Although the initial triggers of pathology and progression are unclear, multiple lines of evidence support therapeutic targeting of asyn in order to limit its prion-like misfolding. Here, we review recent pre-clinical and clinical work that offers promising treatment strategies to sequester, degrade, or silence asyn expression as a means to reduce the levels of seed or substrate. These diverse approaches include removal of aggregated asyn with passive or active immunization or by expression of vectorized antibodies, modulating kinetics of misfolding with small molecule anti-aggregants, lowering asyn gene expression by antisense oligonucleotides or inhibitory RNA, and pharmacological activation of asyn degradation pathways. We also discuss recent technological advances in combining low intensity focused ultrasound with intravenous microbubbles to transiently increase blood-brain barrier permeability for improved brain delivery and target engagement of these large molecule anti-asyn biologics. Pathological accumulation of alpha-synuclein (asyn) is the common distinguishing trait amongst the group of brain disorders known as synucleinopathies, which include Parkinson's disease (PD), Dementia with Lewy bodies (DLB), and Multiple System Atrophy (MSA). These disorders progressively develop neuronal and glial inclusions enriched in misfolded, phosphorylated, and detergent-insoluble asyn , 2. AsynThis process of recruitment and misfolding of native asyn, called permissive templating, is not dependent on cofactors and is readily observed in cell-free conditions with purified recombinant protein. However, the kinetics and range of conformations are constrained by mutations, binding partners, and by oxidative or post-translational modifications. The first evidence of this process in human brain was the recognition that host-to-graft transmission of Lewy bodies (LB) may have occurred in PD patients who had received fetal neuron grafts 10–15 years earlier , 8. ThisThere is also an additional layer of complexity evident in the differential spatio-temporal patterns of the pathological asyn spread between PD, MSA, and DLB, which imply that each disease develops a unique pattern of neuronal vulnerability. Imaging and post-mortem analyses suggest neuronal connectivity between brain regions is an essential but insufficient predictor of the pathological asyn spread . Other lThe multitude of cell and animal models demonstrating intercellular exchange of asyn aggregates have provided a wealth of mechanistic information on asyn internalization and trafficking, and prompted the development and testing of new therapeutic approaches , 34. TheThe possibility that misfolded asyn could be transferred between adjacent neurons and contribute to pathological spread provided the impetus to establish the mechanisms of asyn transfer . Extracevia small extracellular lipid bilayer vesicles known as exosomes, which contain proteins and RNA and are secreted by all cell types (An active mechanism that can enable intercellular asyn transmission is ll types  42). Th. Thvia sll types . Post-moll types . In thisll types , 45–47. ll types , 49.An important side-benefit of CNS-derived exosomes, if they are indeed bona fide mediators of disease, is their potential for use as biomarkers. Analyses of plasma CNS-derived exosomes from PD patients and healthy controls reveal correlations between the amount of exosomal asyn with PD diagnoses and severity –52. In ain vitro  . Pharmac2+ pathway and βCaMKII are involved in the formation of TNTs and the transfer of vesicles and aggregated asyn  also permit asyn exchange through direct cell-to-cell contact –70 Figu. These mted asyn . Drugs tInsights into asyn uptake are relevant to understanding the relationship between neuronal function and pathological spread, and for developing therapeutics. Extracellular aggregated asyn is cytotoxic when added to cell culture and is implicated in disease , 15. IntIn vivo studies also demonstrate intercellular transfer of ectopically-expressed human asyn from rat and mouse host brains to dopaminergic neuron grafts, where the transferred asyn localized to endosomes and seeded aggregation of asyn  inhibits asyn aggregation and reduces its toxicity to neurons in vitro . Inhibitin vitro . Recentlin vitro . PharmacFollowing internalization, asyn fibrils are trafficked through endosomal compartments from early to late endosomes and finally to lysosomes where they can persist for days , 84, 91.in vitro , are a risk factor for sporadic PD and DLB , which becomes fragmented in PD, impairing proteostasis and the activation of unfolded protein response (UPR), and retention of GCase within the ER . InteresRapamycin, an FDA approved drug, increases autophagy levels by inhibiting the mTOR pathway and can reduce dopaminergic cell death, decrease levels of phosphorylated asyn, and improve mitochondrial function in cell and animal models of synucleinopathy –117. RapDirect contact between misfolded and native asyn is necessary for permissive templating. How is this interaction mediated if misfolded asyn is lumenal, enclosed within endocytic vesicles, whereas the native asyn substrate is cytosolic? There is evidence that asyn aggregates can rupture endocytic vesicles following internalization , 129 Fi. The rupThe ability of antibodies to sequester misfolded proteins with high-affinity and high-specificity make them attractive as therapeutics for neurodegenerative diseases . ExtensiActive immunization with peptides mimicking the asyn carboxyl-terminus effectively reduced pathological aggregate formation and significantly mitigated motor deficits in transgenic human asyn mice . These pclinicaltrials.gov).Biogen's BIIB054 (Cinpanemab) monoclonal antibody, on the other hand, did not meet its primary and secondary endpoints in a Phase 2 clinical trial, despite promising pre-clinical results , and theNotwithstanding the significant advances in immunotherapy as a disease modifying strategy, achieving adequate BBB penetrance and target engagement remain key challenges for the use of these therapeutics in the clinic. Retrospective evaluations of multiple immunotherapy clinical trials conducted for Alzheimer's disease (AD) have shown limited benefits , 154. Thin vivo has potential disadvantages including possible neuronal dysfunction, neurotoxicity, and loss of dopaminergic neurons. In vivo studies on the viability of this approach for counteracting PD have been controversial; some reports suggest that >90% suppression of asyn could be neurotoxic and cause the loss of nigral tyrosine hydroxylase expression  and intracellular asyn levels in recipient neurons could also mitigate disease progression by reducing the native substrate available for templated misfolding. RNA interference (RNAi) has been widely used in cell and animal models to effectively counteract asyn accumulation. Small interfering RNA (siRNA) are 21–23 nucleotide long double stranded RNA molecules that drive degradation of target mRNA upon sequence-specific binding. Direct infusion into mouse hippocampus of naked anti-asyn siRNA successfully downregulated SNCA expression . Short hpression –167, whepression , 168. Cupression , 170. HoGenome-wide association analyses (GWAS) have revealed that expression profiles of certain micro RNAs (miRNAs) including include miRNA-30, miRNA- 485, miRNA-29, miRNA-26, and let-7 are altered in the brains of PD patients , 172. MiDelivery vectors for nucleic acid-based therapies can be broadly classified into non-viral and viral vectors. Non-viral vectors display low cytotoxicity and immunogenicity, are cost-effective, and can be easily modified for target binding . For exaViral vectors have been historically preferred for gene therapy targeting neurodegenerative disorders because of their stable and long-term expression profiles. Adeno-associated viral vectors (AAVs) are currently the most advanced vectors for CNS gene therapy, with AAV serotypes 1,2,5 and 9 being the most frequently used , 181. LeSNCA gene in patient-derived human induced pluripotent stem cell (hiPSCs)  mediated genome editing was used to correct point mutations in the (hiPSCs) . Gene ed(hiPSCs) . Recentl(hiPSCs) . This siSNCA in various PD models have been summarized in A central focus of CNS targeted gene therapy is the development of vectors that can efficiently bypass the blood brain barrier (BBB). The BBB is highly selective and restricts the entry of molecules larger than 400 daltons such that it is impermeable to the vast majority of systemically administered small molecule drugs and nearly all biological therapeutics  discuss. It is nThe primary drawback of using antibodies is their high molecular weight, which challenges their therapeutic potential. Intrabodies  are small antibody fragments containing only the antigen-binding domain, derived from either heavy-chain only antibodies (HCAbs) found in all Camelidae and some shark sera or recombinantly produced from conventional antibodies . The twoin vitro. This intrabody was fused with a cell-penetrating peptide, underscoring the ability for genetic engineering to enhance the functionality of intrabodies  of SNCA mRNA, a region involved in regulating translation . The mosin vitro and in vivo, reviewed by  . Based oAlthough these data require replication in independent cohorts and/or investigation in laboratory-based assays or animal models of asyn aggregation, they do provide promising real-world data on the potential of using a computational approach to efficiently identify drugs targeting oligomerization of asyn, that are amenable to repurposing for disease modification in PD. To our knowledge there are still no drugs with regulatory approval that were identified using artificial intelligence, however given the pace of discovery and significant investment in this domain, the true promise of this technology should become apparent in the coming years.A key challenge for asyn therapeutics is their delivery across the BBB, a protective and selectively permeable microvasculature around the CNS composed of tight junctions, endothelial cells, pericytes and astrocytic end foot processes. Receptors, ion channels and transporters on the BBB tightly regulate the transport of molecules into the brain parenchyma, thereby restricting the bioavailability of CNS targeted drugs . Indeed,In the past two decades, the use of MRI-guided focused ultrasound (MRIgFUS) has developed as a revolutionary technology that allows for localized and targeted delivery of CNS therapeutics across the BBB without the need for invasive surgery. Although the first demonstrated use of focused ultrasound waves resulted in BBB opening around a lesion site , a subse3 to 25 cm3 have been safely targeted with MRIgFUS for increased BBB permeability  . ImportaTaken together, growing pre-clinical and clinical evidence indicates that FUS mediated BBB opening offers several advantages for therapeutic delivery. First, the FUS mediated increase in BBB permeability is non-invasive and therefore, repeated therapeutic applications, if required, may be a viable strategy. Second, MRIgFUS allows for precise target engagement, either into discreet or broad brain regions, unilaterally or bilaterally as needed. Third, the FUS mediated BBB opening is transient, with a therapeutic window of ~6 h, thus safely returning the BBB to its prior intact state. Fourth, FUS allows for administration of combinations of therapeutics. Several lines of evidence indicate that neurotrophic factors like GDNF can promote dopaminergic regeneration and may be required in addition to therapeutics that aid in targeting and clearing pathological asyn , 276. InNevertheless, some factors are still unknown and need to be addressed before FUS can be safely employed as a routine therapeutic strategy for brain targeting. For one, more data is required regarding the safety profile of BBB opening, and how it may be affected by age and disease onset. This is especially relevant for neurodegenerative diseases where the BBB integrity may be partially compromised . SecondlNew tests to amplify and identify pathologically misfolded asyn in biopsy tissues are increasing in sensitivity –284 and All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.AT is supported by grants from the Canadian Institutes of Health Research (CIHR PJT180582) and the Weston Brain Institute (RR193223). SM received a postdoctoral fellowship from the Edmond J. Safra foundation. NPV is supported by the Rossy Foundation and Blidner Family Foundation. AH received a studentship from the Canadian Institutes of Health Research (CIHR-CGS-M).SPS is a Sanofi employee and stockholder. The remaining 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.


