are only minor components of the cellular lipidome. Therefore, lipid biosynthesis is a major player in FA metabolism. FAs are incorporated in two major membrane lipid families: glycerophospholipids and sphingolipids.sn-1 and sn-2 positions subsequently, catalyzed by ER localized acyltransferases . PA can be either activated to CDP-diacylglycerol by CDP-DAG synthase or dephosphorylated to diacylglycerol (DAG) by PA hydrolase  (Glycerophospholipids (GPL) are bulk constituents of membranes, thus a major sink for acyl chains. Most steps in GPL biosynthesis take place in the ER. The precursor to all GPLs is phosphatidic acid (PA), which is produced by acylation of glycerol-3-phosphate (G3P) at the mammals) . CDP-DAGmammals) . Alternamammals) . For examammals) .N-acylated to yield the basic sphingolipid ceramide. In metazoans, ceramide is the precursor to simple sphingolipids, such as sphingomyelin, and complex glycosphingolipids such as cerebrosides and gangliosides. In yeast, ceramide is the precursor to inositol-phosphoceramide and derived glycosphingolipids.Sphingolipid synthesis starts with the formation of the sphingosine backbone. First, 3-ketodihydrosphingosine is formed by condensation of serine and palmityl-CoA, catalyzed by serine palmitoyltransferase (SPT). The keto-group of 3-ketosphinganine is reduced to an alcohol, yielding sphinganine, which is O-acylation of their respective precursors. In yeast and mammals these are mainly TAG and SE, synthesized from DAG and sterols by DGAT and SAT enzymes, respectively. In addition, there are minor storage lipids such as O-acyl ceramides  are oxidized in mitochondria, yielding acetyl-CoA. Very long chain FAs (Cn > 22) and branched chain FAs cannot be handled by mitochondria, and are first partially oxidized in peroxisomes, after which intermediate short chain FAs are transferred to mitochondria for the final oxidation steps.To be used for energy production fatty acids must be broken down to acetyl-CoA, which can enter the citric acid cycle. This process is known as β-oxidation, named after oxidation of the acyl-CoA β-carbon. In yeast, β-oxidation takes place in peroxisomes, whereas in higher eukaryotes both peroxisomes and mitochondria are required. In metazoans, medium- and long chain FAs  colocalize with seipin and promote LD biogenesis at specialized microdomains within ER tubules. These proteins mediate stable ER-LD contact sites thus regulating LD biogenesis, number, and size . SeveralThe endoplasmic reticulum is spread throughout the cell, with tubules stretching from the center of the cell toward the plasma membrane, and it forms contacts with all cellular organelles . A twistTo facilitate β-oxidation, FAs have to be released from the NLs stored in LDs and delivered to either peroxisomes or mitochondria . NL cataIn yeast and plants, peroxisomes are the sole organelles that perform β-oxidation, whereas in metazoans they are responsible for β-oxidation of very long-chain FAs. Therefore, LDs and peroxisomes must collaborate to regulate flux and availability of FAs within the cell . Close pThe topological details and molecular machinery that tether LDs and peroxisomes has remained largely unknown. Recent work demonstrated that the AAA-ATPase M1 Spastin found on LDs can directly interact with the peroxisomal ATP binding cassette subfamily D member 1, ABCD1. This interaction regulates LD-peroxisome contacts and promotes FA inter-organelle exchange . InteresUnderstanding of the physiological roles of LD-peroxisome contact sites in localized FA trafficking and metabolism requires more intense investigation. Specifically, understanding how disruption of LD-peroxisome contact sites contributes to human peroxisomal disorders is still unclear. Peroxisomal function is also known to be altered in aging, thus contributing to a host of age-related diseases including diabetes, neurodegeneration, and cancer .In metazoans, mitochondria are a key site for β-oxidation. FA β-oxidation in mitochondria requires release of FAs from the LD through lipolysis and transfer to mitochondria, where they are imported via carnitine shuttling. As such, LD-mitochondria contact sites are highly apparent in cells with high fatty acid oxidation rates, including brown adipose tissue and muscle cells. In skeletal muscle cells, LDs have been observed to be “sandwiched” between mitochondria, likely to increase the contact surface, and these drastic LD-mitochondria contacts have been proposed facilitate the high demand of FA trafficking for rapid energy production .In agreement with LD-contacts facilitating FA handover to mitochondria, LD-mitochondria contacts have been observed to respond to metabolic state and the need for FA oxidation. For example, when cells are acutely starved, organelles are broken down via autophagy, releasing free fatty acids as lipid breakdown products. The released FAs are efficiently incorporated into LDs, and are handed over from LDs to mitochondria for β-oxidation . Under sInterestingly, contacts with LDs may dictate mitochondrial metabolism, as mitochondria that are in contact with LDs have been shown to form a subpopulation that is biochemically distinct from cytosolic mitochondria. The proteome of LD-approximate mitochondria was found to differ from cytosolic mitochondria, and both subpopulations partake in different metabolic processes . StrikinSeveral proteins have been reported to play a role in LD-mitochondria contact site formation , but theLysosomes play a central role in the recycling of organelles and biomolecules, and contain hydrolytic enzymes that can breakdown proteins, nucleic acids, and lipids. In yeast, LDs can be consumed by micro-autophagy (or micro-lipophagy), during which LDs are directly internalized in the vacuole (the yeast lysosome) and degraded by the internal hydrolases . This prIn yeast, interactions between LDs and vacuoles  have been shown to be tightly regulated by metabolic state. During diauxic shift, when glucose is exhausted and yeast cells switch from fermentative growth to aerobic restoration, the size of the vacuole is increased. During this stage, they also form a unique contact site between the nucleus and the vacuole, dubbed the nuclear-vacuole junction (NVJ), and LDs start to localize there . The NVJEnrichment of enzymes responsible for neutral lipid biosynthesis at the NVJ is likely to drive local LD biogenesis. Indeed, during diauxic shift the fatty acid-CoA ligase Faa1 is targeted to the NVJ. By locally activating free fatty acids, yielding acyl CoA, Faa1 likely provided acyl-CoA substrate required for the formation of neutral lipids. In addition, other lipid metabolic enzymes (such as Tsc13 and Pah1) have also been described to be recruited to the NVJ in different conditions . These oin vitro. Therefore, Mdm1 provides a scaffolding function that facilitates the generation of a high local concentration of activated FAs and promotes their incorporation into neutral lipids  as Snazarus (Snz), which is highly expressed in the Drosophila fat body (FB) and was originally associate with a longevity phenotype . SimilarLDs are reservoirs of FAs that are found in organisms from microbes to humans. Survival of organisms in nutrient scarce conditions depends on their ability to mobilize and utilize their stored FAs. FAs have multiple cellular fates and following their mobilization they are targeted to different cellular compartments to be processed. How FAs are trafficked among organelles remains a key question in biology. LDs form inter-organelle contacts that are thought to facilitate efficient trafficking of FAs. At the core of these contacts are tethering proteins that physically hold these organelles together, in addition to lipid metabolic enzymes and transfer proteins that meditate inter-organelle lipid movement. Arguably, the least understood contact site is the one that forms between LDs and peroxisomes. Emerging technologies such as enzyme-mediated proximity labeling will be useful to determine the full proteome of LD contact sites.2 has been shown to be required for trafficking of very long chain FAs from LDs to peroxisomes  tethers possess (putative) lipid binding domains, including the phosphoinositide binding Pleckstrin homology domain found on VPS13A/C, and Phox homology domains on Mdm1 and Snazarus . Peroxisoxisomes , possibloxisomes , giving oxisomes .In vitro reconstitution of FA metabolism at organelle contacts will allow for better understanding of how spatial organization of enzymes fine tune the efficiency and outcome metabolic reactions. Finally, the contribution of LD contact sites to cellular lipid homeostasis and organismal health is currently not completely understood. A key question that remains to be answered is how the disruption of inter-organelle networks leads to metabolic disorders of FA metabolism.Future research will rely on the development of reliable tools to monitor FA trafficking in cells. Beyond their role in facilitating direct FA transfer, LD contact sites are viewed as dynamic platforms that play central roles in regulating FA metabolism. HH and MR contributed to writing and discussing ideas. MR designed the figure. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Vibrio cholerae, Pseudomonas aeruginosa, and Staphylococcus aureus. Techniques used to characterize biofilm adhesion are also reviewed. An understanding of biofilm adhesion strategies can guide the development of novel approaches to inhibit or manipulate biofilm adhesion and growth.Bacterial biofilms are communities of cells enclosed in an extracellular polymeric matrix in which cells adhere to each other and to foreign surfaces. The development of a biofilm is a dynamic process that involves multiple steps, including cell-surface attachment, matrix production, and population expansion. Increasing evidence indicates that biofilm adhesion is one of the main factors contributing to biofilm-associated infections in clinics and biofouling in industrial settings. This review focuses on describing biofilm adhesion strategies among different bacteria, including  Having lived on Earth for billions of years, bacteria thrive in hospitable environments, including rivers, soil, and vegetation, and further inhabit extreme environments, such as hot springs, the deep ocean, and even nuclear waste . BacteriThe study of bacterial biofilms is highly relevant to human health. Many chronic and acute diseases, including cystic fibrosis , tubercuvia divergent mechanisms depending on the species. When biofilm cells face environmental challenges, such as nutrient limitation, they undergo collective dispersal and reinitiate the biofilm developmental cycle on a new favorable surface  parent, Gloag et al. demonstrated that mucoid colony biofilms are more cohesive compared to WT, whereas the adhesion of colony