Concurrent with the development of antibody-drug conjugate (ADC) technology, several recent advances in preclinical research stand to greatly increase the experimental rigor by which promising candidate molecules can be evaluated. These include advances in preclinical tumor modeling with the development of patient-derived tumor organoid models that far better recapitulate many aspects of the human disease than conventional subcutaneous xenograft models. Such models are amenable to genetic manipulation, which will greatly improve our understanding of the relationship between ADC and antigen and stringently evaluate mechanisms of therapeutic response. Finally, tumor development is often not visible in these in vivo models. We discuss how the application of several preclinical molecular imaging techniques will greatly enhance the quality of experimental data, enabling quantitative pre- and post-treatment tumor measurements or the precise assessment of ADCs as effective diagnostics. In our opinion, when taken together, these advances in preclinical cancer research will greatly improve the identification of effective candidate ADC molecules with the best chance of clinical translation and cancer patient benefit.The ability to chemically modify monoclonal antibodies with the attachment of specific functional groups has opened up an enormous range of possibilities for the targeted treatment and diagnosis of cancer in the clinic. As the number of such antibody-based drug candidates has increased, so too has the need for more stringent and robust preclinical evaluation of their  Of broader appeal, however, antibodies can be readily modified to carry a variety of treatment or imaging payloads and target them to practically any tumor-specific cell surface antigen. This effectively concentrates the antibody-drug conjugate (ADC) payload to sites of tumor development, significantly reducing possible treatment-related cytotoxicity in other organs or improving the ability to non-invasively image tumors above background noise.In recent years, monoclonal antibodies have become a hugely promising class of anti-cancer therapeutic molecules, given their high affinity and precise specificity for any number of tumor-specific target antigens. As an effective anti-cancer treatment, antibodies have been used to directly and specifically block cell surface receptor signaling . Given that most ADCs in clinical development recognize and bind to human antigens, IHC staining of frozen human tissue microarrays will most likely be preferable over in vivo mouse models to predict where appreciable levels of the ADC may accumulate in the human body other than tumor sites. However, the relationship between ADC and target antigen in the context of whole-body physiology and measurements of therapeutic effect and ADC biodistribution can now be interrogated to much higher experimental standards.As with the clinical development of any anti-cancer agent, certain aspects of ADC performance must first be rigorously evaluated in an experimental preclinical setting. At a minimum, in addition to showing safety, the target tumor antigen should be identified and then rigorously tested to show that in vitro and in vivo tumor models from patient-derived material , the ability to use CRISPR or inducible transgene technology to specifically manipulate the expression of antigen, and advances in non-invasive imaging that allow dynamic tracking of the ADC molecule or resulting treatment effects. Essentially, these advances greatly improve the quality of experimental control, such that the comparisons of ADC accumulation or therapeutic efficacy can be readily made between matched pairs of normal and tumor cells or between matched tumors that only differ in antigen expression. Imaging further permits many of these effects to be seen in the same individual subject dynamically over time, reducing the need for large experimental cohorts. Imaging also enables the standardization of ADC administration based on measured and not assumed tumor parameters, greatly improving the quality of data.We present here several recent advances in preclinical research that stand to significantly raise the rigor by which candidate ADC molecules and anti-cancer drugs can be assessed prior to clinical application. These include the ability to efficiently establish more representative . However, such cancer models also have significant deficiencies, can be prone to genetic drift over time, and their ability to accurately model human disease and ultimately predict the clinical performance of candidate therapeutics is questionable. The failure rate of establishing an immortalized tumor cell line in culture by traditional means is extremely high. Human tumors did not evolve to grow on tissue culture plastic as a 2D monolayer; thus, it is questionable how representative the low frequency of successfully established cultures are of the original tumor. Further, although subcutaneous engraftment of such cells in vivo is very routine , such models fail to recapitulate the tumor microenvironment that matches their native tissue of origin.The past decade has been transformative for tissue culture technology of patient-derived tumors. Until recently, only a limited number of immortalized 2D cancer cell lines was available to test the preclinical performance of an ADC using xenograft mouse models. Such models remain popular today as they are relatively quick and easy to develop. The cell lines are widely distributed among the research community, and some have been the focus of extensive genomic and gene expression characterization. Tumor samples received fresh from the operating theatre are processed and plated out in vitro in a mixture of growth factors and basement membrane extract such as Matrigel (a viscous matrix resembling a decellularized tissue microenvironment) to provide more natural growth conditions for the cells. As a result, PDOs grown in three dimensions in vitro retain cell polarity and some semblance of structure that can provide biologically relevant cues  are being developed to circumvent such issues, lending greater predictive power to the PET scan.In the absence of a formal biodistribution study, whole-body PET images of an antibody can be used to estimate where in the body the same ADC with a different therapeutic radioisotope attached [such as Yttrium 90 (β-emitter) or Actinium 225 (α-emitter)] would accumulate in the body. As mentioned above, ADCs with different conjugated moieties cannot be assumed to behave in an identical fashion In addition to being able to non-invasively track the biodistribution of a candidate ADC molecule, preclinical imaging can be equally useful in providing a dynamic and quantifiable measure of tumor cell cytotoxicity and therapeutic effect.. The BLI signal is reliant upon first introducing the expression of a luciferase transgene into the tumor model of interest. An expanding list of candidate enzymes and substrates suitable for BLI have now been described; however, enzymes that rely upon ATP to generate light are the most useful for assessing relative tumor cell viability in vivo. A highly relevant feature of these enzymes is that dead cells no longer emit light and so the cytotoxic effects of any drug or ADC can be readily assessed following treatment. This viability measure is often possible at experimental time-points far earlier than when gross anatomic changes to the tumor become evident.The most obvious preclinical modality to mention in this context is bioluminescence imaging (BLI). For reasons of relative sensitivity, speed, safety (does not involve ionizing radiation), and affordability, this optical imaging modality has been widely adopted by the research communityin vivo bioluminescence imaging, whereby cancer cells are first labeled with the expression of a reporter transgene, cells are labeled with Sodium Iodide symporter (NIS) expression. Physiologically, NIS expression is predominantly limited to the salivary gland, thyroid, stomach, and lactating breast in the mouse and transports iodine, an essential component of thyroid hormone biosynthesis. Crucially for imaging, NIS also imports the widely available and cheap gamma-emitting radiotracer 99mpertechnetate, which can be detected by SPECT. In our experience, NIS-SPECT can readily detect and individually resolve multiple sub-cubic-millimeter-sized metastatic tumor lesions in three-dimensional space .We would also like to draw particular attention to a relatively little used imaging technique termed NIS-SPECT. Similar to pace see . As backin vivo performance of any candidate drug or ADC can only stand to enable more informed “go, no go” decisions earlier in their development and ultimately ensure that efforts and resources are focused on those candidate molecules identified as most likely to succeed in the clinic and benefit cancer patients.In the immediate future, it is exciting to consider that the confluence of advances in cancer modeling, genetic manipulation and preclinical imaging discussed here will be able to provide significantly more robust evaluation of candidate ADC performance prior to clinical application. This is especially pertinent given the high attrition rate of promising candidate molecules as they progressively develop from the laboratory to the clinic. For practical reasons, we are not suggesting that these new techniques completely replace existing experimental approaches, but they should be considered and applied to some extent to rigorously explore key ADC development milestones. Improvements in the methods employed to more stringently test and assess the


Regenerative endodontic procedures have been described for over a decade as a paradigm shift in the treatment of immature necrotic permanent teeth, owing to their ability to allow root maturation with subsequent enhancement of the tooth’s fracture resistance in addition to the potential for regeneration of vital intracanal tissues. Concomitantly, minimally invasive endodontics is another rising concept with the main concern of preservation of tooth structure. Stemming from their potential to preserve the original tooth structure, both regenerative and minimally invasive endodontics could be considered as two revolutionary sciences with one common goal. Achieving this goal would entail not only employing the appropriate strategies to recreate the ideal regenerative niche but modifying existing concepts and protocols currently being implemented in regenerative endodontics to address two important challenges affecting the outcome of these procedures; conservation of tooth structure and achieving effective disinfection. Therefore, the search for new biomimetic cell-friendly disinfecting agents and strategies is crucial if such a novel integratory concept is to be foreseen in the future. This could be attainable by advocating a new merged concept of “minimally invasive regenerative endodontic procedures (MIREPs),” through modifying the clinical protocol of REPs by incorporating a minimally invasive access cavity design/preparation and biomimetic disinfection protocol, which could enhance clinical treatment outcomes and in the future; allow for personalized disinfection/regeneration protocols to further optimize the outcomes of MIREPs. In this review, we aim to introduce this new concept, its realization and challenges along with future perspectives for clinical implementation. Intereslatforms .In particular, the dental community faced unique challenges, leading to the development of treatment strategies changes to reduce the risks of virus transmission to patients/dental practitioners . In endoWhile the dental pulp is a unique resilient tissue responsible for the survival of one of the human body’s most important organs; the