Despite their prevalence and reported patient interest in their use, uptake of health-related apps is limited. The Veterans Health Administration (VHA) has developed a variety of apps to support veterans; however, uptake remains low nationally.We examined the prevalence of VHA health-related app use and how veterans learned about these apps in order to identify factors associated with their use.As part of a VHA quality improvement initiative, we recruited a national cohort of veterans to obtain feedback on their use of technology for health and collected data from them via a cross-sectional survey. The survey data were supplemented with VHA administrative data. We used descriptive statistics to examine demographic and health characteristics, health-related technology use, and how veterans learned about apps. We assessed factors associated with app use using bivariate analyses and multiple logistic regression models.We had complete data on 1259 veterans. A majority of the sample was male , aged older than 65 years , White , and non-Hispanic . Most respondents  reported being very comfortable and confident using computers, over half  reported being an early adopter of technology, and almost half  reported having used a VHA health-related app. Just over one-third  reported that their VHA care team members encouraged them to use health-related apps. Respondents reported learning about available VHA health-related apps by reading about them on the VHA’s patient portal , being told about them by their VHA health care team , and reading about them on the VHA’s website . Veterans who self-reported having used VHA health-related apps were more likely to receive care at the VHA , be in worse health , report owning a desktop or laptop computer , have posttraumatic stress disorder , and report having VHA health care team members encourage them to use the apps .We found strong associations between self-reported use by veterans of VHA health-related apps and multiple variables in our survey. The strongest association was observed between a veteran self-reporting app use and having received encouragement from their VHA health care team to use the apps. Veterans who reported receiving encouragement from their VHA care team members had nearly 3 times higher odds of using VHA apps than veterans who did not report receiving such encouragement. Our results add to growing evidence suggesting that endorsement of apps by a health care system or health care team can positively impact patient uptake and use. There is an expanding number of apps available to help patients manage specific health conditions and promote overall well-being . EvidencA variety of barriers to health-related app adoption have been identified in previous work, including concerns about privacy and security -14, app US military veterans are an ideal population in which to examine the adoption of health-related apps. In comparison to the general US adult population, veterans often face significant health-related challenges, including disproportionate rates of physical and behavioral health diagnoses ,21, and In recent years, studies have indicated that integrating apps into care for veterans may improve outcomes . The effDespite the evolving evidence for their effectiveness, as well as recent literature indicating that many veterans are interested in using health-related apps ,27 and hThe VHA Office of Connected Care, in cooperation with investigators from the VHA Quality Enhancement Research Initiative program, developed the Veterans Engagement with Technology Collaborative (VET-C) cohort in 2017, the purpose of which was to engage veterans in the evaluation of VHA technologies that are intended to increase access, enhance coordination, and support self-management . The VETTo be eligible to join the VET-C cohort, veterans had to be users of VHA health care services, have a mobile phone, and be active users of the secure messaging feature of the VHA’s online patient portal, My HealtheVet, a feature that is available to veterans who have a premium portal account. The secure messaging use requirement was intended as a proxy for receptivity to and use of VHA patient-facing technologies more generally. Active use of secure messaging was defined as having sent at least 5 secure messages to VHA clinical team members through the portal in the 12 months prior to cohort recruitment. Veterans who met these inclusion criteria were recruited from VHA facilities across the United States. VHA facilities were chosen as VET-C recruitment sites because they (1) had high rates of secure messaging, (2) served as field test sites for other new VHA patient-facing technologies, (3) were known to serve significant populations of women veterans and veterans from diverse ethnic and minority groups, and (4) had active research and evaluation programs.Recruitment lists to support development of the VET-C cohort were created by querying data from the VHA Corporate Data Warehouse (CDW). Veterans were called once by the evaluation team, and those who answered were told about the purpose of the VET-C cohort and invited to join. Veterans who were interested then completed the cohort baseline telephone survey, and evaluation team members entered their responses directly into an online REDCap database. During 2017 and 2018, 2727 veterans from 14 VHA facilities joined the VET-C cohort and completed the baseline survey. From March 2019 to March 2020, we administered a second survey to all veterans in the VET-C cohort who completed the baseline survey. This follow-up survey was completed by 1418 veterans in the cohort. Both our baseline and second surveys included validated question items, questions used in other studies, and new question items developed specifically for these surveys. They were developed in close consultation with leadership from the VHA Office of Connected Care, which is responsible for the health care system’s digital health strategy. After we excluded veterans for whom there were missing data for the variables used in the analyses, we included a total of 1259 veterans in the current analyses.For this paper, constructs of interest from the surveys included veteran demographics, health and health care use variables, technology ownership and use, VHA care team member encouragement to use VHA apps, and use of VHA health-related apps.We asked all participants to respond to demographic questions on age, gender, race, ethnicity, relationship status, and education; report their perceived health status ; report Additionally, we asked participants to report whether they used VHA health-related apps and how they learned about the VHA health-related apps that are available. We also asked participants to report the perceived extent to which their VHA care team members encouraged them to use health-related apps .We calculated the participants’ prior-year comorbidity index as the Hierarchical Condition Community (HCC) score based on information in the VHA CDW ,32. The t test) to examine differences among veterans who reported using (vs not using) VHA health-related apps. We then assessed factors associated with VHA health-related app use using unadjusted and adjusted multiple logistic regression models. We selected variables for inclusion in the unadjusted model based on significant bivariate associations with the outcome variables and known associations from the existing literature; we selected variables for inclusion in the adjusted model based on significant unadjusted associations at the P<.1 level and known associations from the existing literature. Statistical analyses were performed with Stata MP .We used descriptive statistics  to characterize the demographics of the sample, as well as their reported health and health care use, technology ownership and use, VHA health-related app use, how they learned about VHA health-related apps, and their perceptions of VHA care team member encouragement to use health-related apps. We used bivariate analyses .Descriptive statistics and results of bivariate analyses are presented in Respondents reported having learned about VHA apps through the VHA’s patient portal , their VHA health care team , the VHA’s government website , veteran service organizations , newsletters , other veterans , public app stores , and the VHA mobile app store . These results are presented in P=.004) and veterans who reported owning a desktop or laptop computer , mostly receiving their medical care at the VHA , and being encouraged by their VHA care team to use the apps . In addition, veterans who self-reported using VHA health-related apps had a higher average HCC score than those who did not .Bivariate analyses comparing respondents who reported using (vs not using) VHA health-related apps revealed that the former included greater proportions of veterans with PTSD , and better health status ,18. Our In interpreting these findings, it is important to note that our sample consisted of veterans who were established health-technology users and, as such, they may have had higher levels of technology literacy. It is possible that removing technology literacy as a barrier would affect the relationship between health status and app adoption. This is particularly important because patients who have more chronic conditions may benefit more from using health-related apps and because technology literacy is a modifiable factor , as it cSimilarly, the veterans in our sample reported having learned about VHA health-related apps from a variety of sources, the most frequently reported of which were directly tied to the VHA health care system. Sources included the VHA’s online patient portal, VHA health care team members, and the VHA’s government website. Of note, the VHA understands the importance of its online patient portal in driving adoption of other VHA health-related apps, and leverages the system to promote, market, and direct veterans to these resources in an effort to increase engagement in care and self-management. In this way, use of one patient-facing technology can beget the use of others, suggesting the importance of interventions to support the use of other technologies  and theOf note, we also found that veterans with PTSD had greater odds of self-reporting use of VHA health-related apps. Interestingly, the results of our unadjusted analyses did not suggest that there were differences in app use among veterans with other diagnosed health conditions. While our data cannot speak to the specific reasons for this finding, we suspect that it may be driven in part by the fact that the VHA has more available and established apps relevant to PTSD, as well as conditions highly comorbid with PTSD, including depression, anxiety disorders, and insomnia, and these apps may thus be promoted more frequently than others.Relatedly, a recent systematic review of available VHA and Department of Defense mental health–related apps found that while efficacy data for many such apps were emerging, research did indicate the efficacy of the PTSD Coach and Virtual Hope Box apps . In addiWe cannot infer causal relationships from our analyses, and self-reported survey data are subject to biases. The veterans who compose the VET-C cohort were also intentionally sampled because they were users of another VHA patient-facing technology, the health care system’s patient portal. In addition to potentially being more likely to use technology, previous research has indicated that veterans who use the VHA’s portal are more educated, younger, and have higher income than the overall veteran population ,40, whicIn this survey of veterans, we found that nearly half of respondents self-reported use of VHA health-related apps and that encouragement from a veteran’s health care team was a critical factor associated with self-reported app use. Veterans predominantly reported learning about available health-related apps through other VHA technologies or their VHA health care team members. These results add to growing evidence suggesting that endorsement of apps by a health care system or health care providers can positively impact patient uptake and use. Future work should examine approaches to supporting efforts by health care team members to engage with patients about apps that may be most beneficial to their health, as well as ways to support shared decision-making regarding which apps to use and how best to integrate them as components of care and self-management. Such approaches could be included as part of multicomponent implementation strategies and tested to determine their impacts on the adoption of health-related apps by patients.