biofilms from WT and rugose small-colony variants are comparable , and three matrix proteins, RbmA, Bap1, and RbmC  and rugosity and biofilm structure modulator C (RbmC), have been suggested to contribute to the cell-to-surface adhesion in V. cholerae biofilms as well as to biofilm strength  pore-forming toxin, which is known to recognize host cell surfaces using its β-prism domain  and numerous regulatory two-component systems and transcriptional regulators, making it remarkably capable of adapting to different environments  The Pil-Chp complex senses external signals to induce cAMP production, and cAMP, in turn, activates the transcription of pilY1 gene and (ii) the PilY1 located on the outer membrane can induce activity of the diguanylate cyclase SadC by signaling through type IV pili. SadC has a GGDEF domain that catalyzes c-di-GMP production and therefore promotes biofilm formation , and Pel  proteins that are attached to the thick peptidoglycan cell wall through covalent linkages. Many Gram-positive strains use CWA proteins to bind to components of the mammalian extracellular matrix (ECM), such as fibrinogen, fibronectin, and cytokeratin, which, in turn, associate with proteins found on the surface of mammalian cells . Due to  devices .In vitro, staphylococci can form biofilms on abiotic surfaces, but biofilm adhesion and formation are significantly enhanced if the surfaces are treated with ECM-containing media, or simply fibronectin . MSCRAMMs include a wide variety of adhesins, many of which have the ability to bind multiple ligands resulting in some degree of crosstalk between adhesins and target ligands. MSCRAMMs are present abundantly in many Gram-positive species, such as faecalis . S. aureronments . A commoke folds . The defke folds .S. aureus to attach to a wide range of surfaces of both biotic and abiotic origin. On biotic surfaces, the cell-to-surface interactions are well-defined through specific ligand binding. For example, collagen-binding adhesin, fibronectin-binding proteins, and fibrinogen-binding proteins are some MSCRAMMs that enable S. aureus to attach to the mammalian ECM. Ponnuraj et al. proposed a binding mechanism called “dock, lock, and latch (DLL)” that describes the mode of action for a fibrinogen-binding MSCRAMM protein in S. epidermidis called SdrG , SdrE, and FnBPA, all have such side-chain-independent mechanostability. Such side-chain-independent mechanism might confer an invasive advantage to staphylococci. Similarly, in a subsequent work, Prystopiuk et al. unraveled the molecular interactions underlying the three-component FnBPA-Fn-integrin system  comparable to SdrG-Fgβ binding  with ultra-strong (~2 nN) forces, comparable to covalent interactions . More inn system . S. aure surface . Their r binding , using a binding .S. aureus adhesion can be enhanced by shear forces , including the extracellular fibrinogen binding protein, extracellular matrix binding protein, and extracellular adherence protein . Eap is the most well-studied protein in the SERAMs family. The protein sequence of Eap lacks the LPXTG motif, suggesting that Eap is associated but not covalently linked to the peptidoglycan cell wall. Over the past few decades, Eap has been shown to be an anchorless protein that contributes to staphylococcal adhesion and is capable of modulating the inflammatory response via interactions with the glycoprotein ICAM-1. While Eap has been shown to bind a number of host extracellular components, the exact mechanism of how Eap is involved in bacterial adhesion to these components is still unclear  and the release of eDNA from dead cells. PIA is synthesized by enzymes encoded by the biofilms . Vuong epolymers . More relar mass . Using elar mass . They subiofilms . An inte2+ concentrations and acidic pH, suggesting that Bap also acts as a sensor of the extracellular environment  in S. aureus. Aap and SasG share 54% sequence identity; they both possess a signal peptide at the N-terminus, followed by an A domain and a B region with 3–12 repeating G5 and E domains of 128 amino acids (via two distinct Zn2+-dependent mechanisms: (1) the B region forming twisted rope-like structures between neighboring cells, catalyzed by Zn2+ (Another interesting class of matrix protein in staphylococci biofilms is accumulation-associated protein (Aap) in no acids . These pno acids . Functiono acids . The unpno acids . More ge by Zn2+ , and (2) by Zn2+ . Interes by Zn2+ . This obS. aureus cells use their unique adhesive mechanisms to colonize biotic surfaces in humans, rendering S. aureus a common opportunistic bacterial species in many contexts. Biofilm formation is closely associated with pathogenicity in S. aureus. For example, Parastan et al. reported a significant correlation between the adhesion-related genes and multidrug resistance patterns of S. aureus clinical isolates  Structural biology: More crystal structures of the adhesins need to be solved to build our understanding of bacterial adhesion to specific biotic surfaces. The recent rapid progress in cryo-EM could be harnessed for structural solving , and cla(2) Biophysical characterization: While AFM has become a powerful tool in the study of bacterial adhesion, significant technical expertise is required to perform and interpret AFM experiments. Closer collaboration between microbiologists and biophysicists will certainly lead to many new discoveries regarding bacterial adhesion.V. cholerae biofilms, revealing well-defined cell ordering that is highly dependent on cell-to-surface adhesion. It remains to be seen whether such cell ordering is a general phenomenon in all biofilm-forming species.(3) The connection between cell adhesion and biofilm architecture should be further explored in other species. So far, the single-cell biofilm imaging technique has mainly been applied to (4) Molecular scale information has yet to be connected with bulk adhesion measurements. Many techniques reviewed here reveal the adhesive energy between bulk biofilms (such as a colony biofilm) and a substrate; it is unclear how to compare AFM measurements at the cellular level to these bulk measurements.We believe that with coordinated efforts from microbiologists, biophysicists, and engineers, the secret ingredients of biofilm adhesion will be uncovered in the near future. A comprehensive understanding of biofilm adhesion will pave the way for the discovery of new chemicals that specifically target biofilm-surface interactions and lead to more forward-looking, innovative functional biofilm materials that adhere to various surfaces on demand.ZJ reviewed the literature and complied the review. TN, SM, RO, and JY revised the initial draft and reviewed the final draft. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Earlier use of PSMA-RLT and combinatorial approaches with other novel agents are the promising future of mCRPC management.This review is a summary of recent findings on the role of prostate-specific membrane antigen (PSMA) in metastatic castration-resistant prostate cancer (mCRPC) and how they can be implemented into patient management. The multiple aspects, interactions and functions of PSMA expression should be considered with regard to diagnosis and treatment. Approval of Metastatic castration-resistant prostate cancer (mCRPC) remains an incurable disease, despite multiple novel treatment options. The role of prostate-specific membrane antigen (PSMA) in the process of mCRPC development has long been underestimated. During the last years, a new understanding of the underlying molecular mechanisms of rising PSMA expression and its association with disease progression has emerged. Accurate understanding of these complex interactions is indispensable for a precise diagnostic process and ultimately successful treatment of advanced prostate cancer. The combination of different novel therapeutics such as androgen deprivation agents, 177LU-PSMA radioligand therapy and PARP inhibitors promises a new kind of efficacy. In this review, we summarize the current knowledge about the most relevant molecular mechanisms around PSMA in mCRPC development and how they can be implemented in mCRPC management. Increasing the understanding of the underlying molecular mechanisms of PCA stages and their progression is therefore crucial for optimal and patient-specific treatments.Prostate cancer (PCA) is the most common male cancer with approximately 60,000 new cases in Germany each year . PCA repMost patients recover after prostatectomy after initial diagnosis. However, about 20% present with advanced disease or metastases at initial presentation ,4. AndroThe advent of PSMA-targeted radioligand therapy (PSMA-RLT) for advanced mCRPC almost ten years ago has clearly enriched therapy options to choose from with regard to mCRPC treatment for doctors worldwide . Ever si68Ga-PSMA positron-emission topography (PSMA-PET) allows PSMA detection with high precision and low toxicity, making PSMA the ideal theranostic target. Rising PSMA during evolving castration resistance and a positive correlation with disease progression adds to this favorable biomarker profile of PSMA [ of PSMA ,22,23. H of PSMA .Little was known about the biological function of PSMA in cancer, nor about its role in the progression of prostate cancer into the stage of castration resistance and eventually neuroendocrine PCA, until very recently. Slowly, we begin to fully understand the role of PSMA in recurrent and progressing prostate cancer and its impact on evolving castration resistance in more detail. In this review, we will dissect the role of PSMA in evolving castration resistance and why this makes it not only a valuable therapeutic target in late mCRPC, but also a valid and safe biomarker for imaging and disease staging .PCA develops from a prostatic intraepithelial neoplasia (PIN) as an asymptomatic precursor of adenocarcinoma. Through accumulation of progressive somatic mutations and loss of distinct basal and secretory cell layers, PIN eventually evolves into a local prostate tumor, bearing the potential to metastasize through local invasion or lymphatic and hematogenic pathways. Rising prostate-specific antigen (PSA) serum levels offer the possibility to detect PCA early; however, the PSA blood test does not allow discrimination between low- and high-risk tumors . TherefoThere is a reported relationship with 17–30% incidence between advanced PCAs, which exhibit classical and next-generation ADT resistance, and downstream neuroendocrine-like differentiation (NELD) . Rising Although we are provided multiple modern state-of-the-art screening technologies and opportunities for molecular testing, these are rarely used to tightly monitor ongoing disease during mCRPC patient management. There is reason to use biopsies; however, the effort and violation of the patient to