tooth, it is often undervalued in routine dental practice. While vital pulp therapies have been routinely implemented in daily endodontic practice for years, performing a partial/complete pulpotomy for mature irreversibly inflamed permanent teeth has been a procedure fraught with scepticism over the past years. Recent evidence highlights that these treatments may present a paradigm shift in endodontic therapies in the upcoming era . CongrueAlthough all REPs have been practiced for some time, seasoned endodontists and dental practitioners were always reluctant to apply these techniques in their routine dental practices, dreading the lack of predictability and due to perceptions, on shortcomings of available materials and restorative options. With the advent of advanced tricalcium-silicate cements and sophisticated bonding and restorative materials, successful long-term outcomes are being increasingly documented with reproducibility and obvious advantages for both the patient and the practitioner. This new direction is currently on its way to embedding firm roots in the field with a mental makeshift from a science of “maybe” to a science that has actually transitioned into routine clinical practice; ironically pushed forward by a global pandemic. In this review, we introduce a new concept amalgamating the sciences of MIEs with REPs in the hope of paving the way to strategies that will not only regenerate lost tooth structures but conserve those that still remain as well.Minimally invasive endodontics (MIEs) concept is based on the assumption that tooth structure preservation during access cavity preparation/canal instrumentation is a vital parameter to maintain strength and fracture resistance of the tooth. Contracted endodontic cavity (CEC) designs conserve a larger amount of the coronal tooth structure, thus reducing stress concentration on the occlusal and cervical areas of the tooth . MoreoveRegenerative endodontic procedures (REPs) utilize the concept of tissue engineering to restore the root canal system to a healthy state, allowing for the continued development and regeneration of the root and surrounding tissues . Indeed,A recent suggestion has been to redefine the definition of regenerative endodontics as follows: “The term regenerative endodontics should embrace the repair, replacement and regeneration of dentin–pulp lost due to age, disease, trauma or congenital defects to restore normal function” .Theoretically, if we can sterilize the canal, and recreate a 3D “biological filling” in the form of regenerated vital tissues this would ensure an optimum outcome. Regenerative strategies targeting mature teeth will most likely differ from those for immature permanent teeth, due to the different challenges encountered in both situations regarding the weak natural architecture of immature teeth, in addition to, the complexity of biofilms, toxins, and antigens which could reside in the larger dentinal tubules in these teeth . ExcessiFurthermore, the preservation of peri-cervical dentin in particular which has an anatomical pattern of a lesser inter-tubular dentin and wider dentinal tubule lumen can justify the evidence of greater sequestered growth factors including TGF-β1, FGF2, and VEGF found in this region . This woTherefore, combining MIEs and REPs might be considered a logical and new idea. On the other hand, it may complicate the outcomes of REPs due to the limitation of the cleaning and disinfection of the root canal system which could be encountered through the CEC. Hence, if such a strategy is to be employed, considerations must not only be given to how modification of the access cavity will influence adequate disinfection, but also should encourage single visit treatment modalities via the application of biologically inspired and biomimetic scaffolds to guarantee tissue regeneration in a bacteria-free milieu.The vast majority of failed regenerative endodontic treatment cases have been diagnosed with necrotic pulp with/without some form of apical pathosis which strongly suggests the association between persistent root canal infection and treatment failure . Concomi2 intracanal medication or low concentrations (1–5 mg/ml) of antibiotic pastes, and 17% EDTA is used in the second visit  . On anot2, and the diluted concentrations of sodium hypochlorite and antibiotic pastes currently recommended by the AAE were found to significantly decrease dentin microhardness and cause demineralization , has recently been introduced as a promising antimicrobial agent that could be used in many medical, dental and pharmaceutical fields. The application of NBW in Endodontics is still in its infancy, where it is recommended as a promising agent to upgrade the antimicrobial activity of root canal irrigants at lower non-cytotoxic concentrations. It exerts a kinetic force to dislodge pre-established intracanal biofilm and enhance the delivery of root canal medicaments, where it can be of beneficial in REPs. NBW was shown to be more effective in removing smear layer and at the same time preserves dentin microhardness when compared to 17% EDTA. Moreover, NBW in combination with 1.5% NaOCl was proven to be as effective as 5.25% NaOCl regarding the disinfection ability with enhanced penetration into dentinal tubules .Another promising biomimetic irrigant using catalytic iron-oxide nanoparticles (IO-NPs) has been introduced. The concept was to use IO-NPs in combination with hydrogen peroxide showing potent antimicrobial effects with deep penetration into the entire length of dentinal tubules. However, IO-NPs alone had modest antibacterial effects .2 or antibiotic combinations can cause adverse side effects during REPs. The former is responsible for root weakening even after a short period t period . The latt period . Recentlt period . It was t period .Another promising alternative is the use of probiotics as a potential intracanal medicament. Local delivery of probiotics has shown promising outcomes regarding the eradication of common endodontic pathogens . MoreoveSeveral recent researches have focused on the development of the smart drug delivery scaffolds to both augment root canal disinfection and reduce the risk of stem cell toxicity . An antiin-vitro study has evaluated a novel biomimetic scaffold composed of electrospun poly (lactic acid) nanofibers and electro-sprayed polycaprolactone with tannic acid micro-particles. This 3D cone-shaped scaffold was designed to fill the empty root canal mimicking the architecture of natural extracellular matrix  extracted from dental pulp and dentin tissues. It was reported that these extracts have an antibacterial effect against faecalis . Moreovefaecalis . Furtherfaecalis .The parallel use of immunomodulatory and homing factors in scaffold materials has been shown to induce stem cell homing, modulate cell differentiation and indeed induce regeneration. Cathelicidin peptide (LL37) has been reported to activate the ERK signaling pathway boosting the secretion and expression of vascular endothelial growth factor (VEGF), thus promoting pulp wound healing by inducing angiogenesis . LikewisThe use of cell-type specific exosomes (micro-vesicles) has also recently gained considerable attention in the field of regenerative medicine. These biomimetic cell products have been proven to be a driving force behind the immunomodulatory effects of mesenchymal stem cells due to their direct effect on cytokine release and M2 macrophage polarization . Dental Disinfection is a critical factor to ensure periapical healing in regenerative endodontics where literature has shown that residual infection can lead to either failure or hinder continued root development . Having Based on the previous review of literature, the current review suggests the possible implementation of a new concept of minimally invasive regenerative endodontic procedures (MIREPs). Starting from the access cavity design, CEC for non-decayed traumatized teeth and caries-driven-access cavity for decayed teeth, would represent more suitable tooth preserving designs, where the volume of dentin and enamel removal in case of CEC designs was found to be less than 15% which was significantly less than enamel and dentin removal in TEC . AdditioAs it was agreed that proper root canal disinfection through a cell- and tissue- friendly root canal irrigant and intracanal medicament is crucial for a successful outcome of minimally invasive REPs. NBW is hence recommended as an adjunct irrigant to enhance the anti-microbial activity hindered by the lesser accessibility of CEC designs. It has been proved to be efficient against multiple Gram + ve and Gram -ve bacteria with killing time ranging from 2 to 10 min . A suggePossible scenarios for implementation of a MIREP concept as well as a suggested clinical protocol are provided in


Microbial ecosystem comprises a complex community in which bacteria interact with each other. The potential roles of the intestinal microbiome play in human health have gained considerable attention. The imbalance of gut microbial community has been looked to multiple chronic diseases. Cardiovascular diseases (CVDs) are leading causes of morbidity worldwide and are influenced by genetic and environmental factors. Recent advances have provided scientific evidence that CVD may also be attributed to gut microbiome. In this review, we highlight the complex interplay between microbes, their metabolites, and the potential influence on the generation and development of CVDs. The therapeutic potential of using intestinal microbiomes to treat CVD is also discussed. It is quite possible that gut microbes may be used for clinical treatments of CVD in the near future. Cardiovascular diseases (CVDs) have taken center stage as the leading causes of morbidity and mortality worldwide. The cause of CVD is still under investigation, but thought to be influenced by genetic and environmental factors.Recent studies have shed light on the potential roles of the intestinal microbiome in CVD. Microbes in the intestine vastly outnumber the total number of human cells. They form a complex community of interacting organisms and are even collectively regarded as a human organ  and their potential roles in the generation and development of CVDs. Moreover, possible therapeutic strategies for modulating the intestinal microbes to alter the generation and development of CVDs are discussed.Microbiota dysbiosis refers to the imbalance in host-microbial interactions. Changes in either microbial composition or community-derived factors (such as metabolites or genotoxins) can be directly or indirectly associated with enhanced disease susceptibility. These microbiome based factors may cause activation of signaling pathways and ultimately lead to the pathophysiological diseases of CVDs. Moreover, these components have the potential to be targeted for therapeutic applications.Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria. Bacteroidetes and Firmicutes together account for the majority of the gut community in healthy adults, and the Firmicutes/Bacteroidetes ratio is viewed as a health indicator of intestinal microbiota  and reduced abundance of short-chain fatty acid (SCFA) producing bacteria  have been associated with increased risks for developing CVDs  in atherosclerosis development , and reduced populations of acetate-/butyrate-producing bacteria  more frequently , valvular heart disease (VHD), and ischemic heart disease (IHD) , which is primarily derived from bacterial metabolism of dietary choline and phosphatidylcholine , exists widely in vinegars, red wines and some grape seed oils, has been found to inhibit the microbial choline TMA lyase activity. This compound could inhibit atherosclerotic lesion development in ApoePhenylalanine (Phe), tryptophan (Trp) and tyrosine (Tyr) are aromatic amino acids that may influence immune, metabolic, and neuronal responses. Phe, an essential amino acid, is a metabolic precursor for Tyr, which maybe further processed into neurotransmitters, norepinephrine and adrenaline. Trp, also an essential amino acid, is a precursor for serotonin, another neurotransmitter. In patients with advanced atherosclerosis, the specific microbe-derived metabolite of Trp was significantly lower in plasma , which is produced as the dietary Phe converted into phenylacetic acid, has been reported to be associated with CVDs and major adverse cardiovascular events  cell activity  of hypertension and resistance hypertension showed lower blood pressure after combined antibiotic treatment  or ventricular tachyarrhythmia (VTA)  cholesterol levels, improving blood pressures, and in modulating inflammatory cytokines  significantly reduced the infarct size and improve left ventricular function after MI  mice  is a therapeutic strategy designed to transfer donor fecal samples into the gastrointestinal (GI) tract of a patient with a depleted microbiota, for the aim of restoring normal GI microbiota in patients. Depending on the source of the fecal microbiota for transplantation, FMT can be divided into allogenetic FMT  and autologous FMT . Allogenic FMT are the most commonly used  rodents, and the recipient animals were analyzed for pathological changes  disease -induced hyperlipidemia exhibit reduced Escherichia populations and increased levels of beneficial bacteria   is the dried rhizome of a medicinal plant from the family Ranunculaceae and contains alkaloids such as berberine, coptisine, and palmatine  is a type of condensed tannin highly polymerized and non-absorbed in the intestine. It was reported to modify the intestinal bacterial microbiota composition by directly interacting with the colonic gut microbial ecosystem (Ozdal et al., Increasing evidence suggest a link between the intestinal microbiome and CVD incidence. Studies have indicated that the microbiota interacts with the host through multiple pathways. Abnormal gut microbiota composition or microbial metabolites may be responsible for altering CVDs risks and its related pathological changes. Thus, novel therapeutic targets/strategies for preventing and treating CVD have been developed harnessing the potential use of gut microbiota.Intense efforts are underway to advance the potential use of microbiota in CVD and other human diseases in general. First, identification of specific microbe strains rather than a general bacterial community is helpful to elucidate the contributions of specific microorganisms to disease progression. Second, future research may focus more on microbiome-mediated metabolites and their downstream functional consequences, while the current studies mostly explore the microbial composition. Third, personalized approaches for microbiota modification are urgently needed. This effort may be aided by microbiome profiling of individual patients for their metabolomic biomarker.


Greater than 90% of patients with psoriatic arthritis (PsA) first develop their arthritis on a background of known psoriasis (Pso). Thus, having skin/nail Pso certainly is an important risk factor for PsA but as PsA develops in <30% of those affected with Pso, the presence of Pso alone is insufficient as a means of identifying which patients with Pso will develop PsA. It is hoped that with further molecular assessment of Pso patients who do not have any evidence of inflammatory musculoskeletal disease compared to those with early PsA features, that the “at risk” profile of Pso patients destined to develop PsA can be refined such that disease prevention studies can be designed and a new era of treatment for PsA can emerge. In this article, the early stages in the development of PsA are outlined and what is currently known about clinical features, genetic factors and soluble or tissue biomarkers associated with the development of PsA in patients with Pso is reviewed in detail. Finally, proposals are outlined regarding the approaches required in order to address this important research area. Psoriatic arthritis (PsA) is a chronic immune-mediated condition that results in systemic musculoskeletal (MSK) inflammation and consequent impairment in a person's quality of life and of function . Most paHere, the evidence for the various stages in the development of PsA will be reviewed. Clinical and genetic factors identified as indicating greater risk of PsA development in Pso patients will then be summarised and subsequently tissue and soluble biomarkers which have found some utility in single-centre studies will be introduced. To conclude, we will propose a systematic, unbiased approach to the identification, verification and validation of biomarkers associated with increased risk of development of PsA in patients with Pso.For a patient with inflammatory MSK symptoms or findings associated with skin and/or nail Pso, the experience of having PsA really starts with a diagnosis. There are however no diagnostic criteria or diagnostic tests for PsA and the diagnosis often depends on the clinician's knowledge and experience. A diagnosis of PsA is most commonly reached when there is clear evidence of inflammatory MSK disease, in the presence of Pso and in the absence of rheumatoid factor (RF) and anti-Cyclic Citrullinated Peptide (anti-CCP) antibodies. There are several pitfalls in this approach including the transient nature of symptoms and findings in early disease, the difficulty in separating inflammatory from degenerative findings or from fibromyalgia, the absence of Pso in 10% of patients and the presence of usually low titre RF or anti-CCP in ~10% of patients with clear PsA features. These diagnostic confounders add to diagnostic delay and contribute to the poor outcome experienced by patients when diagnosis is delayed by 6 months or more .Furthermore, in the setting of early, undifferentiated inflammatory arthritis, making a diagnosis of PsA can often be challenging because:Symptoms and signs of PsA in early disease may fluctuate;There is commonly under-recognition of inflammation with imaging studies (US/MRI/PET-CT) often identifying areas of sub-clinical synovio-entheseal involvement;Skin and nail psoriasis may be absent in up to 10% of patients;RF and/or ACPA may be positive in up to 10% of patients; andInflammatory markers (C-reactive protein and Erythrocyte Sedimentation Rate) are commonly normal or only minimally elevated.What is becoming clear over recent years is that PsA likely begins in many if not most patients at a much earlier point, perhaps years before the eventual diagnosis is made. A recent consensus study  defined Current research strongly suggests that the development of PsA is the result of interplay between environmental factors, an individual's phenotype and genotype and both the adaptive and innate immune system. If the early stages of immune activation can be addressed before disease becomes established, it is possible that some of the stages shown in Certain clinical features of Pso such as nail pitting, psoriasis location and the extent/severity of disease might help identify patients with early PsA or those patients with skin/nail Pso only most likely to develop PsA. Whether these variables represent predictors or risk factors is a matter of active investigation .A longitudinal, retrospective, population-based cohort study of subjects with Pso performed by Wilson et al. determined the incidence and disease-specific predictors of clinically recognised PsA in patients with Pso where patients with clinically recognised PsA (determined from a review of the case records) were excluded from the analysis . The psoIn a large, prospective, single-centre cohort study involving Pso patients who were screened annually for arthritis, Eder and colleagues investigated the incidence of PsA in patients with cutaneous psoriasis (PsC), defined as cutaneous psoriasis patients where the presence of PsA has been excluded following full rheumatologic assessment . To idenIn both the studies described above, the findings that psoriatic nail pitting and nail dystrophy are associated with the development of PsA could be explained by the fact that psoriatic nail disease is associated with enthesitis. Inflammation of the enthesis possibly precedes joint involvement, and it may be the reason why nail disease precedes PsA development. Therefore, nail disease may be a marker of increased “immunoreactivity,” which could in turn lead to the development of PsA .In an interesting approach from the UK PROMPT study group, a Bayesian statistical approach was used to identify MSK symptoms influencing the risk of developing PsA in >90,000 people with Pso  for BMIs from 30.0 to 34.9 kg/m2, and 1.48 (1.20–1.81) for BMIs C 35.0 kg/m2 , and this limits their utility . It is iThe possible role of epigenetic factors which might identify Pso patients at risk of developing PsA has been nicely reviewed in 2 recent articles , 19. TheImaging, notably ultrasound (US), magnetic resonance imaging (MRI), high-resolution peripheral quantitative computed tomography (HR-pQCT) and positron emission tomography-computerised tomography PET-CT, might possibly be used as tools to identify asymptomatic Pso patients who will develop PsA. That asymptomatic Pso patients have US-detected abnormalities has been known for some time and is nicely reviewed by Zabotti et al. . Gisondip = 0.005) and synovitis sub-score (p = 0.008) also significantly improved providing some rationale for early intervention studies targeting prevention of the development of PsA in Pso patients considered at high-risk. With the high percentage of at risk Pso having MRI abnormalities, the inclusion of a placebo arm in study design is going to be important so as to study the natural history of these MRI abnormalities.The findings of subclinical synovitis and enthesitis on MRI in Pso patients without evidence of PsA was also described some years ago , 28. SomIn a study by Simon et al., the presence or absence of structural entheseal lesions was assessed by high-resolution peripheral quantitative computed tomography (HR-pQCT) in 114 patients with psoriasis without clinical evidence of MSK involvement . ResultsThe literature regarding the ability of FDG-PET-CT to detect subclinical inflammation is more recent. Takata et al. assessed 18 Pso patients and 28 PsA controls and found evidence of asymptomatic enthesitis in 6/18 patients . To dateIn the Toronto prospective Pso cohort referred to earlier, the authors further assessed whether the presence of non-specific MSK symptoms predicted the development of PsA . They fon = 15) vs. Pso patients (n = 5) and normal control skin (n = 4) identified increased numbers of CD45Ro T-cells, greater vascularity, the presence of B-cells, and increased numbers of DR +epidermal cells as markers for arthritis in patients with Pso  diagnostic tools there have been relatively few biomarker studies undertaken using well clinically phenotyped cohorts of Pso and PsA patients Some studies have led to the identification of candidate biomarkers that may discriminate Pso from PsA and such biomarkers include CRP or inflammatory cytokines such as IL-6, IL-23 and TNF-a, markers of bone or cartilage damage and adipokines –47. A piBased on a growing panel of candidate biomarkers, Rahmati et al., undertook a computational approach to identify nine alternative signatures obtained by combining clinical and protein markers to improve discrimination between PsA and Pso . 