Pattern separation studies suggest age-related declines in discriminating between a newly and a previously encountered overlapping mental representations. However, when similar experiences occur contemporaneously, shared features may serve as intrinsic factors that interfere with the ability to encode and retrieve events as distinct memories. If so, successful inhibition of competitors might also be necessary to maintain memory specificity. Here, we investigated the interplay between memory specificity and inhibitory control in younger  and older  adults by testing whether and how presenting a target item with a lure item (varied in its similarity to the target) influenced recognition accuracy, probed using a 3-item  forced-choice recognition test. Surprisingly, we found no age-related differences, including in target accuracy, which was high in both groups . While not age-specific, we did find that the pattern of errors varied as a function of the level of similarity between the items at encoding. When target and lure were highly similar, a greater proportion of control items were falsely-recognized relative to lure items. It is possible that inhibition of the lure spread to the target, leaving both inaccessible at retrieval . When the target and lure were less similar, participants false alarmed more to lure than control, potentially because the lure provided less competition and thus did not necessitate inhibitory control. These results shed light on how inhibitory processes might be differentially involved, but age-invariant, depending on the similarity of encoded items.


In this editorial, we describe the issues with the current diagnostic process, explore the potential impact of the COVID-19 pandemic, and identify alternative strategies that may improve the early diagnosis of myeloma.Multiple myeloma, the third most common blood cancer, is often discovered following multiple prediagnostic consultations, with delays in diagnosis resulting from the ubiquitous nature of presenting symptoms.1,2 There are various reasons for this. It is a relatively rare cancer; an average GP will see one new case every 8–10 years. Patients present with a range of non-specific symptoms, including back pain, bone pain, fatigue, and blood test abnormalities , which are all common in an ageing population and often attributable to concurrent conditions. Early diagnosis of myeloma is crucial in limiting disease-related complications, such as lytic bone disease, pathological fractures, kidney injury, and severe infection, all of which may impact long-term quality of life, eligibility for routine or experimental therapy, treatment tolerance, response rates, and survival.Myeloma has one of the longest diagnostic intervals of all cancers. Around half of myeloma patients have three or more pre-referral consultations and around one-third are diagnosed through emergency presentation.3 Reductions in overall urgent referrals for suspected cancer were mainly due to changes in patient attendance, rather than GP referral behaviour. Consultation rates for specific symptoms for other cancers, including red-flag symptoms , reduced during the first lockdown and returned to expected rates within months. However, consultation rates for non-specific symptoms common in myeloma patients  remained below expected levels at the end of 2020.4 Given the current workload pressures in general practice, patients with non-specific symptoms may prioritise other health concerns, or fail to present at all due to concerns about wasting doctors’ time. GPs have adopted alternative approaches to triage and consulting, including electronic data capture to triage patients and alternatives to face-to-face consultations. GPs may not prioritise non-specific symptoms, as e-consults will automatically ‘red flag’ some symptoms but not non-specific symptoms. We need to understand how this affects patients’ behaviour with non-specific cancer symptoms and presentation in general practice, especially for older patients and those with lower health literacy.The COVID-19 pandemic resulted in a substantial decrease in myeloma referrals and diagnoses, in parallel with an increase in emergency presentations.5 Once diagnosed, regular monitoring of blood tests and clinical symptoms is recommended, often guided by MGUS risk-stratification strategies. This has cost and workload implications for the NHS, as well as for the mental health of these patients, the majority of whom will never require treatment for MGUS. Any screening programme for myeloma is likely to increase diagnoses of MGUS and increase the pressures outlined above. A population-based screening trial for MGUS is ongoing in Iceland, where participants with MGUS are randomised to different follow-up strategies.6 The results from this will provide vital evidence on the cost-effectiveness of screening and monitoring approaches.Strategies to facilitate the early diagnosis of myeloma must consider the precursor condition monoclonal gammopathy of undetermined significance (MGUS), often diagnosed incidentally via routine blood tests. MGUS is common, with a prevalence of 3–5% and a risk of progression to myeloma of approximately 1% per year.7 A fundamental challenge is prompting the clinician to consider myeloma and initiate appropriate investigations. Once myeloma is suspected, the diagnostic workup is generally straightforward. Serum protein electrophoresis, immunofixation, and serum free light chain assays are sensitive and specific initial tests for the majority of patients, followed by bone marrow biopsy. Therefore, it is essential that high-risk individuals are identified and appropriately investigated. Strategies to do this with minimal impact on existing NHS resources are needed, particularly in the current COVID-19-recovery phase.Many myeloma patients have complex diagnostic journeys and may be seen by other specialists before haematology, potentially delaying diagnosis.8Symptom awareness for both patients and GPs is important to ensure prompt presentation and appropriate referrals. The roll-out of rapid diagnostic centres (RDCs) may provide more timely diagnosis for those with non-specific symptoms by offering a single point of access for further diagnostic investigations, limiting instances of multiple specialty referrals for patients meeting the criteria for multiple site-specific pathways. Evidence emerging from RDC evaluations have shown that around 13% of cancers diagnosed via RDCs are haematological cancers.9,10 There is potential to implement such algorithms into decision support tools within GP electronic systems, but challenges remain before these can be fully integrated, such as how this would trigger alerts prospectively and how these would fit with current GP workflows.11Over recent years there has been a major increase in the development of clinical risk prediction models  based on electronic health records. Specifically for myeloma, clinical prediction models developed from general practice records have combined symptom data and blood test results to identify patients at higher risk of developing myeloma.12 Algorithms based on minor abnormalities and subtle changes in routine blood parameters  may be implemented in hospital laboratories to trigger automatic reflex testing of high-risk individuals for myeloma. This would enable large-scale, low-cost case finding for myeloma, independent of the patient’s ability to articulate their symptoms and the GP considering myeloma as part of the differential diagnosis. Setting appropriate thresholds for testing are challenging and there are legitimate concerns about risk communication, with further research required to explore the acceptability of this approach.Changes in routine blood parameters can be detected several years prior to myeloma diagnosis, before the onset of symptoms or disease complications.https://www.nhs-galleri.org/). This test can identify plasma cell disorders such as myeloma; however, myeloma and MGUS are genetically very similar and it is not yet clear how well the Galleri test differentiates between the two conditions. There therefore remains a risk of overdiagnosis of low-risk MGUS, the consequences of which may be significant and will only be borne out by prospective studies.Cell-free DNA (cfDNA) technologies are potentially paradigm shifting for cancer screening. These tests identify circulating tumour DNA and are often able to identify multiple tumour types with a single blood test. The most widely publicised example of this is the methylation-based Galleri® Test, used as part of the NHS-Galleri [GRAIL] study that is exploring population-level cancer screening in asymptomatic individuals (Improving the timeliness of myeloma diagnosis is vital to improving patient outcomes, but is difficult to achieve because of complex, non-specific, and varied presentations. Improving GP education on the salient features of multiple myeloma presentation and the investigations required for diagnosis, alongside ensuring adequate safety netting for patients with persistent, unexplained symptoms, should be urgent priorities. Changes to general practice consultations following the COVID-19 pandemic have made myeloma diagnosis more difficult, and, over the longer term, research is required to develop intelligent and technological strategies that support physician decision making and reduce diagnostic delay. These approaches must minimise low-risk MGUS diagnosis, demonstrate economic viability, and have prospective evidence of improvements in objective parameters such as diagnostic speed, disease stage at diagnosis, quality of life, and ultimately survival.