receive the material would need more justification if it was to be used for global genomic and gene expression profiling . Additio4), which is known to help with angiogenesis, thus making PSMA an interesting target in antiangiogenic scenarios, which has been shown to be integrin-mediated [PSMA was originally discovered as a folate folypoly glutamate carboxypeptidase (FGCP) and was found in healthy jejunal brush border membrane, prostate, proximal tubules of the kidney and nervous system glia, as well as salivary glands ,49,50. Hmediated ,53,66,67mediated ,68,69,70PSMA expression is associated with early serum PSA recurrence and disease progression. Therefore, it can be considered a valid biomarker for disease monitoring . Further177Lu has been proven to be the safest radionuclide compared to 225Ac in the treatment of mCRPC, and 68Ga is mostly used for imaging [After binding and internalization, the receptor is recycled, as other membrane-bound receptors, via clathrin-coated pits and association with filamin A ,78,79. D imaging ,84. Noneα-dihydroxy-testosterone travels into the cell, is converted to testosterone by the 5α-reductase and binds to the AR. The AR–testosterone complex then binds to its target DNA motif in the nucleus. PSMA expression seems to be suppressed by the AR–testosterone complex, while it is upregulated by unbound AR. Expression of the PSMA-encoding gene FOLH1 is downregulated by androgens which bind the androgen receptor. Therefore, ADT conversely stimulates FOLH1 transcription and translation by non-androgen-bound AR [PSMA expression is inversely modulated by androgens and the androgen receptor (AR) signaling cascade . Normallbound AR . The FOLbound AR ,85,86. Hbound AR .2+ levels via IP3. Ca2+ aids proliferation, migration and vascularization in general, but also activates mTORC2 and thus promotes the whole process of tumor growth and proliferation, which is a hallmark of cancer cells [2+ levels and might support cancer cell survival strategies [Upon androgen deprivation therapy or AR blockade, the PSMA level rises in the tumor tissue and its metastases at the cellular level, thus enabling therapeutic targeting as well as detection by PSMA-based imaging. As an effect of efficient biochemical castration through ADT, however, the overall tumor size may decrease after castration or enzalutamide treatment . After Aer cells ,89,90. Mrategies . Thereforategies ,92. Andrrategies . Howeverrategies ,93,94. Trategies . Althougrategies . Its des3 to PIP2, and thereby antagonizes the function of PI3K. Through this mechanism, PTEN suppresses the activation of the downstream oncogenic RAC-alpha serine/threonine-protein kinase (AKT) and mammalian target of rapamycin (mTOR) signaling pathway. Further, PTEN provides centromere stability and double-strand break repair [Treatment of the AR pathway has long been and is still the treatment of choice for refractory metastatic CRPC. Very recently, however, it was demonstrated by mRNA transcriptome analyses that AR inhibition activates the PI3K pathway ,97. In tk repair . PTEN lok repair . Partialk repair . This lok repair .Enzymatic cleavage of glutamate from poly-glutamated folate by PSMA increases the level of free glutamates, which potentially bind to and activate a G-protein coupled receptor (GPCR). The GPCR in turn then activates PI3K and downstream AKT and mTOR, leading to increased cell growth and proliferation in cancer cells . As if tTaken together, the PSMA molecule presents with a multitude of functions and seems to be involved in more than one important pathway during cancer cell metabolism and signaling. On one side, the natural function of PSMA to nourish the cell with folate and ensure proper prostate organ function accelerates with higher PSMA expression, thus fueling cancer cell growth, proliferation and migration, as well as tumor angiogenesis. On the other side, PSMA expression putatively reaches a level at which it positively influences cancer cell aggressiveness by interfering with regulation (PTEN) and pathway switches, as seen for MAPK/ERK vs. IP3K–AKT–mTOR. As the cherry on the cake, PSMA even combines these two branches, as Kaittanis et al. showed us, by using free glutamate from the folate metabolism to activate the IP3K–AKT–mTOR pathway via stimulation of GPCRs. These interactions are depicted schematically in 68Ga-PSMA-PET imaging allows the detection of the PSMA expression level at different sites of disease progression. Drug-related changes in PSMA expression intensity have to be incorporated as described earlier. Additionally, PSMA imaging enables the detection of tumor mass and heterogeneity and together with FDG-PET it might enable proper staging and the early recognition of tumor dedifferentiation to a more aggressive stage [18F-FDG-PET [18F-FDG-uptake has been found to associate with higher glucose metabolism and PSMA suppression, this requires further investigation for complete understanding of these interactions [As described above, PSMA is involved in a multitude of cell metabolism and tumor-aggravating pathways during prostate cancer progression. However, it is also expressed in benign prostate tissue and a variety of other non-malignant conditions ,106. In ve stage . However-FDG-PET . GLUT1 e-FDG-PET . Based o-FDG-PET ,112. Altractions . It has ractions . Not onlractions . In the 177Lu-PSMA-617-RLT finds application in patient treatment only after therapy failure and the lack of more treatment options until its approval. The first phase III VISION trial was accomplished, meeting both primary endpoints of overall survival and radiographic progression-free survival [177Lu-PSMA-617-RLT an accessible targeted treatment for >80% of mCRPC patients [Currently, survival ,114. Aftpatients . Those ppatients . Althougpatients . Thus, tpatients . With repatients .There is a rising number of studies investigating the options of combining PSMA-targeted therapies with other potent systemic or local treatments. We, for example, have been able to show that patients with visceral hepatic metastases undergoing PSMA-RLT can be treated with local SIRT efficiently and that this significantly prolongs survival . This apAlthough this might be an approach for later and aggressive stages as well as for spontaneous extension of therapies, whenever needed, it represents nonetheless an emergency treatment, as progress had already taken place. One option is the simultaneous use of ADT and PSMA-targeted treatment from the moment the PSMA expression level rises and is detected. This might be at a much earlier timepoint than PSMA imaging and RLT are currently considered. Yet, due to the underlying molecular mechanisms in which PSMA is involved, it might strongly interfere with progression, especially when PTEN is mutated or lost or in the case of BRCA1 and 2 aberrations . It has 68Ga-PSMA-PET is used rarely for this purpose, but only to determine eligibility for 177Lu-PSMA-617 therapy after complete failure and refractory PCA in daily clinical business. This might, however, more be an issue of feasibility and financial support by compulsory health insurance funding than of clinical justification, which might be an international issue. Even though PET imaging is a complex procedure, which wants to be planned and conducted within a certain time frame and requires the patient’s compliance and consent, it is yet less invasive than taking a biopsy. PSMA can also be detected in circulating tumor cells, which does not require more than an additional blood sample from the patient, also called liquid biopsy. A general rise in PSMA expression can therefore be easily detected from this blood sample. However, although it has been shown that PSMA can be detected in CTCs and additionally correlates with disease, this does not localize the tumor’s distribution nor allow grading of local re- or progression, heterogeneity or success of localized therapies [68Ga-PSMA-PET should be included in the diagnostic panel more often and earlier according to the rate of progression and depending on the treatment strategy to improve staging [The principle of theranostics combines the features of a biomarker that is eligible for both targeting and detection. This enables a highly patient- and disease-stage-specific monitoring of biomarker distribution and abundance, as well as direct targeting of the marker-expressing sites with few side effects. As PSMA expression on tumor cells correlates with disease progression, its detection by labeling allows close to real-time detection of tumor growth and migration ,122. Regherapies ,124. An herapies . This miherapies . In the  staging .68Ga-DOTATATE-PSMA and 177Lu-DOTATATE-PSMA-617 are extensively used for imaging and radioligand therapy, respectively. A 12-center study from Germany with 145 enrolled patients revealed a biochemical response, determined by a ≥50% decline in serum PSA in >45% of the patients [225Ac which exhibit unfavorable side effects, such as unspecific binding and significantly higher toxicity [177Lu seems to be the radionuclide of choice and 177Lu-DOTATATE-PSMA-617 the soon-to-be only approved PSMA-RLT [68Ga-PSMA-11 uptake of >1.5 times greater than normal liver uptake was determined as predictive for therapy response by Michael Hofman and his colleagues [The feature of binding a ligand as a receptor and internalizing it paved the way to use PSMA as a specific ferry to transfer antibodies, small molecules bound to radionuclides directly into the tumor. Although antibodies were the first approved imaging agents and seem to not be toxic on the salivary glands, they require longer clearance and seem to have higher hematologic toxicity, as well as sensitivity and specificity issues . As PSMApatients . Comparepatients ,114. Thitoxicity . While tPSMA-RLT . While, PSMA-RLT ,131. 