192 PsADespite all these initial studies suggesting there may be different levels of serum proteins in patients with Pso compared to those with PsA, a broad screen of 951 serum proteins using an affinity-based proteomic platform applied to 18 Pso patient samples compared to 20 PsA patient samples concluded that these patients shared a broadly similar serum proteomic signature . In thisIndeed, in a study conducted by Cretu et al.  skin bioThis summary of selected protein biomarker studies suggests that there remains an important opportunity to continue to develop protein biomarkers that might have clinical utility in the management of patients with Pso who may progress to PsA. These future studies will undoubtedly benefit from incorporation of:i) large well-characterised cohorts of patients with longitudinal sampling;ii) the application of a range of proteomic strategies including unbiased LC-MS approaches as well broad panel screens for candidate discovery;iii) continued use of targeted proteomic methods for evaluation of multiple protein candidate;iv) application of existing and more sophisticated multivariate statistical methods including machine learning and random forest analysis;v) combination of clinical, genetic and soluble biomarker measurement in the multivariate statistical analysis.To date, studies of the risk factors and mechanisms underpinning progression of Pso to PsA have struggled to make progress due both to the relatively small data sets available at individual centres and also to the considerable heterogeneity of disease presentation and progression. While exciting recent work has highlighted biomarkers of interest (including those presented above), there is an urgent need for studies that include sufficiently large cohorts of patients.Advancing from biomarker identification to validation and ultimately to use in the routine clinical setting presents its significant challenges but we suggest that this is most likely to be achieved successfully if future studies are undertaken using collaborative multi-group, multi-cohort approaches and multi-analyte investigations incorporating relevant quality controls focussed on the development of new diagnostic tools.Confirming the features characteristic of patients with psoriasis at increased risk of PsA transition and validating proteins that may distinguish PsA from PsC should help narrow a fundamental research gap as the PsA field matures. Several conditions are essential to successfully conduct investigations to identify transition risk, predict progression, and prevent PsA. The longitudinal characterisation of a large cohort of psoriasis patients at increased risk of PsA transition; close cooperation between rheumatologists and dermatologists with experience in various psoriatic disease subareas; and the participation of researchers as well as patient research partners . The orgAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


However, this debate on therapeutics is based on a false premise. The onus for justifying a new, non-approved prescription treatment lies unilaterally with proponents to establish a convincing case for efficacy and safety in treating a specific disease. It does not rest with the unconvinced to rebut such presumptions. While this onus is clearly defined in legal terms for commercial sponsors seeking marketing for a drug, the logic is equally true for enthusiasts promoting a novel medical treatment. In this case, asserting a justification for testosterone treatment for male ageing fails on all three counts - unconvincing efficacy, unproven and doubtful safety for the mirage of an invented “disease”. This review will outline the scant, inconclusive evidence for efficacy and safety while highlighting the invented “disease” is poorly defined, motivated by vague, subjective, wishful thinking about rejuvenation. Yet experience shows that once ingrained by simple repetition supplemented by confirmation bias, such an abstract illusion becomes reified into unshakeable delusion, impervious to evidence. This combination of circumstances has culminated in a quasi-epidemic increase in testosterone prescribing over three decades evident in the 100-fold increase in testosterone prescription sales by 2010  has focused exclusively on testosterone, reflecting communal perceptions of testosterone as an elixir of youthful vigour. Above all the successes of medicine in the 20th century reaffirm that legitimate medical therapies must focus on a specific, well defined organic disorder so that the requisite clinical trials for efficacy and safety have a firm foundation. That is not, nor ever likely to be, true for medicalizing of ageing itself and remains contentious for the tunnel-visioned surrogates such as reduced blood testosterone without regard to underlying systemic diseases or definitive linkage to symptoms.Debates are usually understood as contests between two opposing cases, each argued with reasonable Even when history does not exactly repeat itself, it usually rhymes (Mark Twain) but what we learn most from history is that we do not learn from history (GWF Hegel).th Century  introduced to give this drive an upmarket medical gravitas. The key to this process is redefining the term “hypogonadism”. In men, this term has always been defined as pathological disorders of the reproductive system including organic disease of the testis (primary hypogonadism) and the hypothalamus or pituitary (secondary hypogonadism) known collectively as the hypothalamo-pituitary testicular (HPT) axis. These disorders impair the twin functions of the testis - steroidogenesis and spermatogenesis – possibly leading to androgen deficiency and subfertility, respectively  , 20 toge“ androgen deficiency in the aging male [(P)ADAM], “LowT”, and the most recent, upmarket makeover to create greater medical gravitas, “late-onset hypogonadism”. The terminology adopted in the title of this debate “late-onset hypogonadism” is only among the latest descriptors of a series of neologisms for what may be most neutrally described [e.g. by FDA  –50. NTISMore broadly, the decline in blood testosterone in male ageing is neither inevitable nor related to age per se. Rather as the accumulation of comorbidities of ageing, it is inherently at least partially avoidable or reversible by alleviation of those co-morbidities. For example, the Healthy Man Study showed that among 325 men studied 9 times over 3 months recruited for their excellent, symptom-free health there was no age-related decline in serum testosterone (or its metabolites dihydrotestosterone and estradiol) despite the study sensitive enough to detect changes from fasting, obesity and smoking . The intvs placebo) transdermal gel over a 12-month trial  study . In thisSuch minimal symptomatic benefits of testosterone for men without pathologic hypogonadism may explain the high discontinuation rates of men with only non-specific symptoms (but not impaired intrinsic reproductive function) who start treatment with testosterone gels for “hypogonadism”, by the expansive redefinition , 68. UndAn ironic, alternative perspective on this issue derives from the well-established findings that supraphysiological testosterone or synthetic androgen dosage can achieve dose-dependent increases in muscle mass and strength from below to well above the natural physiological limits in healthy eugonadal men –72. One There is no convincing evidence for the safety of testosterone treatment of men without pathologic hypogonadism. Without such data, testosterone treatment cannot be recommended. There are at least three well defined major areas of public health concern about the unknown safety impact of testosterone treatment on male ageing; others may yet appear. These major issues comprise risks of testosterone-initiated or accelerated cardiovascular and prostate health and androgen dependence.In the absence of pathologic hypogonadism, clinical studies of safety for testosterone treatment for cardiovascular events [including heart failure  or venouAnother concern for the long-term safety of testosterone treatment of men without pathologic hypogonadism is whether it will increase the risk of testosterone-induced or accelerated benign or malignant prostate disease. Sustained exposure to adult male blood testosterone concentration from puberty onwards is required for completion of prostate development, a necessary but not sufficient condition for the development of late-life prostate diseases. Available meta-analyses of observational studies suggest minimal risk of subsequent prostate cancer associated with exposure to endogenous or exogenous testosterone exposure over many years of observation , 93. SimThe prevalence of benign prostate disease increases steeply with age with a majority of men over age 50 years having benign prostate hyperplasia –98. HencConsequently, population surveillance of prostate diseases is needed to detect any impact of the recent epidemic of testosterone prescribing. For prostate cancer, this requires making the distinction between screened-detected, organ-confined and life-threatening advanced or metastatic cancers. Due to the much longer disease latency, it is doubtful that the TRAVERSE study can evaluate safety for the risk of testosterone-induced or accelerated prostate disease. Hence for both benign and malignant prostate diseases of older men, there is minimal evidence that testosterone will have no adverse effects on prevalence of late-life prostate diseases.A further and less considered public health concern is the creation of iatrogenic androgen dependence when testosterone treatment is administered to men without reproductive pathology . In men It is not easy in the present environment for endocrinologists to avoid being drawn, however reluctantly, into testosterone misuse. Many endocrinologists are referred patients with a single, marginally low blood testosterone measurement seeking testosterone treatment for “hypogonadism”. Under the misguidance of numerous extant guidelines or other manifestos, encourage excessive testosterone prescribing where there is any clinical doubt, which is almost always. Like family doctors or GPs, they may fear that if they do not succumb to prescribing on demand, the patient will go doctor shopping and get the testosterone they think they need or demand elsewhere. These dilemmas are enlivened, if not enlightened, by concerted marketing and papers emanating from pharma, upscale single-issue men’s health clinics and academic enthusiasts. These rarely highlight the vested commercial interests, where present, promoting testosterone use outside approved indications under the disease-mongering rubric of “hypogonadism”.Testosterone is unique among hormones for its high level of public recognition, which unfortunately is imbued with fantasies and fictions unrelated to endocrine reality, an enchantment that easily unlocks latent but irrational wishes for rejuvenation. Reproductive medicine is unique in that, unlike other medical specialties, virtually everyone’s personal experience of sex and reproduction provides them with the subjective confidence they possess sound insight into reproductive biology and medicine without needing recourse to the established objective facts. This particularly extends to beliefs about what testosterone is and does biologically. This illusion of sophisticated expertise forms a powerful coupling with tenacious wishful thinking. This latent demand is readily entrained by clever marketing from pharma and other commercial enterprises that promotes testosterone’s use as an anti-ageing or sexual dysfunction tonic, reinforced by cyberchondria. The net effect is to expand the horizons of prescription drug misuse from opiates and benzodiazepines to include testosterone. Under the disease-mongering expanded definition of “hypogonadism” , as covest century whereby targeted efforts aimed to shift testosterone prescribing onto family doctors or GPs, but away from