Brain cancer and leukemia are the most common cancers diagnosed in the pediatric population and are often treated with lifesaving chemotherapy. However, chemotherapy causes severe adverse effects and chemotherapy-induced peripheral neuropathy (CIPN) is a major dose-limiting and debilitating side effect. CIPN can greatly impair quality of life and increases morbidity of pediatric patients with cancer, with the accompanying symptoms frequently remaining underdiagnosed. Little is known about the incidence of CIPN, its impact on the pediatric population, and the underlying pathophysiological mechanisms, as most existing information stems from studies in animal models or adult cancer patients. Herein, we aim to provide an understanding of CIPN in the pediatric population and focus on the 6 main substance groups that frequently cause CIPN, namely the vinca alkaloids (vincristine), platinum-based antineoplastics , taxanes (paclitaxel and docetaxel), epothilones (ixabepilone), proteasome inhibitors (bortezomib) and immunomodulatory drugs . We discuss the clinical manifestations, assessments and diagnostic tools, as well as risk factors, pathophysiological processes and current pharmacological and non-pharmacological approaches for the prevention and treatment of CIPN. Chemotherapy-induced peripheral neuropathy (CIPN) is defined as dysfunction of peripheral nerves caused by the use of one or more chemotherapeutic agents. The prevalence of CIPN amongst pediatric patients and cancer survivors can approach >90% depending on the patient population, chemotherapeutic agent used, the cumulative dose administered, and the criteria used to diagnose CIPN . While aThis review focusses on the 6 main substance classes of chemotherapeutics that frequently cause CIPN, the vinca alkaloids (vincristine), platinum-based antineoplastics , taxanes (paclitaxel and docetaxel), epothilones (ixabepilone), proteasome inhibitors (bortezomib), and immunomodulatory drugs . While there is data to suggest that the drugs doxorubicin and cyclophosphamide, often used in the treatment of childhood leukemia or brain cancers, may cause neuropathy, there is insufficient evidence of this in pediatric populations and therefore will not be included in this review . We discThe most common types of cancer diagnosed in children <14 years include leukemias and cancers of the central nervous system (CNS) , vinblastine (first generation), vindesine (second generation) and vinorelbine (third generation). The third-generation agents are reportedly less neurotoxic and associated with a decreased frequency of adverse events  inhibitor, and it is frequently used to treat multiple myeloma in adults . It is uPediatric CIPN is a common, debilitating side effect of chemotherapy and the actual incidence in pediatric populations is probably higher than the reported incidence . Especia2 of vincristine experienced severe peripheral neurotoxicity compared to 20%–52% of patients who received cumulative doses 30 mg/m2 , 50% experienced an improvement in electrophysiological abnormalities with the gradual reduction in thalidomide dose .Pediatric CIPN is often characterized by a combination of sensory, motor, and autonomic symptoms of different intensities . CIPN syVincristine-induced neurotoxicity occurs during the early stages of treatment, typically within the first month . VincrisOxaliplatin is a third-generation platinum derivative that causes acute and chronic neuropathy. Phase I and II studies in pediatric patients have shown that oxaliplatin causes neurotoxicity that manifested as cold dysesthesia, laryngopharyngeal and limb paresthesia and dysesthesia, muscle cramps and jaw pain. Additionally, oxaliplatin causes diarrhea and constipation . CisplatPaclitaxel causes dose limiting neuropathy that occurs early, within 1 week of the infusion, and is characterized predominantly by sensory symptoms, such as tingling, numbness and burning pain in a “stocking and glove” distribution. Myalgia and gastrointestinal disturbances are also common . Adult pThere is limited data describing the development of CIPN symptoms following ixabepilone and bortezomib treatment in the pediatric populations. However, in a phase I study of ixabepilone in children and adolescents with refractory solid tumors, the neuropathy reported was mostly mild, characterized predominantly by sensory symptoms . BortezoThalidomide causes sensory, motor and autonomic neuropathy that is characterized by paresthesia, numbness, mild motor impairment and constipation . ThalidoAssessment and diagnosis of CIPN among pediatric cancer patients and survivors can be challenging. Children generally are either unable, or find it difficult, to recognize and describe symptoms associated with CIPN. Additionally, the signs and symptoms of CIPN experienced by individuals can be highly variable, and often change over the course of treatment as well as the transition from the pediatric, to adolescent, and adult years. To date, there have been no systematic studies that have specifically examined the temporal course of CIPN symptoms over the course of treatment, nor specifically compared these between different age groups. Accordingly, further research will be needed that integrates factors such as individual susceptibility to CIPN, age, time- and chemotherapy-related factors to determine the most suitable methods for diagnosis and assessment of CIPN for different chemotherapeutics and age groups.Nevertheless, a range of screening instruments have been developed to diagnose and assess the severity of CIPN symptoms in clinical settings, which can be divided into three major groups: subjective assessments, objective assessments and combined subjective and objective assessments . Howevervia a 20-item questionnaire assessing CIPN symptoms and functional impairment  questionnaires and pain/neuropathy scales, such as the Pediatric Quality of Life Inventory and Wong-Baker FACES pain scale , current perception threshold (CPT) and tactile perception threshold (TPT) tests, to name a few (for full list see Another objective test that can be used to assess CIPN in children is the Movement Assessment Battery for Children—Second Edition (MABC-2), which is a functional test that has been developed to assess functional and motor impairment in children . The MABCombined subjective and objective assessments are tests that utilize questionnaires and combine these with functional, neurological, or electrophysiological assessments. They include tests such as the Pediatric–modified Total Neuropathy Scale (ped-m TNS) and the Total Neuropathy Scale–Pediatric Version (TNS-PV)  inflammasome is predominantly expressed by macrophages and neutrophils and to some extent by dendritic cells, microglia and possibly by dorsal root ganglia . NLRP3 wHow chemotherapeutics activate NLRP3 is unclear. One possible activation pathway could be via increased production of reactive oxygen species caused by mitochondrial disfunction . Mitocho2+) is a ubiquitous signaling molecule and particularly important for function of sensory neurons, it is perhaps not surprising that evidence of dysregulated Ca2+ homeostasis has been reported for several chemotherapeutics, including oxaliplatin, cisplatin, paclitaxel, bortezomib and vincristine , voltage-gated potassium (KV), voltage-gated calcium (CaV) and transient receptor potential family (TRP) channels are believed to contribute substantially to the cascade of events that lead to neuronal hyperexcitability  being expressed in dorsal root ganglion neurons. Although involvement of tetrodotoxin (TTX)-resistant NaV isoforms, most notably NaV1.8 and NaV1.9, in CIPN has been suggested  channels regulate the excitability and resting membrane potential of a cell and shape the action potential waveforms in neurons. They are classified into 4 groups, namely the voltage-gated K (KV) channels, calcium-activated potassium (KCa) channels, inwardly rectifying potassium channels (Kir) and two-pore domain potassium (K2P) channels. Enhanced neuronal excitability attributable to altered expression and/or function of K+ channels has been suggested as a putative pathophysiological mechanism for some chemotherapeutic agents, although the functional heterogeneity of these channels, which can assemble as homo- or heteromultimers, has made dissection of specific subtype contributions comparatively difficult. Evidence of functionally altered K+ conductances arose from observations that oxaliplatin treatment leads to broadening of the repolarization phase as well as repetitive firing and afterhyperpolarization (AHP) in isolated rat sciatic nerve  channels function as stimulus transducers and often respond to thermal or chemical signals—including protons and reactive oxygen species—leading to activation and sensitization of peripheral nerve terminals. The intense interest in the role of TRP channels in various pain states has also extended to CIPN, often with conflicting results that may arise due to species, dose, or other experimental differences. Particular interest in pathognomic cold hypersensitivity induced by oxaliplatin has seen the cold-thermosensors TRPA1 or TRPM8 implicated by several studies , althougThe management of pediatric CIPN includes preventative strategies as well as pharmacological and non-pharmacological measures to control symptoms. The future of CIPN prevention and treatment arguably lies in the development of mechanism-based treatment strategies, targeting specific molecular structures and preventing peripheral nerve damage caused by chemotherapy agents. Currently, pharmaceutical agents used for the prevention and/or management of CIPN include supplements, such as calcium, acetyl carnitine, glutamine, and omega 3, and medications, such as amifostine, amitriptyline, venlafaxine, oxcarbazepine, and carbamazepine . The benSome studies have also explored topical administration of drugs to manage CIPN. Topical gel containing baclofen (10 mg), amitriptyline HCL (40 mg), and ketamine (20 mg) alleviated CIPN symptoms such as cramping and tingling in patients but this outcome was associated with higher systemic toxicity .®) in in vivo rodent models  which has effects at the molecular/genetic, cellular and systems levels. PBM has found some support in recent placebo/sham-controlled studies for reducing and/or controlling neuropathy pain, function and symptoms . ElectriThe diversity of effectiveness of both pharmacological and non-pharmacological approaches to the prevention and management of CIPN suggests that a precision medicine approach tailored to an individual’s CIPN phenotype, physical capabilities, co-morbidities and willingness to comply with treatment may be needed, therefore accurate profiling of the individual’s CIPN would need to be developed .Our understanding of the pathomechanisms leading to CIPN have improved greatly in recent years. In particular, combination of optimized chemotherapy regimens with molecular or personalized treatment approaches could lead to development of improved treatments for CIPN with the promise to deliver improved patient outcomes. Some of the key considerations that have become apparent include the need for agent-specific, mechanisms-based treatments. These will likely include approaches targeting both aberrant neuronal function, as well as pathological processes such as inflammation that may indirectly affect sensory systems. Although adult rodent models remain invaluable in this regard, there is also a clear need for juvenile rodent models and humanized, or human, model systems that will facilitate clinical translation. One such approach may include the use of human DRGs, or stem-cell derived sensory neurons that can facilitate investigation of pathological mechanisms in more directly relevant model systems. Importantly, this review has highlighted the dearth of evidence specific to CIPN mechanisms and treatments in the pediatric population. Additional research is desperately needed, particularly in light of the clear physiological differences between children and adults that are likely to contribute to divergent mechanisms, and accordingly divergent treatment approaches, that are needed for individuals suffering from CIPN.