68Glleagues . While alleagues . There illeagues .Covering all promising treatment strategies under investigation for all stages of PCA is out of the scope of this review. As PSMA is most relevant to mCRPC, and to meet the scope of this review, we will focus on treatments that target this stage specifically. Further, only approved or close to-approval treatments will be discussed.The stage of castration resistance is characterized by lack of sensitivity to androgen deprivation, rising PSMA expression and the accumulation of mutations in DNA damage repair (DDR) genes such as BRCA1/2. Dysregulation in DDR pathways leads to mismatch mutations, aberrant gene expression and cancer cells escaping apoptosis. Therefore, it seems reasonable to target these pathways in a combinatorial, consecutive or alternating treatment sequence with novel and potent agents.177Lu-PSMA-617-RLT. PSMA-RLT functions at two sites: binding of PSMA leads to internalization and suppresses the enzymatic function of PSMA. This leads to less folate uptake and less free glutamates that in turn might activate downstream pathways such as IP3K–AKT–mTOR signaling. Further, the radionuclide, in this case 177Lu, that is internalized with the PSMA receptor induces double-strand breaks and downstream apoptosis of the tumor cells. The combination of ADT and PSMA-RLT therefore offers to prevent or at least delay disease progression and the development of resistance to agents such as AA and enzalutamide. By interrupting the downstream effects on tumor cell proliferation and growth via enhanced PSMA expression, this treatment combination interferes with the accumulation of gain-of-function point mutations in the AR gene and mismatch repair.The most obvious and best investigated to date is the combination of PSMA-RLT with novel ADTs such as abiraterone acetate (AA) and enzalutamide. Although the outcome of the VISION trial should be taken into consideration, before application outside compassionate use, this option seems more than realistic for future application ,20. The Intact DDR mechanisms mediated by the BRCA2 gene might hamper the success of induced double-strand breaks, by simply maintaining genomic stability in the tumor cell. Mutations of the BRCA1/2 gene are associated with a higher risk of certain tumors, including prostate cancer. Moreover, BRCA mutations are a strong prognosticator of response to poly-ADP ribose polymerase inhibitors (PARPi) such as olaparib and platinum ,135. TheThese studies and data are very promising; however, none of these treatments should be applied blind and without awareness of the patient’s gene status. Therefore, genomic profiling and gene expression analyses should be considered an important part of routine management of mCRPC patients for true precision medicine . FurtherMetastatic CRPC is a complex disease and during its progression tumor cells activate multiple pathways to escape common selective mechanisms such as senescence and apoptosis. The increased expression of PSMA is part of cancer cell survival strategies and the activation of oncogenic pathways. There is a huge need for accurate profiling on the basis of the molecular interactions. This could be met by using global genomic and gene expression profiling of patient material. Biopsies, once taken, should be subjected to global screenings for suspected key players in defining disease severity and enable a precise stage classification. Additionally, circulating tumor cells as well as peripheral blood should be used to detect biomarker expression and reveal correlations with progression. Indispensable for this ultimate patient screening is imaging, whether it may be global imaging tracers, such as FDG to quantify tumor mass and identify metastases, or specific ligands that allow stage-specific expression, such as PSMA. PSMA and SSRs are definitely targets that allow detection of tumor heterogeneity and prediction of progression and therapy efficacy. As there are multiple ways for tumors to progress and inter-patient fluctuations in marker expression, clinicians need a precise profile of each individual patient. Besides genome and transcriptome multi-omics, screening approaches should be taken into account. Current approved and impending treatment options provide powerful tools to fight the uncurable stage of mCRPC. Yet, potent treatments such as PSMA-RLT, once approved, and PARPi should be considered for earlier use, and our current understanding of rigid treatment lines should be discussed. Combinatorial, consecutive and alternating treatment strategies fitted to the patient’s disease profile are the future of mCRPC treatment.


Ultrasound (US) could become a standard of care imaging modality for the quantitative assessment of femoral cartilage thickness for the early diagnosis of knee osteoarthritis. However, low contrast, high levels of speckle noise, and various imaging artefacts hinder the analysis of collected data. Accurate, robust, and fully automatic US image-enhancement and cartilage-segmentation methods are needed in order to improve the widespread deployment of this imaging modality for knee-osteoarthritis diagnosis and monitoring. In this work, we propose a method based on local-phase-based image processing for automatic knee-cartilage image enhancement, segmentation, and thickness measurement. A local-phase feature-guided dynamic-programming approach is used for the fully automatic localization of knee-bone surfaces. The localized bone surfaces are used as seed points for automating the seed-guided segmentation of the cartilage. We evaluated the Random Walker (RW), watershed, and graph-cut-based segmentation methods from 200 scans obtained from ten healthy volunteers. Validation against manual expert segmentation achieved a mean dice similarity coefficient of 0.90, 0.86, and 0.84 for the RW, watershed, and graph-cut segmentation methods, respectively. Automatically segmented cartilage regions achieved 0.18 mm localization accuracy compared to manual expert thickness measurement. Osteoarthritis (OA) of the knee joint is the most common type of arthritis in elderly people . It occuImaging plays an important role during OA detection and management. Currently, X-ray planar radiography is the standard imaging modality used in clinical practice for diagnosing OA and monitoring disease progression . OsteophIn order to provide a cost-effective and real-time imaging alternative to MRI, ultrasound (US) was investigated to diagnose and monitor cartilage degeneration ,18,19,20As a means of decreasing inter- and intra-user measurement errors, various research groups have focused on developing automated US image-enhancement and cartilage-segmentation methods for accurate and robust cartilage-thickness measurement ,22,23. IIn order to provide a robust solution to some of these imaging conditions, in this work we propose an intensity-invariant cartilage US image-enhancement and segmentation framework. During the first stage, B-mode US images are enhanced using local-phase-based image features. During the second stage, knee-bone surfaces are automatically localized from enhanced US images using a local-phase image-feature-guided dynamic-programming approach. Localized bone surfaces are used as seeds for automatic segmentation. The final stage involves automatic mean cartilage thickness measurement. We evaluated the performance of three different seed-based segmentation methods. A preliminary study of this approach was reported in Reference . In thisWritten consent was obtained prior to the collection of US scans. A total of 200 2D images from 10 healthy volunteers were collected during this study (20 scans per subject). The scans were acquired using a Sonix-Touch US machine  with a 14–5 MHz linear US transducer with a depth setting of 3.5 cm and image resolution of 0.15 mm. During the scans, the knee was positioned at 90 deg of flexion, and the US transducer was placed transversely in line with the medial and femoral condyle above the superior edge of the patella. Different scans of the cartilage were obtained from both the left and right knee joints. An ultrasound technician with 20 years of clinical experience collected all the data.The proposed image-processing framework consists of four main subprocesses: (1) cartilage image enhancement, (2) knee-bone localization for automatic seed initialization, (3) cartilage segmentation, and (4) mean thickness computation .The orientation of the US transducer with respect to the imaged knee surface and the 3D anatomy of the knee affects the cartilage response profile in the acquired US data. If the transducer is perfectly aligned, and attenuation from the soft-tissue interface is low, then the cartilage interface response profile appears as a dominant ridge edge along the scan-line direction. However, due to the inaccurate alignment of the US transducer, this response profile was degraded during data collection, which affected consecutive image analysis. The first step in our framework involves the enhancement of the low-intensity knee-bone surface and cartilage interface by performing image filtering in a frequency domain similar to :(1)USELPT(xIn Equation  ϕ represfined as :(4)TevenH, ∇, and Here The SM, ,29 is foIn Equation , h1S images ,30. LPEAcoustic bone-shadow information in US is important during bone imaging. Real-time feedback of bone-shadow information can guide the clinician to a standardized diagnostic viewing plane with minimal artefacts, and can provide additional information for bone localization. The proposed bone-shadow region enhancement method is based on the confidence-map (CM) approach  using anbined as :(9)CMLP, is achieved by minimizing a cost function composed of two energy functions, internal energy , watershed, and graph-cut, as they showed better performance with prior shape knowledge. RW segmentation is advantageous over the nonsmoothness of the boundaries (metrication error), preference for shorter boundaries (shrinking bias), boundary length regularization, and number of initial seeds ,33,34,35The ideal seed points for the above mentioned segmentation methods must lie inside the region and should be near the center of the region of interest. The distance from the foreground seed pixel to its neighboring pixels should be small enough to allow continuous growing. Automatically extracted bone surfaces are used as initial seeds for automatic cartilage-segmentation algorithms. In Reference  mean carFor the RW segmentation algorithm, background regions were initialized by translating localized bone surfaces V corresponds to pixels and E are the edges connecting each pair of adjacent pixels [In RW, the input image is represented as graph t pixels . Edges aL represents the Laplacian of the graph, I is the identity matrix, x is the probability vector of each pixel, Here, In the watershed algorithm, the gray image is transformed as a topographic relief. The objective of watershed transform is to find the `catchment basins’ and `watershed ridge lines’ that divide the neighboring catchment basin in the image . In a trf is defined as A marker-controlled watershed algorithm is an enhancement of the traditional watershed algorithm which defines a marker and a segmentation function for efficient segmentation of objects with boundaries expressed as ridges. Markers are placed as an internal marker (foreground) associated with the region of interest, and external marker (background) associated with the backgrounds. In traditional watershed, the catchment basin of image function f at specific locations denoted as Markers (M). New image function g is defined asIn the above equation, p represents the pixel co-ordinates, and Here, E), where each pair of the connected node is represented by a single edge S(source), and T(sink) that represents the foreground and background labels. Each