endocrinologists. However, busy family doctors or GPs who find it easy to write prescriptions giving patients something they go away with, but those doctors are less familiar with the critical basis for, and consequences of, initiating testosterone treatment in the absence of genuine hypogonadism. Reversing this misdirection for the initiation of testosterone prescribing away from specialists was one of the key reforms created by the 2015 reimbursement restrictions of Australia’s Pharmaceutical Benefits Scheme which required a specialist to initiate new testosterone treatment though, once started by a specialist, it could be continued by a family doctor or GP  under the expanded disease-mongering definition, these ageing-comorbidities are a fiction in search of a definition. Finally, there is no basis for population or individual patient screening (“case finding”) by measuring blood testosterone without a genuine clinical suspicion of underlying male reproductive pathology based on the clinical presentation including examination of testes. When measurement of blood testosterone is justified by the clinical presentation including physical examination, it should be accompanied by blood LH, FSH and SHBG to clarify interpretation and assays should be performed multiple times. Pathologists should be encouraged to switch to more accurate LCMS-based measurements of testosterone and stop reporting the misleading imaginary fractions of testosterone , a numerical artifice signifying nothing for clinical guidance .Testosterone prescribing for men without pathological hypogonadism is a therapeutic illusion in search of a definition. It is fostered by wishful thinking of an affluent populace with eyes mistily focused on the mirage of rejuvenation. Although cultivated by the disease-mongering expanded definition of “hypogonadism” as any lowering of blood testosterone coupled with any non-specific complaint, it is nowhere near justified by current standards of efficacy and safety evidence. As such, testosterone treatment other than replacement therapy for authentic hypogonadism should be confined to adequately powered, well-designed and placebo-controlled clinical trials geared to determining the efficacy and safety of testosterone prescribing for functional states, including but not limited to, age-related hypogonadism. Available evidence gives minimal grounds for expecting such proof in the foreseeable future. In the meantime, doctors need to be supported resisting the popular but misguided demand for testosterone. In many respects this is an educational challenge like smoking cessation, which was equally driven by clever advertising confecting public demand for the product without health benefit.st century.The public health consequences of the recent epidemic-like increase in testosterone prescribing on cardiovascular and prostate health and iatrogenic androgen dependence remain to be evaluated over coming decades. At best it may have little adverse impact but there could be detrimental changes in cardiovascular and/or prostate health. It may be claimed in defence of unjustified off-label testosterone prescribing that men treated with testosterone for no good reason often stop treatment after a short period. While short-term evidence suggests that this is true, it comes at a cost of undermining the credibility and confidence in the integrity of medical care. Furthermore, some evidence suggests that significant numbers of men who start testosterone treatment may have difficulty stopping it even if it proves ineffective as they become androgen dependent from androgen deficiency withdrawal symptoms while their endogenous testosterone production resumes, albeit slowly. In any case this is an indictment on the judgement of those prescribing T without proper evidence-based indication and the potential harm and waste this creates. Adverse outcomes would grievously erode public confidence in medical integrity and might eventually curb doctors’ traditional freedom to prescribe any registered drug off-label. The permissive disease-mongering redefinition of “hypogonadism”, an elastic broadening of the diagnosis facilitates much more testosterone prescribing resulting in a deterioration in clinical evaluation of androgen status creating a churn market in testosterone products and disappointment in misled patients. While this may favour pharma and other commercial enthusiasts with self-serving benefits, this misadventure is a net loss for the highest standards of medical care in the 21The author confirms being the sole contributor of this work and has approved it for publication.The author declares 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.


Wider and more profitable legume crop cultivation is an indispensable step for the agroecological transition of global agri-food systems but represents a challenge especially in Europe. Plant breeding is pivotal in this context. Research areas of key interest are represented by innovative phenotypic and genome-based selection procedures for crop yield, tolerance to abiotic and biotic stresses enhanced by the changing climate, intercropping, and emerging crop quality traits. We see outmost priority in the exploration of genomic selection (GS) opportunities and limitations, to ease genetic gains and to limit the costs of multi-trait selection. Reducing the profitability gap of legumes relative to major cereals will not be possible in Europe without public funding devoted to crop improvement research, pre-breeding, and, in various circumstances, public breeding. While most of these activities may profit of significant public-private partnerships, all of them can provide substantial benefits to seed companies. A favorable institutional context may comprise some changes to variety registration tests and procedures. Better There is increasing interest by some farmers of the organic sector for greater cultivation of seeds of genetically heterogeneous material of inbred crops rather than pure line cultivars, because of the advantages in terms of crop resilience and yield stability that genetic diversity is expected to provide. This conflicts with the genetic uniformity required for cultivar registration. Recently, the EU has allowed to market “Organic Heterogeneous Materials,” not fulfilling the cultivar requirements as regards uniformity under certain conditions , 2021. TBreeding needs to identify and exploit genetic variation for relevant traits supported by innovative phenotyping tools and molecular markers-based procedures to develop “smart” cultivars that yield more with low inputs. Key traits for grain or forage legume breeding were summarized in previous reviews e.g., . HereaftThe agroecological transition requires not only greater legume crop cultivation in rotations but also greater exploitation of legume-based intercrops, which, in modern agriculture, has been widespread only for some perennial legumes, especially white clover. The association of annual legumes with cereals, or that of perennial legumes with forage grasses, can exploit plant functional diversity to raise crop yields, yield stability, and/or crop quality, while simultaneously enhancing ecosystem services and reducing adverse environmental impacts . BreedinGreater adaptation to low input conditions will be a leading priority in legume breeding, particularly for organic systems that are on the rise in EU with a mandate to reach 25% of the EU cropping area by 2030. Improved symbiosis and nitrogen use efficiency are therefore becoming compulsory . PhosphoThe global change and increasing instability of the climate pose additional challenges to breeders . It emphClimate change-related stresses will have a negative impact not only on yield but also on nutrient quality of grain legumes  with a cThe predicted increase of legume-based food consumption due to the rise of plant-based and flexitarian diets and the planned EU reduction of meat consumption will widen the economic opportunities for domestic cultivation of grain legumes, since food use provides greater added value than feed use. Until recently, food legume breeders’ efforts have focused mainly on improving protein yield and reducing “undesirable” compounds contents , while largely neglecting sensory or processing important traits . The insFor forage crops, useful new quality traits may include increased Omega-3 polyunsaturated fatty acids, vaccenic acid, and conjugated linoleic acid, with reduced levels of omega-6 fatty acids and palmitic acid in milk . ConsideFarmer’s acceptability of cultivars cannot be considered a new trait by itself, but farmer-participatory selection is gaining increasing attention especially for organic farming. There is a paucity of formal assessments of its value for countries with developed agriculture, but its application to a large-scale pea breeding program in Italy produced greater yield gains than ordinary breeder’s selection .Adaptation for use as a service  crop is a further new trait of interest for some legume species in the European agriculture. Two main situations are encountered as: Either a crop (or a crop mixture) is grown in summer just after the last harvest until the new sowing  to catch residual nitrogen, control weeds and pests, avoid soil erosion, and improve the soil carbon content, or a crop is grown together with a cash-crop as a service-provider only. Annual legumes for these uses should possess rapid summer establishment and autumn growth . Perennial legumes may offer a living mulch for grain crops over several years . The breeding of these crops is challenged by greater interest of minor species or plant types and the use of test conditions and target traits that are completely different from those currently used as grain or forage legumes . In addition, their seed cost ought to be very low.As outlined in the earlier sections, legume breeders have to cope with ever-increasing quantitative target traits and the need to reduce the profitability gap with major cereals by drastic yield improvements, in the presence of modest budgets. Field phenotyping remains a bottleneck for crop genetic improvement. Legume improvement can today be supported by the integration of modern genomics approaches, high-throughput phenomics, and simulation modeling . The chaThe use of markers in breeding programs got impulse from the development of high-throughput genotyping techniques, such as genotyping-by-sequencing (GBS), and large SNP array tools. Besides the QTL identified in restricted parental crosses that are hardly used in breeding programs, genome-wide association studies (GWAS) provided markers that explain trait variation in a chosen population. These QTL explain a portion of genetic variance, useful to track major alleles. Most agronomically important traits are polygenic and, as such, can greatly profit of genomic selection (GS), which enables small-effect loci to be incorporated into prediction equations. An interesting option would be to combine in a single model both QTL and all the markers covering the genome, through procedures that may also allow to define a subset of highly explanatory SNPs for inclusion in less expensive selection tools .A key question for legume breeders is whether GS may have greater selection efficiency than phenotypic selection for crop yield improvement. Pioneer studies highlighted greater predicted yield gain per unit time or unit cost for alfalfa , soybeanVariant detection  is also a way to identify genes involved in trait variation and alleles conferring a positive effect on a trait . For autWider and more profitable legume crop cultivation is an indispensable step for the agroecological transition of global agri-food systems but represents a challenge especially in Europe. Plant breeding is pivotal in this