Insects are associated with a plethora of different microbes of which we are only starting to understand their role in shaping insect–plant interactions. Besides directly benefitting from symbiotic microbial metabolism, insects obtain and transmit microbes within their environment, making them ideal vectors and potential beneficiaries of plant diseases and microbes that alter plant defenses. To prevent damage, plants elicit stress-specific defenses to ward off insects and their microbiota. However, both insects and microbes harbor a wealth of adaptations that allow them to circumvent effective plant defense activation. In the past decades, it has become apparent that the enormous diversity and metabolic potential of insect-associated microbes may play a far more important role in shaping insect–plant interactions than previously anticipated. The latter may have implications for the development of sustainable pest control strategies. Therefore, this review sheds light on the current knowledge on multitrophic insect–microbe–plant interactions in a rapidly expanding field of research. A review on insect-associated microbes and their role in insect-plant interactions Microbes have attained substantial attention in the past decades because of their importance in interactions with both animals and plants. Besides their ability to initiate serious infectious diseases with detrimental effects on their hosts, beneficial effects of microbes are also well-known. Microbes can support their animal hosts with digestion and the production of essential amino acids and vitamins. For plants, microbes have similar roles. Microbes in the soil decompose organic material and release nutrients, they can promote plant growth by facilitating efficient nutrient uptake and support the plant’s defensive system by priming plant responsiveness to potential threats , and symbionts that can have a relationship with their host in which both benefit , none benefit , or one benefits while the other is harmed  causes bacterial canker in kiwifruit plants , plant beneficial and growth promoting microbes, and pathogens  bacteria  are intracellular Gram-negative bacteria that are present in insects and fulfill roles in primary nutrition. They can manipulate insect reproduction and are transmitted to plants where they are pathogenic. Insects transmitting RLOs are white flies, cicadas, leafhoppers, and psyllids symbiotic bacteria that are transmitted by insects including thrips, flea beetles, shield bugs, and false potato beetle (Leptinotarsa juncta), and are present in different life stages of their insect vector  can cause center rot of sweet onion  and causes Stewart’s bacterial wilt and leaf blight in maize  or citrus greening disease is associated with three different Ca. Liberibacter species, namely Ca. Liberibacter asiaticus, Ca. L. africanus, and Ca. L. americanus. Ca. L. asiaticus is the most severe pathogen, spread by Asian citrus psyllid Diaphorina citri and causes devastating epidemics in several countries. Ca. L. africanus occurs in Africa, where it is spread by the African citrus psyllid Trioza erytreae. Ca. L. solanacearum is associated with diseases, e.g. causing Zebra chip symptoms in several solanaceous plants , and transmitted by potato psyllid Bactericera cockerelli, and the psyllids Trioza apicalis and Bactericera trigonica . Sulcia provides its host with 8 out of 10 essential amino acids  their microbiota was shown to be essential for enabling efficient reproduction on specific plants , whereas they lost almost 50% fecundity on white clover (Trifolium repens) in comparison with control offspring, again showing the importance of the insect microbiota for host–plant interactions  bacteria were found likely supporting their host with breakdown of cellulose  Enterobacteriaceae were found to be most active in cellulose degradation, shown by high 13C isotope-labeled carbon incorporation into bacterial DNA after insect feeding from 13C-cellulose  that are meant to ward off invading insects and microbes  influx, and phosphorylation of downstream receptor-like cytoplasmic kinases (RLCKs) and calcium-dependent protein kinases (CPKs) that recruit and phosphorylate respiratory burst oxidase homologue D  in Arabidopsis, by suppressing the ROS-burst after recognition -induced signaling, plant receptor activation leads to downstream receptor like kinase (RLK) and mitogen activated kinase (MAPK) signaling that activates transcription factors (TFs) involved in stress signaling regulation, cross-communication between different stress signaling pathways, and amplification of phytohormone-driven and plant stress responsive pathways  induce plant salicylic acid defenses followed by jasmonic acid in their necrotrophic stage. In response to chewing herbivores jasmonic acid and abscisic acid are induced, whereas with abiotic stress abscisic acid is the responsive hormone. Jasmonic acid signaling results in two distinct antagonizing signaling branches modulated by ethylene and abscisic acid  and DAMP-induced plant stress signaling with the help of its AvrPto effector molecule  or modulation of plant hormones such as auxin, cytokinin, and gibberellin  that produces cytokinins that cause a “green island” phenotype of photosynthetically active green patches and, thereby increase the viability of its insect host due to an increase in plant chlorophyll content  was shown to harbor other microbiota with the potential to form plant galls, including Agrobacterium, Rhodococcus, Pseudomonas, and Erwinia  and its closely related false potato beetle (L. juncta) it was demonstrated that Stenotrophomonas, Pseudomonas, Enterobacter, and Pantoea bacteria within oral secretions induce plant salicylic acid defenses, thereby repressing jasmonic acid-defenses, leading to optimal growth of the beetles , priming plants for jasmonic acid and ethylene defenses that protect against necrotrophic pathogens and insects  larvae  endosymbionts H. defensa reduced the systemic release of plant volatiles, decreasing parasitoid (Aphidius ervi) recruitment  are attracted to oviposit on fruit with volatile terpenes, like 2-methylisoborneol, produced by Streptomyces bacteria  and toxic secondary metabolites  that predominantly target the insects’ digestive system and affect the insects’ gut barrier (Miya et al. In the interaction between insects and plants, insect-associated microbes play important roles in shaping plant responses (Atkinson and Urwin Multitrophic interactions are distinct from single interactions, making them hard to predict and lead to unexpected outcomes (Atkinson et al. Considering future perspectives, plants and crops are likely to encounter complex multitrophic interactions more frequently, as our changing climate with extreme weather conditions will support insects and pathogens to spread more easily and even beyond seasons where they normally occur (Chakraborty and Newton


Long non-coding RNAs (lncRNAs) exert central pathophysiological roles through the regulation of gene expression both at transcriptional and post-transcriptional levels. The characterization of lncRNAs’ interactome is disclosing several new mechanisms that control disease onset and progression thus opening the way to the development of new pioneering therapeutic approaches. Regarding the lncRNA HOTAIR, found upregulated in several cancers and in liver fibrosis, it has been proved as a potential therapeutic target. HOTAIR acts as a ceRNA for several miRNAs and it directly interacts with chromatin remodelling complexes (e.g. PRC2 and LSD1/NuRD complexes). In this regard, we recently reported the transcription factor SNAIL-mediated recruitment of HOTAIR/PRC2 complex on specific chromatin sites causing epithelial genes’ repression through epigenetic chromatin modifications. Conversely, HOTAIR is repressed by the liver-enriched transcriptional factor HNF4a that binds to both HOTAIR promoter and distant enhancer and impairs the formation of a chromatin loop between these genomic regions.In a therapeutic perspective, we design and validated the first example of a dominant negative lncRNA molecule (HOTAIR-sbid) that covers the HOTAIR portion involved in the interaction with SNAIL while is devoid of the domain of interaction with EZH2. Functionally, HOTAIR-sbid expression impairs SNAIL/EZH2/endogenous HOTAIR interaction; thus, PRC2 complex is not recruited on SNAIL-target chromatin sites . Accordingly, the cells rescue an epithelial phenotype, reduce EMT and, in turn, migratory, invasive and anchorage independent growth abilities. This approach promises high level of specificity and limited off-target effects. Future investigations should enhance RNAs’ stability and should design strategies for the delivery of these molecules to specific target cells. Long non-coding RNAs (lncRNAs), an important class of RNAs more than 200 nucleotides long, that acquired attention only in the last decades due to their subtle phenotype, are now recognized to exert central pathophysiological roles. The efforts in highlighting their functions, structures and interactors  and in the development of experimental approaches to detect these interactomes, revealed their pivotal role in regulating gene expression both at transcriptional and post transcriptional level. In particular this apply to lncRNAs involved in several tumours’ onset and dissemination where a remarking contribution of