edge S and T are separated as The graph-cut segmentation algorithm  is similIn order to automatically measure cartilage thickness, we calculate the Euclidean distance map from the segmented cartilage region. The distance values corresponding to the automatically extracted cartilage boundary were averaged for the final thickness calculation. This analysis was repeated for manually segmented and all automatically segmented cartilage regions during quantitative validation. We also performed a second manual operation by drawing a normal line between the cartilage–bone interface and the synovial space on original B-mode US images at ten different points and the mean thickness was computed for each B-mode US image . Figure Automatically segmented cartilage regions and thickness values were compared with manual segmentation and thickness measurements provided by an expert ultrasound technician. Segmentation validation was obtained by calculating DSC. Automatically computed thickness values were compared with manually measured expert thickness values. We also provide quantitative and qualitative results if B-mode US data were used as input to segmentation methods rather than the enhanced Parameter settings: The Log-Gabor filter was designed using the filter parameters provided in Reference . LPT, and 10.53 and 11.51 (±0.3) seconds, respectively. These computation times include the required time for the image-enhancement and bone-surface localization steps.In The Bland–Altman plots shown in p values as shown in p values are shown in A paired t-test between manual landmark-based cartilage-thickness measurements and measurement obtained from manual segmentation, RW, watershed, and graph-cut segmentation methods at a 5% significance level achieved Knee-cartilage region segmentation and thickness analysis from 2D US scans has potential for the clinical assessment of cartilage degeneration, a clinical indication used for OA diagnosis and monitoring. We presented a fully automatic and accurate method for cartilage image enhancement, segmentation, and thickness measurement from 2D US data. Quantitative evaluations demonstrated that there was no significant agreement between manual landmark-based cartilage-thickness measurement, and thickness measured from manually segmented cartilage regions. During this work, we evaluated three different segmentation methods. The overall qualitative and quantitative results indicate that, between the RW, watershed, and graph-cut algorithms, RW segmentation is more consistent with the manual results. Quantitative evaluations showed that there is no significant agreement between manual landmark-based cartilage thickness measurement, and thickness measured from manually segmented cartilage regions. This further proves the manual segmentation process of US data is an errorprone procedure. Furthermore, manual measurements and segmentations were performed by a single experienced US technician. Intra- and inter-user variability errors need to be evaluated in order to fully understand the challenges involved during the manual segmentation process. In order to fully overcome the errors introduced during the manual segmentation of US data, gold-standard thickness measurements obtained from an MRI scan should be investigated. Furthermore, thickness calculations were performed using the distance function. A more accurate thickness computation method is the star-line-based method proposed in Reference , which wThe proposed framework requires bone-surface translation to mark the initial seeds for the segmentation algorithm. The seeds were translated on the basis of prior shape knowledge of healthy cartilage and were kept constant for the whole dataset during validation. The method will still be successful for segmenting cartilage from subjects with slight-to-moderate OA with thinned (but connected) cartilage. For segmenting broken cartilages associated with severe OA, automatic seed initialization might be problematic. However, since seed extraction is based on the localization of knee-bone surfaces, the seed-selection process is not affected by the severity of the OA. More in-depth analysis is necessary in order to assess the full clinical usability of the proposed work for segmenting cartilage regions from OA patients.The quality of cartilage segmentation depends on the collected image data and seed initialization for the segmentation algorithm. As US is user-dependent modality, an important consideration while evaluating articular cartilage is the inclination and the positioning of the US transducer on the proper plane. During data collection, specific attention was given to collect clinically adequate knee scans. In the future, we plan to develop methods based on deep learning for automatic adequate scan plane selection. In order to improve accuracy and robustness, we plan to extend our work for processing 3D US scans. Recently, medical-image segmentation methods based on deep-learning theory have had successful results. Further comparison of deep-learning-based segmentation methods is required in order to assess the full potential of the proposed framework.In this work, we were interested in the development of a general cartilage enhancement and segmentation method that could be applied to any B-mode US image collected from a standard US machine or point-of-care US device for widespread applicability in a standard clinical setting. In recent years, researchers have been looking into designing segmentation or enhancement methods based on extracted information from raw radio-frequency (RF) US data. Although access to RF data is only available in dedicated research machines, it appears that RF signal information could provide important information about the cartilage and should be further investigated. Elastography and shear-wave elastography (SWE) has also been investigated for imaging cartilage ,45. In Rartilage . Therefo


The Colorectal cancer Study of Austria (CORSA), an ongoing multicenter prospective case‒control study, was initiated to discover prognostic as well as diagnostic biomarkers for colorectal cancer (CRC) risk prediction mainly based on OMICS research, such as genomics, metabolomics, and metagenomics centered at the Institute of Cancer Research at the Medical University of Vienna; recruitment for CORSA started in 2003. Until now, we have generated genomics data, untargeted and targeted metabolomics data, folate-dependent one-carbon metabolism data, and leukocyte telomere length data using the CORSA biobank. The generated data, the collection of biological samples  and the comprehensive CORSA database represents a valuable resource for ongoing and future national and international cooperation projects on CRC research.The Colorectal cancer Study of Austria (CORSA) is comprised more than 13,500 newly diagnosed colorectal cancer (CRC) patients, patients with high- and low-risk adenomas as well as population-based controls. The recruitment for the CORSA biobank is performed in close cooperation with the invited two-stage CRC screening project “Burgenland PREvention trial of colorectal Disease with ImmunologiCal Testing” (B-PREDICT). Annually, more than 150,000 inhabitants of the Austrian federal state Burgenland aged between 40 and 80 are invited to participate using FIT-tests as an initial screening. FIT-positive tested participants are offered a diagnostic colonoscopy and are asked to take part in CORSA, sign a written informed consent, complete questionnaires concerning dietary and lifestyle habits and provide an ethylenediaminetetraacetic acid (EDTA) blood sample as well as a stool sample. Additional CRC cases have been recruited at four hospitals in Vienna and a hospital in lower Austria. A major strength of CORSA is the population-based controls who are FIT-positive and colonoscopy-confirmed to be free of polyps and/or CRC. Colorectal cancer (CRC) is the fourth most common cancer and the third highest cause of cancer-related deaths in Europe, thereby representing a severe public health problem . In AustScreening programs have the potential to detect early precancerous lesions and perform endoscopic removal of adenomas, thereby contributing to the reduction of CRC incidence and mortality . TherefoIn Austria, the organization of screening programs is not regulated nationwide; it is a matter of the federal states. To date, only two invited screening programs are performed in Austria, in the federal states Vorarlberg and Burgenland. In Vorarlberg, a colonoscopy-based screening program is offered to the insured population older than 50 years since 2007. In Burgenland, the screening program “Burgenland Prevention Trial of Colorectal Cancer Disease with Immunological Testing” (BPREDICT), initiated in 2003, is a two-stage invited screening program for individuals aged between 40 and 80 using FIT as the initial screening. Participants with a positive FIT were invited for further diagnostic work-up such as a diagnostic colonoscopy. These participants were asked to take part in the “Colorectal Cancer Study of Austria” (CORSA), sign a written informed consent, complete questionnaires, and provide an ethylenediaminetetraacetic acid (EDTA) blood sample as well as a stool sample for the CORSA biobank. In cooperation with B-PREDICT, several hospitals in Vienna and the Hospital Wiener Neustadt, we have established the CORSA biobank, which currently comprises more than 13,500 participants. From each participant, genomic DNA and plasma samples are stored at −80 °C. Two years ago, we also started recruitment of stool samples within the CRC screening program B-PREDICT.The recruitment of participants for the ongoing CORSA started over 18 years ago at the Medical University of Vienna, Austria. The initial motivation of CORSA was to identify genetic risk variants that modify individual predisposition to CRC. At the beginning, the aim was the identification of single nucleotide polymorphisms (SNPs) as well as tandem repeat minisatellites and their association with CRC risk in a case‒control study design . With thThe aim of CORSA is to identify new robust diagnostic biomarkers for advanced adenomas and early-stage CRC by using state-of-the-art OMICS technologies such as metabolomics, proteomics, and metagenomics in order to improve the identification of patients at higher CRC risk.CORSA is an ongoing population case‒control study of women and men recruiting newly diagnosed CRC patients , histopathologically confirmed invasive cancer of any stage), high-/low-risk adenomas and population-based colonoscopy-negative controls, with an age range between 30 and 90 years. Blood and fecal samples, dietary/lifestyle questionnaires, and anthropometrics/demographics were obtained from each participant.Since 2003, more than 13,500 participants have been recruited across nine