context. Variety breeding is reputed to be a fruitful business, enabling economic activity through seed marketing and genetic progress. However, when the surfaces are low, the return of the breeding activity is not sufficient to support strong breeding programs. Taking the number of registered legume cultivars ; Table 1Reducing the profitability gap of legumes relative to major cereals will not be possible in Europe without public funding devoted to crop improvement research and pre-breeding activities and, in various circumstances and especially for minor crops, public breeding. While most of these activities may profit of significant public-private partnerships, all of them can provide substantial benefits to seed companies . A favorable institutional context ought to comprise variety registration procedures and marketing regulations able to value innovative variety traits and to reverse, under specific circumstances, the trend toward the cultivation of genetically uniform varieties. In Europe, only France has a levy system on grain legume production  when collected by a cooperative and allocated to a fund that supports plant breeder’s projects. This tax, known as “Cotisation Volontaire Obligatoire” (Mandatory Voluntary Contribution), is not applied (yet) to other grain legumes and does not apply to forage legumes, which are mostly not collected but used directly on farm to feed cattle. Similar systems apply in Canada and Australia, for instance, with returns to public breeding programs, mainly held by Universities, leading to continuous funder-led breeding improvements. Europe’s policy makers should explore this further, facilitating public-private breeding interaction for the good of all. The lack of sufficient economic returns also explains the fact that public breeding programs still are the main contributors to legume crop improvement in most European countries. In any case, the support of public research institutions is crucial for the development of pre-breeding or breeding activities and innovative selection procedures (such as GS), as a form of research policy-supported public-private partnership.At the moment, Genetically Modified Organisms (GMO) in Europe are submitted to complex regulation although are they are used for scientific research. Moreover, plants derived from genome editing enter into GMO category at the moment . ChangesAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.This work was supported by Spanish AEI project PID2020-11468RB-100, Italian MIPAAF project GENLEG, EU-H2020-EUCLEG project 727312, and Portuguese FCT project UID/04551/2020.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.


Existing quantitative imaging biomarkers (QIBs) are associated with known biological tissue characteristics and follow a well-understood path of technical, biological and clinical validation before incorporation into clinical trials. In radiomics, novel data-driven processes extract numerous visually imperceptible statistical features from the imaging data with no a priori assumptions on their correlation with biological processes. The selection of relevant features (radiomic signature) and incorporation into clinical trials therefore requires additional considerations to ensure meaningful imaging endpoints. Also, the number of radiomic features tested means that power calculations would result in sample sizes impossible to achieve within clinical trials. This article examines how the process of standardising and validating data-driven imaging biomarkers differs from those based on biological associations. Radiomic signatures are best developed initially on datasets that represent diversity of acquisition protocols as well as diversity of disease and of normal findings, rather than within clinical trials with standardised and optimised protocols as this would risk the selection of radiomic features being linked to the imaging process rather than the pathology. Normalisation through discretisation and feature harmonisation are essential pre-processing steps. Biological correlation may be performed after the technical and clinical validity of a radiomic signature is established, but is not mandatory. Feature selection may be part of discovery within a radiomics-specific trial or represent exploratory endpoints within an established trial; a previously validated radiomic signature may even be used as a primary/secondary endpoint, particularly if associations are demonstrated with specific biological processes and pathways being targeted within clinical trials.• Data-driven processes like radiomics risk false discoveries due to high-dimensionality of the dataset compared to sample size, making adequate diversity of the data, cross-validation and external validation essential to mitigate the risks of spurious associations and overfitting.• Use of radiomic signatures within clinical trials requires multistep standardisation of image acquisition, image analysis and data mining processes.• Biological correlation may be established after clinical validation but is not mandatory. Quantitative imaging biomarkers (QIBs) are associated with tissue characteristics that are altered by disease and its treatment. Necrosis decreases tissue cellularity and increases water content manifesting as an increase in T2 , a reducRecently, a new approach to derive imaging biomarkers has been advocated through the concept of radiomics , 9. ThisA major disadvantage of a non-mechanistic data-driven approach is that random chance associations may occur. Most studies look at the associations between a large number of features extracted from discretised images and prognosis/response/outcome in an inadequate number of samples. For biomarker profiles that rely on statistical rather than biological associations, generalisation and scalability to multicentre trials requires more than a simple standardisation process. Also, their validation pathway needs to incorporate measures that may differ substantially from traditionally accepted methods. This article prepared by imaging experts from the European Society of Radiology EIBALL (European Imaging Biomarker ALLiance) and the EORTC (European Organisation for Research and Treatment of Cancer) Imaging Group with representatives from QIBA (Quantitative Imaging Biomarkers Alliance) examines how the process of standardising and validating data-driven imaging biomarkers differs from those based on biological associations, and what measures need to be considered when implementing them into clinical trials and, eventually, into clinical routine. Structured discussions were conducted via teleconferencing and written communications.Radiomics analyses rely on image acquisition, image analysis and computational statistics , so stanAn element of diversity of acquisition protocols or machines is advantageous at the discovery phase of data-driven biomarkers so that the identified radiomic signatures used in clinical trials are robust enough across a range of platforms . DatasetBasic methods of image normalisation include pixel size resampling by filtering  and/or rThe Image Biomarker Standardization Initiative (IBSI)  offers aAs with biologically driven biomarkers, manual region of interest delineation introduces inter- and intra-observer variability because of variation in border perception. Observer training and working to protocol assists in this regard. Semi-automated segmentation methods, e.g. region-growing or level set active contour models  and deeptrans) or clinical parameters may be added to the radiomic signature if pertinent.‘Hand-crafted’ radiomics extracts predefined human-engineered features from the volume-of-interest (VOI) . These iSeveral tools are described –72. To iAfter dimension reduction, selected features are investigated for their association with clinical outcome using tools such as univariable or multivariable logistic regression, decision tree, random forest, support vector machine, neural networks, all described extensively in previous publications –68 and uA recent evolution has been the integration of radiomics with deep learning (DL) –78. ‘DisFollowing identification of a radiomics signature associated with disease/outcome, two fully independent datasets are needed, one for training and cross-validation , and at least one other to test the final model and confirm generalisability and performance . Both training and testing datasets should be of sufficient uniform quality  and representative for the patient population for which the radiomics model is intended. An adequate sample (size and diversity) is essential for the training and validation datasets, with respect to the number and type of features (‘signature’) considered. Testing the model with a dataset containing a different prevalence of cases and/or a high degree of imbalance may result in overoptimistic conclusions. Feature selection avoids over-parameterised models, reduces dimensionality of the feature space (data dimension reduction) and ensures that only a small and stable subset of original features relevant to the task are retained. A strategy to cross-validate the structure of the model requires careful considerations regarding sample size, accuracy estimation and the choice of the validation method . Grid searches pose the danger of overfitting, leading to overoptimistic model performance that is not reproduced on other datasets or in clinical practice. Finally, repeatability and reproducibility of the signature in a multicentre context  is a crucial step in technical validation –81. As wBiological correlation with liquid/tissue biopsies may be performed after the technical and clinical validity of a radiomic signature is established but is not mandatory. A radiomic signature that is related to survival outcomes may potentially reflect a tissue phenotype associated with a specific biology. Biological validation reduces the likelihood that radiomic features are selected by statistical chance or may be attributed to the nature of the data sample used for model development. It also offers the opportunity to reduce the number of selected features.The process by which the clinical utility of a single quantitative feature, or multiple features embedded in a statistical model is demonstrated, allowing improvement of health outcomes  is being addressed slowly for radiomics. Following initial ‘discovery’, new and independent datasets are required to replicate the performance of the identified model and validate it clinically. Performance metrics, e.g. sensitivity and specificity, should be evaluated ideally in prospective trials, or prospectively in the clinic using routinely obtained clinical data  in order to avoid bias. Table Images provide an averaged macroscopic view  of the geometry and/or function of the tissue. Radiomic features are statistical descriptors characterising the macroscopic visual aspect of images and only indirectly relate to the microscopic histological characteristics of the imaged tissue. Such features are then used as a statistical/phenomenological description of the outcome, and not embedded into an actual biological/physical model of this outcome that would unambiguously establish causality between features and outcome.Radiomic information on visually imperceptible phenotypic characteristics such as intensity, shape, size and texture distinguish benign and malignant tumours, likely reflecting different cellular morphology . In cervAlthough it is possible to extract mathematically hundreds or thousands of radiomic features from digital images, most studies to date suggest that less than 20 are indicative of unfavourable biology, and these largely relate to shape and textural uniformity. 