basic and applied research now opens the way to the development of new pioneering therapeutic approaches.The lncRNA Hox Antisense Intergenic RNA (HOTAIR) represents one of the most studied and characterized lncRNAs; it is encoded within the Homeobox C (HOXC) locus and is found upregulated in several cancers , 2, in CIn parallel, HOTAIR was found to directly interact with chromatin remodelling complexes like Polycomb repressive complex 2 (PRC2) and the LSD1/NuRD complex . In thisConversely, the liver-enriched transcriptional factor HNF4a, in order to drive hepatocyte differentiation and promote mesenchymal to epithelial transition (MET), negatively regulates HOTAIR expression through its binding to both HOTAIR promoter and distant enhancer. This in turn impairs the formation of a chromatin loop between these genomic regions that promotes HOTAIR transcription .In a therapeutic perspective, accumulating evidence revealed that ncRNAs are potential druggable targets, due to their functions and structure. In this frame, the majority of the reports evaluated the effect of lncRNA knockdown in preventing disease onset and progression, including cancer. The most recognized and shared approach is based on the use of shRNAs that induce a downregulation of lncRNA expression by RNA interference. This strategy allows a general silencing of the lncRNA but does not selectively target specific functions. Differently, we recently reported the first example of a dominant negative RNA molecule that competes with the endogenous HOTAIR lncRNA in a specific function. This molecule, named HOTAIR-sbid (for Snail-binding domain), covers the HOTAIR portion involved in the interaction with SNAIL while is devoid of the domain of interaction with EZH2. Molecularly, HOTAIR-sbid was shown to impair SNAIL/EZH2 interaction and the SNAIL binding to the endogenous HOTAIR. Therefore, PRC2 complex is not recruited on SNAIL/HOTAIR target sites and SNAIL/HOTAIR-dependent epigenetic transcriptional repression of epithelial genes  is impeded. Functionally, this dominant negative-based strategy, reflects on the rescue of an epithelial phenotype, with reduction of migratory, invasive and anchorage-independent growth in HCC cells and notably prevents TGFβ-induced EMT in hepatocytes . These r


A standardized objective evaluation method is needed to compare machine learning (ML) algorithms as these tools become available for clinical use. Therefore, we designed, built, and tested an evaluation pipeline with the goal of normalizing performance measurement of independently developed algorithms, using a common test dataset of our clinical imaging. Three vendor applications for detecting solid, part-solid, and groundglass lung nodules in chest CT examinations were assessed in this retrospective study using our data-preprocessing and algorithm assessment chain. The pipeline included tools for image cohort creation and de-identification; report and image annotation for ground-truth labeling; server partitioning to receive vendor “black box” algorithms and to enable model testing on our internal clinical data (100 chest CTs with 243 nodules) from within our security firewall; model validation and result visualization; and performance assessment calculating algorithm recall, precision, and receiver operating characteristic curves (ROC). Algorithm true positives, false positives, false negatives, recall, and precision for detecting lung nodules were as follows: Vendor-1 ; Vendor-2 ; Vendor-3 . The AUCs for detection of solid (0.61–0.74), groundglass (0.66–0.86) and part-solid (0.52–0.86) nodules varied between the three vendors. Our ML model validation pipeline enabled testing of multi-vendor algorithms within the institutional firewall. Wide variations in algorithm performance for detection as well as classification of lung nodules justifies the premise for a standardized objective ML algorithm evaluation process. Interest in machine learning (ML) algorithms for use in radiology is growing rapidly. As algorithms become available for clinical use, an objective method for evaluating these tools is required. ML challenges are one mechanism for assessing algorithm performance, and such competitions involving medical images are increasing. Recent examples include the RSNA Pediatric Bone Age Challenge , Data ScSuch open challenges are an objective way for the research community to develop, test, compare, and advance state-of-the-art data science and ML techniques. However, a large gap exists between algorithm performance evaluated on a highly-curated research dataset and a clinically usable product that provides valuable advice in real-world use. Success of ML algorithms on research datasets from specific site (s), timepoints, imaging equipment and acquisition parameters, as well as disease type, spectrum, severity, and presentation, is not a guarantee of its generalizability, explainability, robustness, or performance in clinical environment. To address these issues, it is important to develop and incorporate objective processes to validate ML algorithms on representative local clinical data.There is an urgent need for an objective, standardized evaluation process to compare algorithms on an even clinical plane. For this purpose, we designed, built, and tested an evaluation pipeline for the use case of lung nodule detection on chest CT studies.Lung cancer is currently the leading cause of cancer death in the US . There iAlong with the socio-economic reasons, the high vendor interest in systems for automatic detection of lung nodules makes assessing such tools a valuable use case. However, the heterogeneity of existing systems’ capabilities, the substantial variability among radiologists on what constitutes a lung nodule , and theThis retrospective study was approved by Mass General Brigham Institutional Review Board . The need for consent was waived by the ethics committee. It was performed at an urban tertiary care academic medical center. Our data-preprocessing and algorithm assessment chain included the following components 1. an image cohort creation and de-identification tool 2. report and image annotation tools for ground-truth labeling 3. server partitioning for vendor “black box” algorithms, and model testing on our internal clinical data within our security firewall 4. model validation and result visualization tool and 5. assessment of algorithm recall and precision. This section provides a brief description of each step required for building, implementing, and testing the evaluation pipeline.Cohort selection was made on picture archiving and communication system (PACS) . Two thousand four hundred fifty-nine (2459 CTs) consecutive chest CT studies and reports were reviewed to include 100 CT studies done for lung nodule surveillance or detection . The CTsTo select the final cohort of 100 chest CT studies with lung nodules from the larger cohort of 2459 chest CT studies retrieved from our PACS archive, two infrastructure software applications were built in-house: a report annotation tool and an image annotation-visualization tool to facilitate the process in a semi-automated fashion.The web-based interactive report annotation tool consisted of a graphical user interface that enabled report loading and user-configurable search terms. The tool allows for the assignment of multiple study-level labels per report and keyword or phrase search. The presence of specific wording in the study report can be used as an initial filter to select studies likely to present determinate imaging findings. Specifically, using the tool, a radiologist views a report, highlights a phrase or keyword of interest, and associates it with a given label. Any report containing this phrase or keyword, and not previously matched to another phrase, is marked with a “soft label.” This process allows the radiologist to cull many reports and group them efficiently. Next, manual confirmation of the initial soft labels is performed on a study-by-study basis, and exclusion criteria are applied. This preliminary grouping of reports facilitates and expedites further processing.Most current ML algorithms  are limited in detecting a single imaging finding—so-called “narrow AI” . Therefore, studies containing other non-nodule-relevant imaging findings were not included in the final cohort to properly assess the nodule detection capabilities of the evaluated models. Instead, study-level labels were created in the report annotation tool, and keyword searches of related terms were used to identify and consequently exclude reports containing variations of the following excluded criteria: consolidation, moderate/severe pleural effusion, emphysematous change, architectural distortion, and surgery. After removing study reports containing these findings, the remaining reports were assessed for a first pass filtering of studies with mention of lung nodules in the reports .This method was applied to all 2459 study reports, and 100 studies containing nodules of at least one composition and without keywords or phrases related to the exclusion criteria were randomly selected. A radiologist reviewed all images to confirm the presence of lung nodule(s) without significant possible confounding factors (excluding cases that had findings described in the exclusion criteria that might not have been described in the radiology report text but were evident on image review) and assessed for