sites in Austria. The multicenter recruitment within CORSA follows standardized protocols, resulting in consistent data from all recruitment sites. These sites include the Medical University of Vienna (Department of Surgery), three further hospitals in Vienna , and the Hospital Wiener Neustadt in Lower Austria. Furthermore, the recruitment for CORSA was performed in four hospitals in Burgenland  within the population-based screening program “Burgenland PREvention Trial of colorectal cancer DIsease with ImmunologiCal Testing” (B-PREDICT) .B-PREDICT is an invited two-stage screening project initiated by gastroenterologist Karl Mach at the Hospital Oberpullendorf, Burgenland. Annually, more than 150,000 inhabitants of Burgenland aged between 40 and 80 are invited to participate in this program using a fecal immunochemical test (FIT) as an initial screening. Nowadays, FIT is the preferred approach in testing for occult blood in feces used for CRC screening programs . FIT-posFIT-positive participants from the B-PREDICT screening are recruited when undergoing colonoscopy and comprise CRC patients, high- and low-risk polyps and colonoscopy-negative controls. CORSA participants from other centers are recruited before surgery or at the time of their clinical follow-up and are mainly CRC cases. All subjects gave written informed consent, and the study was approved by the institutional review boards.Genomic DNA, plasma  and stool samples are collected at each CORSA site using harmonized protocols and stored at −80 °C. Paraffin-embedded (FFPE) tissue from CRC cases is stored at room temperature.Within CORSA, no regular sequential follow-up recruitment is performed.Blood draws are performed at least at baseline (first participation in CORSA). In addition, 2263 CORSA participants (17%) have been repeatedly recruited. On average, these participants were recruited about 2.3 times. The mean time period between single participations amounts to 2.9 years. Some participants have a follow-up of about 15 years.Survival data, representing a comprehensive output file comprising confirmed or unconfirmed date of death, date of last contact, and information on missing or incorrect data input, are provided through a biennial clinical data abstraction from the IT database of the Medical University of Vienna, “Allgemeines Krankenhaus Information Management” (AKIM) in cooperation with Statistics Austria. In addition, we abstract survival data in cooperation with the Main Association of Austrian Social Insurance Institutions . Therefore, the social insurance number, first and last name, date of birth, and sex of all participants were processed through a database pipeline. For a subset of the data, the cause of death in terms of ICD-10 category codes of the World Health Organization can be obtained.Clinical data were abstracted from medical records and processed in a structured database following standardized documentation guidelines and according to the General Data Protection Regulation (GDPR). The CORSA databank comprises structured information on diagnosis, treatment, histology, progression (recurrence and metastasis), and survival data . Follow-CORSA participants provided at least a basic CORSA questionnaire assessing data on body mass index (BMI), smoking history, alcohol consumption, education level, family status, profession, basic dietary habits, and diabetes . PatientAdditionally, CORSA participants were asked to complete a slightly modified version of the VITamins And Lifestyle cohort study (VITAL) questionnaire and the European Prospective Investigation into Cancer and Nutrition Food Frequency Questionnaire (EPIC-Potsdam FFQ2). The VITAL questionnaire assesses the use of nutritional supplements, vitamins, medication, physical activity, and social environment . The EPISo far, we have generated genomics data, metabolomics data, folate-dependent one-carbon metabolism data, and leukocyte telomere length data from the CORSA biobank . GWAS dan = 218), 6 months (n = 10), and 12 months (n = 17). The analytical measurements were performed at Bevital AS, Bergen, Norway. Conducting liquid chromatography tandem mass spectrometry (LC-MS-MS) circulating folate, folate derivatives, and folic acid were detected. Gas chromatography tandem mass spectrometry (GC-MS-MS) was performed to analyze other amino acids. C-reactive protein, cystatin C, and its variants were measured by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometric analysis.Furthermore, data on folate and biomarkers related to one-carbon metabolism are available from 245 CRC patients at three follow-up time points: baseline  were determined using monochrome multiplex quantitative PCR (MMQPCR).CORSA key publications are based on genomics and metabolomics: “Bayesian and frequentist analysis of an Austrian genome-wide association study of colorectal cancer and advanced adenomas”, “Plasma metabolites associated with colorectal cancer: a discovery replication study” and “Plasma metabolites associated with colorectal cancer stage: findings from an international consortium” ,11,12. AWe have performed a GWAS of CORSA participants comprising 1060 CRC patients, 689 patients with advanced colorectal adenomas, 928 colonoscopy-negative controls, and additionally, 3439 controls from the “Cooperative Health Research in the Region of Augsburg” (KORA) using Axiom Arrays CEU 1 . We pursued a dual approach to investigate genome-wide associations with disease risk applying both single marker analysis as well as model selection based on the modified Bayesian information criterion, mBIC2, implemented in the software package MOSGWA. The advantage of the model selection approach is its larger power to detect candidate SNPs compared to single marker tests. Furthermore, 56 SNPs that are already known to influence CRC susceptibility from previous studies were tested in a hypothesis-driven approach and some of them were also found to be relevant in CORSA. Furthermore, we found some so-far unreported SNPs [This GWAS was the basis for many international cooperation projects: the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO) at the Fred Hutchinson Cancer Center, Seattle, Washington, two European Cooperation in Science and Technology (COST) Actions “Cooperation studies on inherited susceptibility to colorectal cancer” (BMI1206) and “Identifying biomarkers through translation research and prevention and stratification of colorectal cancer” (CA17118).Within the “Metabolomic profiles throughout the continuum of colorectal carcinogenesis” (MetaboCCC) consortium, we performed an untargeted metabolomics study comprising 268 CRC patients and 353 controls using independent discovery and replication sets from two European cohorts, the ColoCare Study from Germany and CORSA. The aim of this study was to identify circulating plasma metabolites associated with CRC and to improve knowledge regarding CRC etiology. Multiple logistic regression models were used to test the association between disease state and metabolic features. We identified 691 statistically significant features in the discovery cohort. Testing the second cohort narrowed it to 97. These corresponded to 28 metabolites, of which 15 could be identified .Furthermore, we performed a targeted metabolomics profiling within the MetaboCCC consortium aiming to identify biomarkers related to CRC progression. We investigated plasma concentrations of 130 metabolites from 744 CRC patients (stages I-IV) from two Dutch cohorts , the ColoCare study from Germany, and CORSA. Our results suggest that metabolic pathway involving citrulline, histidine, and other molecules that have been previously implicated in CRC development may also be linked to progression .CORSA has multiple strengths bringing together a multidisciplinary team of molecular biologists, clinicians, laboratory scientists, and statisticians to foster research on CRC. The close collaboration with clinicians and clinical centers allows a regular and comprehensive clinical follow-up as well as the automated abstraction of data such as survival information. The multicenter cohort standardized the recruitment of patients, with uniform protocols for the collection, processing, and management of biospecimens. Participants were mainly Caucasian and geographically and ethnically uniform. The comprehensive biological samples and harmonized data provides the basis for national and international cooperation projects on CRC research. Therefore, CORSA represents an active and leading player in cooperation, support actions, and consortia. So far, CORSA contributed to multiple international consortia such as Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO) ,47,48,49A major strength of the biobank is the colonoscopy-negative control group, known to be free of polyps and CRC because all participants underwent a complete colonoscopy within the screening project B-PREDICT.Nevertheless, some limitations have to be mentioned. CORSA high-risk and low-risk adenomas as well as controls are mainly recruited within the screening program B-PREDICT from centers in Burgenland rather than from centers in Vienna. The reason is that in Vienna no organized CRC screening program has been performed so far; therefore, no colonoscopy-negative controls can be recruited. In Austria, the organization of CRC screening programs is not regulated nationwide; it is a matter of the federal states. Within CORSA, no regularly invited, targeted recruitment at defined follow-up time points is performed.A new research focus of CORSA besides genomics and metabolomics is the gut microbiome. Recently, the project “Gut MICRObiome-based approach for incorporating new biomarkers into COLOrectal cancer screening” (MICROCOLO) was funded by the Austrian Research Promotion Agency (FFG). There is evidence that changes in the gut microbiome occur during different stages of colorectal neoplasia, supporting an etiologic and diagnostic role for the microbiome. Harnessing knowledge of the microbiota may lead to new preventive strategies and diagnostics. myBioma GmbH, our company partner within this FFG-funded project, will use their already established data processing and statistical analysis workflow to process the resulting microbiome data. Predictive microbiome-based signatures specific for CRC and high-risk adenomas will be defined by multivariate statistical modelling. The combination of conventional screening methods such as FIT with microbiome-based methods is a promising tool for early detection of CRC and could improve diagnostic accuracy.The CORSA biobank, comprising genomic DNA, plasma, fecal samples, and a comprehensive CORSA database, represents a valuable resource for ongoing and future OMICS-based CRC projects. CORSA is open for cooperative research projects; expressions of interest are more than welcome.