2D shape features indicate more rapidly progressive disease with reduced overall survival in glioblastoma multiforme . Shape aWhen defining training datasets for radiomic feature extraction and selection in clinical trials, case-control data may be considered but may underrepresent the disease. Enrichment of training datasets with normal and abnormal cases of varying disease severity is mandatory to achieve appropriate balance. Bias in the training datasets limits generalisability. For example, a radiomic signature developed on lung nodules detected on chest x-rays in a population with a high prevalence of tuberculosis and few cancers will overdiagnose tuberculosis in a population with a high prevalence of cancer. Image acquisition bias (cases recognised as disease acquired with a specific protocol or device) where selected features are linked to image acquisition rather than to image content may fail to predict disease when applied to an independent population. Manual VOI segmentation and use of locally developed methodology risks discovery of features that are not generalisable and may be influenced by hardware or software-related factors rather than the disease itself. Diverse but balanced image acquisition conditions in the training dataset should counteract these effects. Though balance and diversity are necessary at the discovery stage, it is crucial to evaluate performance only on populations representative of the natural prevalence.p values impossible to reach. Strategies to reduce spurious correlations and overfitting include artificially increasing the number of samples by data augmentation . Cross-validation or bootstrapping are alternative strategies, but an independent dataset to confirm the findings is always required.The radiomic process, which tests combinations of hundreds and thousands of parameters, risks false discovery. Traditional statistical corrections for multiple tests would lead to Firstly, where radiomic signature discovery is the objective, a trial should follow the steps described and illustrated  to identify the most promising feature(s) and a subsequent phase using the remaining patients (independent cohort) to evaluate the performance of the identified radiomic signature. Thirdly, where a previously validated radiomic signature is used, this could be incorporated into a clinical trial as a primary or secondary endpoint. In this last case, the pathway of a data-driven biomarker does not differ from a QIB.Although the discovery phase requires image acquisition diversity, standardised protocols, pre- and post-processing methods, tools and algorithms for feature extraction are needed for incorporating into clinical trials and facilitated by centralised data analyses and publicly available analysis software Table . To incoted Fig. . SecondlData-driven imaging biomarkers provide information beyond that perceived by human readers. Their benefits may be exploited if specific standardisation and validation pathways are defined and the different/additional hurdles compared to more traditional QIBs are addressed. Effects of different types of processing on subsequent extracted feature variability and predictive model performance is an open area of research . AvailabWhile radiomics may eventually encompass all quantitative image-derived information into a common framework, current implementations mostly relate to intensity, shape and textural features within a VOI. In the future, quantitative  functional information, e.g. derived from PET, SPECT, pharmacokinetic modelling and other parametric imaging modalities, may form part of the radiomic signature, and require a smaller or biologically more meaningful set of parameters. Deep radiomics may also be deployed in trials, and recent studies have already demonstrated the potential of such approaches –111.Regardless of definitive biological correlation, once adopted and properly deployed, data-driven biomarkers may be combined with clinical data and other biomarkers . Such expanded use of radiomics should eventually improve disease characterisation, prognostic stratification and response prediction in clinical trials, ultimately advancing precision medicine.


As of 2021, the biothreat policy and research communities organize their efforts around lists of priority agents, which elides consideration of novel pathogens and biotoxins. For example, the Select Agents and Toxins list is composed of agents that historic biological warfare programs had weaponized or that have previously caused great harm during natural outbreaks. Similarly, lists of priority agents promulgated by the World Health Organization and the National Institute of Allergy and Infectious Diseases are composed of previously known pathogens and biotoxins. To fill this gap, we argue that the research/scientific and biodefense/biosecurity communities should categorize agents based on how they impact their hosts to augment current list-based paradigms. Specifically, we propose integrating the results of multi-omics studies to identify bioagent-agnostic signatures (BASs) of disease—namely, patterns of biomarkers that accurately and reproducibly predict the impacts of infection or intoxication without prior knowledge of the causative agent. Here, we highlight three pathways that investigators might exploit as sources of signals to construct BASs and their applicability to this framework. The research community will need to forge robust interdisciplinary teams to surmount substantial experimental, technical, and data analytic challenges that stand in the way of our long-term vision. However, if successful, our functionality-based BAS model could present a means to more effectively surveil for and treat known and novel agents alike. In the United States, biodefense and biosecurity policy as well as biological threat research largely center around several lists of priority agents including the Federal Select Agent Program (FSAP) Select Agents and Toxins list  which isWe argue that the biodefense and emerging infectious diseases communities should adopt a new strategy to augment—but not replace—list-based approaches. Others in the biodefense community have recognized how too heavily relying on lists limits our biothreat detection, diagnostic, and countermeasure capabilities; the 2018 National Academies of Sciences report on Biodefense in the Age of Synthetic Biology  states “The outcomes of this research would lay the foundation for identifying bioagent-agnostic signatures (BASs) of disease; we defined a BAS as a clinically measurable suite of biomarkers that accurately and reproducibly predicts the impacts of infection without a priori knowledge of an agent. Bioagent-agnostic signatures would incorporate various forms of data  that describe host characteristics and responses or broadly identify the presence of a class of pathogen . These signatures would comprise multiple “readout” molecules such as RNAs, proteins, and immune metabolites. For example, the ‘omics analysis of peripheral blood mononuclear cells and plasma from Ebola virus-infected patients identified subsets of host responses that correlated with either survival or severe disease outcomes ,6.We believe that this strategy shift could help unify existing research efforts; it could also serve as a basis for host-focused biosurveillance to complement pathogen-focused biosurveillance programs and determining which countermeasures to deploy or develop against a disease. Employing a BAS-based approach to biodefense would shift the community’s focus from characterizing specific pathogenic taxa to a framework that hinges on identifying functional interactions in host networks which drive or enable pathogenesis; recentering our biodefense posture in this way could make it easier to control future biological threats. These signatures would (1) be demonstrative of the specific interactions a pathogen employs to usurp host processes and (2) represent evolutionarily conserved host-response patterns to a wide range of pathogens and toxins that elicit disease.Because this approach would push the community to delineate broader and more consistent themes in host response to identify and mitigate disease, it might also help dampen rapid priority swings as new threats emerge  ,8. AlthoWe recognize that our proposal constitutes a major shift in the U.S. biodefense posture and will likely take many years to realize. Biologists must make fundamental advances in experimental design, technology, and data analysis to identify BASs that robustly classify similar courses of disease elicited by disparate pathogens and toxins. With BASs in hand, translational scientists and engineers will need to develop assays and procedures to operationalize them to protect human health and to conduct biosurveillance. Some BASs may only describe correlations to disease; scientists will likely need to understand the causal factors driving pathogenesis to fully leverage BASs as tools for countermeasure deployment and development and to triage cases based on predicted disease severity.We will briefly outline representative research with the potential to identify signals that scientists could incorporate into BASs and current impediments to implementing this approach. We will focus on humans as the host of interest, but this concept could be equally valid in categorizing animal and plant host responses.Bacillus anthracis infection in humans); however, commonalities exist in host responses to disparate pathogens, which could serve to nucleate the development of BASs  in response to stimuli such as lipopolysaccharide, protozoal extract, and viral infection, which promotes a Th1 adaptive response to clear intracellular pathogens, including bacteria, protozoa, and viruses ,33,34,35Similar opportunities could exist for viral pathogens. Hosts activate interferon signaling pathways in response to viral infection, which many viruses in turn manipulate as part of their pathogeneses and makes these pathways attractive as BAS inputs. Multiple cell types can sense viral PAMPs and subsequently activate the innate immune system to produce interferons and other cytokines ,42,43,44Viruses also reprogram innate immune response networks to establish productive infections. For example, herpesviruses encode proteins that inhibit different forms of programmed cell death ,49,50. UIron homeostasis underpins many processes in both healthy and diseased host cells as well as pathogens, which suggests that associated pathways could be a rich source of information for creating BASs. Hosts closely control iron levels, which protects them against both infection  and reacIron acquisition is a key virulence mechanism in both Gram-positive and Gram-negative bacteria that hosts actively resist. Bacterial pathogens express iron-scavenging systems  to suppress autophagy in a way that also suppresses the interferon response [Staphylococcus aureus induces host autophagy as a mechanism to scavenge nutrients during infection, through extensive remodeling of central carbon metabolism [Bacteria such as proteins ,78 or esproteins  to avoidresponse . In conttabolism . In thisViruses also exploit autophagy in a complex manner during infection; host cell autophagy can exhibit both pro-viral and antiviral roles during infection . VirusesStreptococcus pneumoniae infection [Haemophilus ducreyi and human hosts and characterizing the transcriptional response in both organisms. Studies such as these demonstrate the utility of large-scale computational methods to integrate ‘omic data to dissect complex host/pathogen interaction networks.Systems biology approaches can define “interactomes” of interconnecting proteins and molecules between host cells and pathogens; we anticipate higher-order analyses that compare interactomes across pathogeneses induced by different agents will reveal similarities that scientists could exploit in