quality assurance. Studies with inconsistent slice spacing, missing slices, or significant artifacts from motion or metal were excluded. All images were stripped of identifying patient information and re-coded with study-specific numbers, with the mapping blind to all but the study project manager.Usable studies were loaded into our image annotation tool. Three radiologists annotated each CT using the web-based application developed using open-source and commercial tools. The Cornerstone Javascript library was used to render images and annotation markup, Microsoft. NET 4.5 application programming interface for endpoint development, Microsoft’s Internet Information Services for hosting, and Microsoft’s SQL Server Express for application persistence. The resulting image display tool included window-width/level manipulation, zoom/pan, study/series navigation, and measurements. The measuring tools allowed bi-directional measurement  and two sets of measurements for part-solid nodules (independent of the whole nodule and separately for the solid component) that are automatically recorded and linked to identified nodules. Associated with each annotation are configurable labels defined by an Extensible Markup Language (XML) schema based on the computer-assisted reporting and decision support (CAR/DS) schema  that allGround-truth was consensus-driven: only nodules accepted by the radiologist majority (at least 2 out of 3) were eligible for evaluation. Nodules with different compositions , size ≥ 4 mm and ≤ 30 mm were annotated. Nodule measurements were performed using the lung window display. We excluded diffusely calcified nodules from the annotation process and communicated to the vendors that their system should not detect these nodules. Nevertheless, a total of eight diffusely calcified nodules were detected . These detected granulomas were not included in the statistical analysis. The radiologists annotated the following data elements for each nodule in the ground truth data set: size , presence of spiculation (yes/no), and laterality and lobar location. The ground truth radiologists did not have access to radiology reports before or during annotating the ground truth.We received each vendor algorithm wrapped in a Docker container that was loaded on individual servers. Our chest CT dataset is input to the vendor algorithms for processing. Vendors were instructed to provide their output using our defined XML data schema format . All stuManual evaluation of vendor model output versus ground-truth was performed using the image annotation visualization tool to navigate ground-truth and vendor-detected nodules Figs  and 5. EStatistical analysis was performed in Microsoft Excel and SPSS Statistical Software  We assessed the performance of individual models with recall and precision for overall lung nodule detection. In addition, we calculated the receiver operating characteristic curves (ROC) area under the curve (AUC) with 95% confidence intervals for attenuation-based stratification of lung nodules.A total of 243 nodules were annotated on our ground-truth dataset . We used medians for lung nodule size due to non-normal distribution of data. The mean average nodule size (regardless of nodule type) was 7.00 mm .There was a significant difference in sizes of solid , part-solid , and groundglass nodules  (p<0.001). The detailed distribution of annotated features of the nodules is described in Vendor model performance varied widely, as can be seen in Detection of nodules of different composition was also assessed individually . When exThere were wide variations in the ROC AUCs of different vendor algorithms for classifying lung nodules based on their attenuation. The ROC AUCs  for detection of solid, groundglass, and part-solid nodules are summarized in We were successful in designing, building, and testing a validation pipeline on three vendor applications with the goal of normalizing the performance measurement of independently developed algorithms, using a common test dataset of local clinical imaging studies and an evaluation module. Our evaluation successfully tested our data-preprocessing and algorithm assessment pipeline of tools from perspective of an image cohort creation and de-identification tool ; report and image annotation tools for ground-truth labeling (with multi-reader ground-truthing capabilities); server partitioning to receive vendor “black box” algorithm, and to enable model testing on our internal clinical data from within our security firewall ; model validation and result visualization tool ; and a performance assessment tool for calculating algorithm recall and precision .There are several publications on development and evaluation of validation pipelines reported for both imaging and non-imaging ML algorithms –13. ThesVendor algorithm performance varied greatly, justifying our premise that a standardized objective ML algorithm evaluation process is needed. One vendor algorithm (vendor 3) was not able to detect ground-glass nodules, and as a result, their general performance suffered. However, even when assessing only solid nodule detection, this same vendor’s performance did not improve greatly. Another possible explanation for poor performance could be due to overfitting of the vendor model to a limited or homogeneous dataset upon which they trained their ML algorithm. Moreover, there is an obvious trade-off between sensitivity and specificity that requires adjustment during each algorithm model design, typically based on specific clinical needs. Vendors were instructed to tune their algorithms to an optimal intermediate configuration that would allow potential users to “plug and play” their tools without any kind of sensitivity-specificity calibration, allowing for a fair baseline comparison among the tools.The major implication of our study included creation of validation pipeline for seamless processing and integration of AI outputs from multi-vendor AI systems. With an increasing number of the United States’ Food and Drug Administration (FDA)-cleared AI algorithms , it is iOur validation pipeline pertained to the detection and characterization tasks of AI; therefore, modifications will be necessary to assess AI systems for triage, quantification, segmentation, and image processing. Scalability of such pipelines can also aid in evaluation of real-world surveillance and on-ground clinical performance of AI algorithms. The FDA sets minimum “pass criteria” or performance thresholds for AI algorithm based on their target application or predicate devices (AUC >0.95 for triage applications under the QFM product code) . We recoThe most difficult part of the validation pipeline is case selection and ground truthing. The former requires review of both radiology report, a robust radiology report search engine and an unbiased radiologist to further confirm appropriateness of selected cohorts both with and without target and distracting findings. Balancing the number of cases required for validation across a multitude of scanners, scan parameters with and without distractors, and the ground truth radiologists could be challenging at non-academic or extremely busy academic sites. The AI vendors in our study were pliant and cooperated with us to ensure a standardized output. However, standardization of AI output across multiple vendors from multiple parts of the world is not easy and might require intervention from the FDA or other stakeholder entities.Limitations of our study include a data cohort of imaging examinations from one institution, although multiple scanner vendors, model, and protocol types were included. Exclusion criteria consisted of studies with findings of consolidation, significant pleural effusion or emphysematous change, architectural distortion, and post-surgical findings. While these findings can be commonly seen and should not be confounding or limiting factor for automatically detecting lung nodules, due to limitations of currently available narrow AI systems capable of detecting only single imaging features or findings, or systems trained using publicly available data sets that do not commonly contain studies presenting these types of findings, we opted to exclude studies with these findings. Since our objective was to assess differential detection abilities between solid, part-solid and groundglass nodules, we did not include distracting or confounding findings. However, for AI algorithms that target triage and lesion characterization, it is prudent to have representation of true negative (without any findings), potentially false positive (with distracting findings such as pneumonia or atelectasis for lung nodules), and cases where target findings exist with other imaging findings. In addition, lung nodule detection performance for diffusely calcified nodules was not assessed on this analysis since these findings are usually suggestive of benign etiology , although also seen with metastases . We acknIn summary, evaluation was done on our own clinical images without any data leaving our institutional firewall and without requiring third-party algorithm disclosure. The machine learning model validation pipeline we describe was tested in the specific use case of detecting lung nodules in chest CT examinations, but the developed techniques are easily generalizable to other types of tools now starting to be available from commercial entities.