Prolonged sitting non-significantly increased HR (weighted mean difference (WMD) = 0 bpm, 95% CI: −2, 3) and HRV (standardized mean difference (SMD) = 0.12, 95% CI: −0.08, 0.33) compared to pre-sitting baseline. Interrupting prolonged sitting yielded a non-significant small increase in HR  compared to pre-sitting baseline. Sub-group analyses investigating interrupting prolonged sitting revealed small-to-moderate increases in HR in healthy populations  and following walking interruptions . In conclusion, prolonged sitting does not significantly affect HR or HRV. However, interrupting prolonged sitting yielded a small non-significant increase in HR, potentially indicative of increased metabolic demand. Further research is needed to investigate poor CVD outcomes via autonomic disruption from prolonged sitting.Prolonged sitting increases cardiovascular disease (CVD) risk, however the physiological mechanisms contributing to CVD from acute sitting exposure are not well-understood. Therefore, this study investigated the heart rate (HR) and variability (HRV) responses to prolonged sitting and after interrupting prolonged sitting . Electronic databases were searched (inception-August 2020) for studies which exposed adults to prolonged (≥1 h) sitting with and/or without interruptions. Twenty-one articles (27 trials,  Regular sedentary behavior exposure, defined as low energy expenditure  in a seated, reclined, or lying posture  responses  guidelines  were searched by two authors  utilizing the following search terms: (sitting or sedentarism or sedentary or television time or screen time) AND (HRV or heart rate variability or heart rate).The reference lists of all identified trials and relevant reviews or editorials were also examined. The search was limited titles and abstracts written in English language with human adult (18 years of age and older) populations published between database inception and August 31st, 2020.Two researchers (LB and AA) completed the study selection independently. For the purpose of this meta-analysis, the term “article” is used synonymously with “study” and “trial” is the unit included in the meta-analysis. A given article may have resulted in more than one eligible “trial” if the article included more than one HR and HRV measure and/or more than one intervention group or condition. Initially, article titles and abstracts were screened for relevance by two reviewers (LB and AA) and a third reviewer was used to settle a potential split-decision (LS). The full-text versions of potentially eligible articles were obtained to evaluate for eligibility, also by two reviewers (LB and AA). The following criteria were used to select trials for inclusion in this review: (i) English language, (ii) human studies, (iii) adults (18 years of age and older), (iv) inclusion of an uninterrupted prolonged (>1 h) sitting bout. The following criteria were used to select trials for the secondary objective of interrupting prolonged sitting (i) prolonged sitting occurred prior to interruption, (ii) subjects returned to sitting following interruption. Repeated publications from the same studies were excluded.Data extraction, quality assessment, and investigation of the sitting interruption were completed by two reviewers (LB and LS). Data extracted for each eligible trial included bibliographic information , baseline participant characteristics, details of prolonged sitting bout , and reported outcomes.Study quality was assessed using the RoB2: revised Cochrane risk-of-bias tool for randomized trials (range 1–3), which includes items related to randomization, deviations from intended interventions, missing data, measurement of the outcome, selection of the reported result, and overall bias  and calculation of final results was conducted by one author (LS). For each outcome of interest, the pre- and post-prolonged sitting bout values (mean and standard deviation) were extracted. When mean differences and associated standard deviations were not published, they were estimated from the pre- and post-intervention values based on methods  from the Cochrane Handbook for Systematic Reviews of Interventions  conducted all statistical analysis using the MetaXL software , which may be explained partially by sample population differences. However, sub-group analysis did not reveal any effects of age, sex, cardiometabolic status (CMD), or sitting duration on the pooled estimate . Sensitivity analysis indicated that none of the trials unduly influenced the pooled estimate. Visual inspection of the funnel plot revealed asymmetry , which may be explained by variation in the strategy for interrupting prolonged sitting, age or biological sex differences, or prolonged sitting duration variation between studies.Overall and sub-group analyses are reported in symmetry , which wsymmetry . Additiop = 0.060) and a negligible change for those aged under <40 years . For women, there was a moderate increase in HR  but non-significant change for men (p = 0.585) and mixed sex (p = 0.088). For populations without CMDs, there was a moderate increase in HR , and, for populations with CMDs, there was a non-significant change (p = 1.000). When the sitting bout length was >4 h, there was a moderate increase in HR , and when the bout was <4 h there was a small, non-significant (p = 1.000) change. Last, there was a moderate increase in HR for walking-based interruptions , but non-significant changes for standing (p = 1.000) or other (p = 0.339) interruptions.When the populations were grouped by age, there was a non-significant moderate increase in HR for those aged >40 years , which was potentially explained by the differences in biological sex differences or the use of different HRV outcomes. When the populations were grouped by sex for sub-group analysis, there were negligible-small, non-significant changes in HRV for men  and mixed-sex . When the trials were grouped by HRV outcome type, there were negligible-small and non-significant changes for out outcomes .Overall and sub-group analyses are reported in We attempted to account multiple HRV estimates  within a single trial by using two approaches: (i) sub-group analysis by HRV estimate  as reported in the previous paragraph; and (ii) a multi-level meta-analysis model. The findings were consistent when we examined the data with the multi-level model, which revelated that all the heterogeneity was at level 1, suggesting our first approach was robust.The aim of this meta-analysis was to synthesize the existing literature investigating the effects of acute exposure to prolonged sitting, with and without sitting interruptions, on HR and HRV. The main findings were that prolonged sitting without interruption does not significantly affect HR or HRV. Interrupting prolonged sitting with walking increased HR while standing or other  interruption modalities had trivial-small, non-significant effects, respectively. There were not enough HRV studies with an interruption condition to examine HRV response to interrupting prolonged sitting.n = 7). The trials that reported HRV utilized different HRV assessment methodology and reported a variety of outcomes, resulting in different units of frequency and time components. This was addressed by standardizing the unit of measure and by performing sub-group analysis by HRV assessment type  and conducting a multi-level analysis. Second, the small number of HRV trials limits the conclusions drawn from the sub-group analysis. We were unable to investigate the effect of interrupting prolonged sitting on HRV as only two HRV trials reported interruption conditions. It should be noted that we attempted to perform an additional sub-group analysis of age using study population recruitment criteria (for example: 25–65 years old) instead of average age because mean age includes a large age range. However, this was not possible as only a few studies reported recruitment criteria. Lastly, the majority of the HRV trials were mixed sex, and it remains unclear whether both sexes respond similarly.Several limitations should be borne in mind when considering these findings. First, the sample size of included trials was small, with a limited number of trials reporting HRV . Our hypothesized mechanism proposed that blood pooling in the lower extremities as a consequence of prolonged sitting , for females (p = 0.007), and when interrupting longer bouts of sitting (p = 0.007). Additionally, walking as the interruption strategy resulted in the greatest and only significant increase in HR (p = 0.001), as compared to standing or other strategies. Since this meta-analysis measured the change in HR from before and after the sitting bout with interruption conditions (and not immediately after the interruption), we can speculate that an increase in HR is a result of the cardio-autonomic system acclimating to the lack of increase in blood pooling, therefore preserving stroke volume in order to maintain venous return. The aforementioned mechanism may also explain the lack of effect of sitting interruptions on HR in populations with CMDs. That is, in populations with CMD, sitting interruptions may be ineffective at preventing blood pooling in the lower extremities – at least with the strategies that have been trialed thus far. Additionally, the increase in HR for female populations is potentially indictive of the protective effect of estrogen receptors in the endothelium. The 17 β-estradiol (E2) receptor increases the production of nitric oxide (vasodilator) in endothelium and preserve healthy endothelial function . An increase in HR with interruption was expected and represents an increased metabolic demand following muscle contractions. Sub-group analyses revealed moderate effects (increase) of sitting interruption on HR in healthy populations  alone . Our findings are summarized in There are important gaps in the current literature which were identified. For example, due to the low number of included trails which investigated the effect of interrupting sitting on HRV, the current meta-analysis was unable to draw any firm conclusions. Additionally, the wide variety in sitting interruption strategies make it difficult to determine the optimal prescription for sedentary behavior guidelines. Thus, direct within-study comparisons of interruption strategies as well as standardized frequency, intensity, and duration guidelines for prolonged sitting interruptions across studies that facilitate comparisons may result in a better understanding of the cardio-autonomic mechanisms contributing to the CVD risk associated with sedentary behavior. It is also worth noting that, like exercise guidelines, sedentary behavior guidelines should consider whether specific guidelines are required for special populations, including those with CMDs or disabilities.Sedentary behaviors, like prolonged sitting, are associated with CVD incidence and mortality. This meta-analysis consolidated the existing literature on the effects of prolonged sitting, with and without interruption, on HR and HRV. Furthermore, gaps in the existing literature were identified in order to better inform investigators in establishing the optimum guidelines to mitigate prolonged sitting. The results of this analysis suggest that there are not meaningful changes in HR or HRV during uninterrupted sitting bouts (>1 h). Interrupting sitting resulted in a non-significant increase in HR, which may be indicative of the increase in metabolic demand. Future research is needed in order to optimize interruption strategies including frequency, duration, and intensity and more sitting interruption studies investigating HRV are needed to better inform sedentary behavior guidelines.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.LB, AA, BG, and LS: conceptualization, methodology, and formal analysis and investigation. LB and LS: writing - original draft preparation. All authors: writing - review and editing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


ABSTRACT IMPACT: Given the association between lower time to treatment and better clinical outcomes in stroke patients, identifying factors correlated with reduced proximity and thus greater time to stroke care can aid efforts to reduce disparities in stroke outcomes. OBJECTIVES/GOALS: The objective of this study is to quantify the relationship between distance to the nearest certified stroke hospital and census-derived demographics of age, race/ethnicity, income, and insurance status. METHODS/STUDY POPULATION: This is a cross-sectional study. Population data for all census tracts in the contiguous United States were obtained from the US Census Bureau’s 2014-2018 American Community Survey. Stroke hospitals were identified from national or state level certification databases and were required to offer at least IV tPA. The main outcome is driving distance in kilometers from each census tract to the nearest certified stroke center, which was calculated using OSMnx, a Python package to retrieve, model and analyze real-world street networks. Quantile regression analysis was used to compare relationships between distances and tract-level demographics of age, race/ethnicity, income, and insurance status. RESULTS/ANTICIPATED RESULTS: 2,423 stroke centers and 71,929 census tracts containing 316,995,649 individuals were included. 49,918 (69%) tracts were urban. Demographic disparities in proximity to certified stroke care were greater in non-urban areas than in urban areas. Higher representation of individuals with age ≤65 years were associated with increased median distance to a certified stroke center in non-urban areas, but not urban areas. Median distance was greater with greater representation of American Indian or uninsured populations in urban and non-urban census tracts. Higher median income was associated with decreased median distance in non-urban census tracts and greater median distance in urban census tracts. DISCUSSION/SIGNIFICANCE OF FINDINGS: Reduced proximity to stroke care exists in areas with greater representation of elderly, American Indian, or uninsured persons; and low median income. These disparities are magnified in non-urban settings. Such knowledge can aid efforts to address and reduce disparities in stroke outcomes.