BASs ,93. For nfection . Griesennfection  demonstrIndividual ‘omics technologies may suffice to generate some BASs; however, we expect that as multi-omics technologies develop, they will provide an increased resolution of different disease states. Individual ‘omics technologies  only measure events in a single type of analyte , which might not be fully indicative of the state of the larger biological system. In contrast, multi-omics technologies promise to connect events that transpire among different analyte layers  and bettAs of 2021, multi-omics approaches are relatively immature; consequently, elucidating BASs will be experimentally and computationally challenging. Although scientists are actively researching advances in throughput and multimodal integration ,101,102,Scientists will need to overcome substantial experimental, technical, and data-analytic challenges to identify robust BASs and implement them in practice. These hurdles will take time and focused effort to resolve. However, the research community is beginning to take steps in this direction. For example, the US Food and Drug Administration (FDA) recently granted 501(k) approval for a diagnostic that measures host responses to distinguish between bacterial and viral infections . The toxUsing BASs to define disease states and provide an early warning system for infection and outbreaks represents an important addition to the current list-based biosurveillance and biodetection model. In contrast to list-based models of biosurveillance, the BAS model focuses on identifying host responses that are diagnostic of disease severity; health authorities would not need a priori knowledge of an infectious agent to identify a disease outbreak. In this way, the BAS model proposed here extends syndromic monitoring approaches to describe the nature of identified diseases in greater detail and towards a more functionality-based perspective. Host signature-based approaches have previously been proposed for specific infectious organisms  and as


The prevalence of gambling harm among active duty military personnel is a largely unexplored topic. With different forms of social gambling often found within (or in close proximity to) military bases around the world, understanding the extent of gambling activities and consequent harms occurring within military contexts warrants further attention. This review aims to identify, describe and thematically synthesise published literature on gambling harm and related issues among active duty military personnel. Scoping review methods were applied in order to understand this relatively under-researched population and understand appropriate avenues for future research. A systematic multi-database text word search, incorporating search results from Scopus, Pubmed, Web of Science, PsychInfo, and the Journal Military Medicine, was conducted. A total of 11 sources met inclusion criteria, all originating from the United States of America. The results suggest a distinct gap in the current international literature on this topic. Despite gambling’s long and colourful association with defence downtime, research into gambling harm prevalence rates in relation to what could be considered a high-risk group is limited. Findings reveal that strategies to identify and address gambling harm within this population are severely lacking from the published literature and non-existent outside North America. Implications for understanding and addressing gambling harm among active duty personnel and directions for future research are discussed. National Defence Authorization Act passed into United States (U.S.) federal law in 2018 mandated GD screening into the routine health checks of the U.S. Department of Defence (DoD).Wagering and games involving games of chance are widely accepted within Western defence cultures, as in broader society, as legitimate ‘recreational activities’. Gambling has historically been popular among defence members as a means to combat the stress, boredom, and isolation that can be experienced once deployed or upon returning home from active duty. These particular traits and past-times relating to gambling and culture are not unique to military contexts, as they are relevant in other similar sittings . These settings can be demographically skewed to young, male, high-risk-taking populations. In the United States, there is well-accepted support for gambling disordersThe international research understanding gambling as an issue within the military has emerged predominantly from North America, and almost exclusively focuses on veterans  and to provide a systematic synthesis of the disparate sources. English-speaking defence settings were targeted because of the similar socio-cultural settings and understandings of gambling and gambling related harm.A scoping review methodology was employed to capture the breadth of information available on the topic . Details of the Boolean searches applied in each database are described in Table To be considered eligible for inclusion, sources had to: focus on gambling prevalence; have active-duty military personnel from English speaking geographic regions  as the target population, and; be available online in full text. No restrictions were placed on study design or publication dates and no preference was given to qualitative or quantitative methodology. Foreign-language material was excluded because of the parameters of the focus of the review and potential translation costs. Therefore, sources were included if they were primary studies , secondary studies involving the analysis and interpretation of primary research, or discussion papers.After the initial database search, duplicates were removed. The screening of titles and abstracts, and the selection of articles from retrieved potentially relevant full manuscripts, were conducted by two reviewers (PL and MW) using the selection criteria described above. The reviewers independently classified the articles as ‘include’, ‘unclear’ or ‘exclude’, with discrepancies being resolved by discussion or referral to a third reviewer (MP). Full manuscripts that did not fulfil all of the criteria were excluded, with reasons for their exclusion documented. See Fig. Two reviewers independently conducted the data extraction. The data extraction form used was adapted from the ‘Data collection form for intervention review – RCTs and non-RCTs’ of The Cochrane CollaborationThematic synthesis was chosen to analyse these data collectively. Therefore, data analysis was undertaken in three stages: (1) evidence mapping, (2) identification of evidence gaps and (3) synthesis of selected research areas. In other words, iterative coding was applied to the extracted data which was organized into descriptive themes that were then used to generate broader analytical discussion.As outlined, search terms were intentionally broad to capture as much relevant literature as possible, yielding 317 references for further processing. Our informal search strategy yielded 54 studies for further review. After accounting for duplicates our search of online databases yielded 294 unique references See Fig.  and one Qualitative or text-based sources were analysed separately. Tables Examination of the extracted data revealed three key concepts spanning the literature (represented by the included studies) represented by the umbrella terms; ‘military culture’, ‘risk factors’ and ‘comorbidity’. Major themes (n = 6 +), mid-range n = 4–5), and minor themes (n = 3–4) were organized into their associated category and tabulated in Table –5, and mFeatures unique to military environments may inadvertently encourage engagement with gambling activities and at the same time contribute to concealment of potential issues or deterring access to treatment. Review findings highlight several related issues.The hierarchical (chain-of-command) structure of military forces is problematic in that supervisors are promoted as a primary resource that can provide advice, referral and support, however these authority figures also have the power to discipline, demote, and even discharge personnel with GD related issues. Soldiers in a Naval medical centre in the U.S. reported failing to disclose problematic gambling behaviour due to shame and confusion about the military’s confidentiality policies GSU . Other sFurther to this, studies suggest that the military is quick to reprimand people with GD while being slow to offer meaningful assistance, arguing that defence culture has little tolerance for behavioural problems. Clearly, the risk of prosecution for those who had committed crimes related to their gambling could discourage those with GD from seeking treatment and appropriate counselling. Thirty years ago, Little and Hecker  examinedopportunities to intervene when appropriate. Such interventions may well reduce the incidence and prevalence of GD, as well as alleviate associated negative impacts to public health (Korn and Reynolds 2009). Furthermore, confidential ways of seeking treatment should be put in place to address barriers to self-referral. Raising awareness of gambling harm would also assist the implementation of effective screening and referral processes.Many sources in this review state or otherwise imply that GD diagnosis is not common in defence populations, echoing the broader literature  released a report that examined GD in the military and recommended that questions specific to gambling be incorporated as part of DoD’s screening process GAO . DoD agrThe second overarching theme relates to the numerous risk factors for GD among military personnel relative to the general population.Recent studies show gambling is increasingly available and impactful in military life Whiting . As in tAshley  claim thThere is also the potential need to assess the impact of online gambling on military personnel  indicating that GD occurs with roughly similar frequency to the general population, though use of a less sensitive measure of GD (DSM-III & DSM IV instead of the more commonly used SOGS) indicates that comparable prevalence rates may be higher than reported. In addition, concerns on the presence or lack of confidentiality for individuals in the military reporting GD . Sources found associations between GD and heavy drinking Bray , 2002, aThe association between disordered gambling and PTSD in active duty populations is particularly under studied relative to other military population research. A review by Gates et al.,  suggestsThe secondary review objective the review highlighted some of the most salient research opportunities for understanding gambling in military settings. First, comprehensive prevalence studies of active duty defence personal to understand levels of gambling participation and harm in military populations is a high priority. Second, there is a need for dedicated gambling services for active-duty personnel and research is needed to evaluate any programs or services robustly Ashley . OverallThis is the first systematic review investigating GD among members in active service in a specific set of defence populations. A significant research gap exists internationally regarding the prevalence of gambling harm among active-duty defence personnel. What is consistently strong (in U.S. studies) is measuring GD in the context of other behaviours that jeopardise physical and mental health among active duty service personnel. However, structural and cultural barriers appear to be a primary reason for military forces and active-duty personnel refraining from recognising or engaging with GD. Although it will be of great interest to see the flow on effects of mandated GD screening in the US military, perhaps in other military settings, screening or prevalence studies should be conducted externally and independently.