Uveitis is the most common extra-articular manifestation in axial spondyloarthritis (axSpa) and can affect up to a third of patients. It usually presents acutely and unilaterally in the anterior aspect as iritis. It is treated with topical steroids, mydriatics and immunosuppresants. Anti-TNF has been used successfully to treat uveitis in the context of axSpa and is the cornerstone of long-term control of recurrent uveitis. In severe cases, surgery in the form of vitrectomy is done. In axSpa patients who have failure or adverse effects from anti-TNF, alternative treatments are required to prevent the complications of recurrent uveitis and visual loss.A 68-year-old female patient with axSpA presented with recurrent anterior uveitis in the left eye. She had radiographic axSpA (ankylosing spondylitis) for 6 years and was HLA-B27 positive. Her past medical history included hypertension which was treated with amlodipine and ramipril. She was treated with adalimumab for 3 months for her axSpa and this was stopped due to intolerance. She was then switch to etanercept which controlled her axial and peripheral joints symptoms but she developed recurrent anterior uveitis. She remained on etanercept for 2 years. The uveitis was treated with Maxidex, dorzolamide and brimonidine eye drops. Despite this, her uveitis progressed and closed angle glaucoma and cataract. She was then switched to golimumab in order to gain control of her uveitis. She developed cutaneous vasculitis after starting treatment. She presented with a widespread vasculitic rash over her calves with areas of central necrosis. Her investigations revealed a WCC 15.04, plts 456, neut 9.17, eosin 0.30, ESR 84, CRP 45, U&E normal, ALP 178, ALT 36. Urine dip was negative for protein/blood/leuc/nitrites. Chest X-ray showed mild bronchial wall thickening in the right lower zone with a few ill-defined small opacites of indeterminate significance. Blood cultures x3 were negative. Echocardiogram was normal with no vegetations. CT thorax was normal. ANA 1:160, ENA negative, RF/anti-CCP negative, ANCA negative, C3 1.88, C4 0.38. She was commenced on prednisolone 20mg once a day weaning regime once infection was excluded. Her anti-TNF golimumab was stopped. She made a dramatic improvement in her cutaneous vasculitis after withdrawal of anti-TNF but developed attacks of uveitis in the left eye. This was treated trabeculectomy, intravitreal injection of Ozurdex and a YAG capsulotomy. She was commenced on secukinumab to treat her active axSpa and this has stabilised her uveitis.Anti-TNF biologic drugs are effective in treating axial, peripheral joint as well as extra-articular manifestations (EAMs) in axSpa. Uveitis is the commonest EAM in axSpa and responds very well to anti-TNF drugs. The presence of HLA-B27 positivity as in our case, predisposes the patient to uveitis and EAMs. Uveitis may lead to complications such as glaucoma, cataracts, cystoid macular oedema, detached retina and posterior synechiae. In our case, the patient had the first three complications as a result of recurrent uveitis and withdrawal of anti-TNF due to adverse events. She required surgery include trabeculectomy, intra-vitreal injection and capusulotomy. Ongoing systemic treatment is required in her to avoid any further loss of vision.In a third of axSpa patients there may be failure or adverse reaction to anti-TNF. In patients who have failed anti-TNF they may be switched to another anti-TNF such as in our case. In patients with adverse reactions to anti-TNF, the choice is to either re-challenge with anti-TNF or switch to another drug with a different mode of action. For axSpa, the options include anti-IL17 and JAK inhibitors. Adverse reactions to anti-TNF include cutaneous manifestations such as cutaneous vasculitis as in our case, cutaneous lupus, psoriasis, hidradenitis suppurativa, lichen planus, vitiligo and alopecia areata. Having stopped anti-TNF due to the cutaneous vasculitis, we switched her to secukinumab for control of her axSpa. This also resulted in improved control of her uveitis with reduced frequency and severity of uveitis.In three phase 3 RCTs  aiming to determine efficacy of secukinumab in non-infectious uveitis (Behcet's and non-Behcet's) against placebo, there was no significant differences in uveitis recurrence between secukinumab treatment groups and placebo groups in any study (primary end-point). More studies are required to assess the efficacy of IL-17 inhibitors in treating and preventing uveitis in axSpa.Uveitis is the most common extra-articular manifestation (EAM) in axSpa. Early assessment and treatment are required to prevent complications such as visual loss. Close liaison and working with ophthalmologists is essential in order to improve patient outcome in axSpa. In severe cases of uveitis with complications, surgery may be required to prevent visual loss.Anti-TNF drugs are very effective in treating axSpa and are also beneficial in treating uveitis. A third of axSpa patients may have failure or adverse reaction to anti-TNF. The adverse event may include cutaneous manifestations such as vasculitis. This may necessitate withdrawal of anti-TNF.In patients who are not able to take anti-TNF, suitable alternatives such as anti-IL-17 and JAK inhibitors are used to treat axSpa. The impact of anti-IL17 and JAK inhibitors on uveitis in axSpa is yet to be determined. In our case, anti-IL17 secukinumab was shown to be effective in reducing the recurrence of uveitis. This requires further research to study its efficacy in treating and preventing uveitis in axSpa.


Editorial on the Research TopicMechanisms of Fluorescent ProteinsThis collection of papers for the Research Topic “Mechanisms of Fluorescent Proteins” (FPs) samples a broad range of research on physical mechanisms, applications, and molecular engineering strategies. The papers demonstrate a combination of experimental and computational approaches and are of broad interest to researchers working on FPs, microscopy, and spectroscopy.In bioimaging with FPs, increasing the penetration depth and decreasing unwanted scattering are desirable, which have motivated efforts for engineered FPs with redder emission and higher brightness . In a coEx and Ey) at the protein chromophore enables separation of contributing factors to the nonradiative relaxation rate. A small range of positive or negative values for Ex and Ey (–10 to +10 MV/cm) was revealed to facilitate both a red-shifted absorption and a high FQY, providing rational design principles for site-specific mutagenesis using RFP scaffold like DsRed.To brighten the generally dim red and far-red FPs, a systematic study of nonradiative relaxation in red FPs (RFPs)  reveals ab initio molecular dynamics simulations in hybrid form with density functional theory (DFT) to bridge local and larger-scale effects in FPs.On a fundamental level, the fluorescence mechanism via excited-state proton transfer  is elucip-hydroxybenzylidene-imidazolinone (HBDI). Typical examples are GFP from Aequorea victoria, and DsRed from Discosoma sp. wherein the HBDI chromophore is amended by an acylimine tail that lengthens π-conjugation and red-shifts the absorption. Targeted engineering for brighter variants was typically driven by linear, one-photon absorption and fluorescence. With the advent of two-photon laser microscopy, the need for bright and photostable FPs has increased. Yet two-photon absorption obeys different quantum-mechanical rules compared with one-photon absorption. Consequently, the brightest FPs with canonical chromophores for one-photon imaging are not necessarily optimized for two-photon excitation. The two-photon cross-sections of ten non-canonical chromophores (nCCs), inserting substituted tyrosines into the DsRed scaffold, were calculated using QM/MM schemes with polarizable embedding and external effective field correction  . The FSRAmong the most exciting applications of FPs is their use in single-molecule and super-resolution fluorescence microscopy. Such applications necessitate FPs with superior properties such as high FQY, fraction of time remaining fluorescent, outstanding photostability, and structural stability . In thisFront. Mol. Biosci. (https://www.frontiersin.org/research-topics/10542/mechanisms-of-fluorescent-proteins#articles) has captured the essence of this field and will inspire future innovations and breakthroughs in both the understanding and applications of FPs.From this exciting line of inquiries decoding fluorescence mechanisms of FPs, particularly targeting redder and brighter emissions, we foresee the interdisciplinary spectroscopy, microscopy, theoretical, and computational communities to continue joining forces to paint a comprehensive portrait of FPs and implement these molecular machines in ever-expanding applications. Nature has evolved FPs for millions of years, while GFP has revolutionized molecular and cellular biology just for several decades . Much re