Even though these models can capture cardiorenal interaction, they are difficult to manipulate and control. Moreover, interspecies differences may limit extrapolation to patients. The questions we address here are what would it take to model CRS in vitro and how far are we? There are already multiple independent in vitro (human) models of heart and kidney, but none have so far captured their dynamic organ-organ crosstalk. Advanced in vitro human models can provide an insight in disease mechanisms and offer a platform for therapy development. CRS represents an exemplary disease illustrating the need to develop more complex models to study organ-organ interaction in-a-dish. Human induced pluripotent stem cells in combination with microfluidic chips are one powerful tool with potential to recapitulate the characteristics of CRS in vitro. In this review, we provide an overview of the existing in vivo and in vitro models to study CRS, their limitations and new perspectives on how heart-kidney physiological and pathological interaction could be investigated in vitro for future applications.Heart and kidney diseases cause high morbidity and mortality. Heart and kidneys have vital functions in the human body and, interestingly, reciprocally influence each other’s behavior: pathological changes in one organ can damage the other. Cardiorenal syndrome (CRS) is a group of disorders in which there is combined dysfunction of both heart and kidney, but its underlying biological mechanisms are not fully understood. This is because complex, multifactorial, and dynamic mechanisms are likely involved. Effective treatments are currently unavailable, but this may be resolved if more was known about how the disease develops and progresses. To date, CRS has actually only been modeled in mice and rats  Heart ace versa .“Cardiorenal syndrome” (CRS) is an umbrella term used to describe the dysfunction between the two organs , 4 and iin vitro one can control and investigate the role of the initiator and its consequence.However, the practicality and clinical applicability of this classification system has been recently questioned, as pathological cascades involving heart, kidney, and neurohormonal systems are already initiated by the time clinical manifestations are detected. Hence, it is almost impossible to define which factor was the initiator and which was its consequence. Another classification system was proposed where CRS is categorized on the response to various treatment modalities . Althougin vivo. Although animal models can capture the interaction between the organs, they remain difficult to manipulate and control. Moreover, interspecies differences in genetics, anatomy, vascular neural conduction may limit extrapolation to the human condition. Heart and kidney have each been modeled separately in vitro but models so far have had insufficient complexity to capture the dynamic interactions between the organs. For this reason, CRS is clearly a condition that would benefit from increased complexity of in vitro models that reflect organ-organ interaction “in-a-dish.”Cardiorenal syndrome causes high morbidity and mortality, yet the underlying biological mechanisms are not fully understood because complex, multifactorial, and dynamic mechanisms are involved. So far, the disease has only been modeled in vivo and in vitro models available to recapitulate CRS and, more generally, models of either of the two organs that demonstrate hallmarks of CRS such as oxidative stress, inflammation, and fibrosis. Importantly, we discuss new perspectives on how CRS can be investigated in vitro using these state-of-the-art organ models.In this review, we describe the state of the art regarding Cardiorenal syndrome develops through hemodynamic and non-hemodynamic mechanisms. Hemodynamic mechanisms are defined by the interactions of blood pressure, renal function, extracellular fluid volumes, and cardiac contractility. Non-hemodynamic mechanisms include cardiorenal connectors such as RAAS, SNS, nitric oxide (NO), and reactive oxygen species (ROS) . While h2+ metabolism, increases myocardial oxygen demand and myocardial cell β1-adrenergic mediated apoptosis and stimulates α1 receptors  oxidase causes inflammation. Chronic inflammation is a hallmark of both heart and kidney failure. Sera from CRS patients were characterized by high levels of proinflammatory cytokines and proapoptotic factors .2– reacts with NO and inactivates its beneficial effect by forming peroxynitrite, which oxidizes lipids, DNA and proteins . Patients had increased levels of NADPH oxidase and myeloperoxidase—an enzyme catalyzing the formation of ROS—with upregulation of proinflammatory mediators . In AKI,nd COX-2 .Fibrosis is a typical tissue-remodeling process following stress and injury. It is the result of several phenomena including the epithelial-to-mesenchymal transition (EMT), fibroblast activation to produce extracellular matrix (ECM), and the recruitment of inflammatory cells with cellular regeneration at the site of damage. EMT in particular is one of the major causes of renal fibrosis: tubular epithelial cells lose epithelial properties and acquire myofibroblast characteristics by producing excessive deposition of ECM . IncreasIn AKI patients, cardiac injury is characterized by myocyte apoptosis and neutrophil infiltration. The upregulation of beta-galactoside-binding lectin galectin-3  expression in renal ischemia links AKI to cardiac fibrosis . Gal-3 iCardiac fibrosis and left ventricular hypertrophy (LVH) are common features of cardiomyopathies contributing to mortality in CKD patients. Excessive cardiac fibrosis and enhanced accumulation of collagen fibrils occurs as the result of increased ECM protein synthesis, in parallel with ECM degradation regulated by matrix metalloproteinases (MMPs) and TIMPs . In the Cardiac hypertrophy and fibrosis were linked to increased serum levels of FGF23 in CKD patients . FGF23 iAnimal models have a pivotal role in the identification of molecules related to cardiac and renal injury, helping in the elucidation of the mechanisms underlying heart-kidney interactions. Because of their availability and possibility to introduce targeted genetic mutations , the large majority of preclinical studies has been performed in either rats or mice. However, the predictive value of murine models is limited, mainly because of species-specific differences; as a result, translation of results to patients has also been limited . In the To model acute cardiac dysfunction, intravenous injection of potassium chloride in mice has been widely used to induce cardiac arrest followed by cardiopulmonary resuscitation. The acute response resulted in AKI, characterized by reduced GFR and increased serum creatinine levels . CardiacExperimental myocardial infarction (MI) is used to induce regional injury and chronic HF though the sudden occlusion of the left anterior descending coronary artery or one of its branches . In a moIn murine models of CRS type 1, circulating levels of TNF-α and monocyte chemoattractant protein-1 (MCP-1) decreased upon treatment with exogenous apela, a secretory hormone playing an important role in embryonic CV system development. Apela inhibited apoptosis, DNA damage, inflammation, and fibrosis in renal cells of AKI mice and also inhibited the expression of adhesion molecules in renal tissue of CRS mice , 38. In Transverse aortic constriction was used to examine renal response to chronic cardiac pressure overload. A stable pressure gradient across the thoracic aorta of the mouse resulted in ventricle hypertrophy and renal inflammation. Elevated serum creatinine and tubulointerstitial fibrosis characterized the resultant CKD. This model can be considered as clinically relevant for CRS type 2, as it showed the development of CKD in a non-ischemic, hypertrophic chronic HF model . Renal rWarm ischemia-reperfusion is characterized by a sudden decline in renal function with severe injury in the straight segment of the proximal tubules  and it i+ T-cells mediated post-ischemic renal injury, which was associated with increased inflammation. While both Toll-like receptor 4 mutant mice and T-cell deficient mice were resistant to AKI, expression of proinflammatory genes such as IL-6 was increased in the kidneys of mice when subjected to cardiac arrest , that can impair cardiac function by decreasing contractility and increasing myocyte apoptosis. Mitochondrial fragmentation could lead to cardiomyocyte apoptosis and cardiac dysfunction , 49.in vivo and in vitro through restoration of PGC1α expression or genetic knockdown of interferon regulatory factor 1 . Alterations were attenuated factor 1 .Metabolomic analysis following ischemic cardiac injury showed inadequate oxidative phosphorylation and oxidative stress. A shift of energy depletion and oxidative stress, reflected systemically in the plasma, was detected in the metabolomic analysis of the kidney .Heart remodeling in CKD is characterized by interstitial fibrosis and capillary loss. The potential pleiotropic effects on heart remodeling of erythropoietin, used to correct renal anemia, was investigated in rats. In combination with enalapril, erythropoietin caused reduced cardiac fibrosis and microvessel disease subtotal nephrectomy (STNx) rats, presumably by decreasing myocardial oxidative stress .Uremic cardiomyopathy and atrial fibrillation in CKD may be caused by inadequate renal clearance and the subsequent accumulation of protein-bound uremic toxins . IndoxylDdah1 and Ddah2 increased the amount of ADMA in the kidneys (Other major uremic toxins contributing to CV disease are asymmetric dimethylarginine (ADMA), advanced glycation end products (AGE), and trimethyl amine N-oxide (TMAO) that induce both heart and kidney damage , 59. Ind kidneys .The development of uremic cardiomyopathy is characterized by declined renal function linked to changes in circulating levels and activities of physiological phosphate regulators FGF23 and Klotho . AdminisImpaired endothelial and renal NO production causes systemic NO reduction in CKD. In rats, chronic NO depletion by the administration of an NO synthase inhibitor induced hypertension, proteinuria, glomerulosclerosis, tubulointerstitial fibrosis, systolic dysfunction, and cardiac hypertrophy. Bilateral renal denervation ameliorated these changes and this was associated with decreased RAAS activation . Cardiacvia the A2A receptor, ameliorating cardiorenal fibrosis  in the pathogenesis of fibrotic disorders was reviewed some years ago  and an efibrosis . The sigfibrosis . Myofibrfibrosis .The investigation of experimental models of dual insults combining elements of chronic cardiac and renal dysfunction is key to mimicking the complex disease processes that occur in individuals with comorbid conditions. To understand the pathophysiology of concomitant organ dysfunction, a rat model of MI, followed by STNx was used. ANP, TGF-β1 and collagen type I were increased. STNx induced a significant decrease in GFR, while MI accelerated STNx-resulting renal cortical tubulointerstitial fibrosis. Altogether, decreased cardiac function as well as increasing cardiac remodeling and renal tubulointerstitial fibrosis was observed in MI/STNx rats, nicely recapitulating the features of the clinical disease manifestations . AnotherMyocardial infarction accelerated renal fibrosis in STNx rats and increased the expression of kidney injury molecule 1 . STNx inin vivo models have led to detailed mechanistic understanding of many human diseases. However, biological processes that are specific to humans cannot be modeled in other animals. Current models of heart and kidney have been studied separately in vitro but are presently often too simplistic to capture complex organ crosstalk. The following section focuses on in vitro models used to study heart and kidney, which are summarized in in vivo disease modeling in animals can be potentially overcome through human in vitro (3D) cell culture approaches using stem cells from different organs  and influences several physiologic processes. The primary source of ROS in the kidney cortex and medulla are the NADPH oxidase (NOX) enzymes. Upon angiotensin II and aldosterone stimulation, ROS production increases. The function of NOX-derived ROS in the kidneys can be classified into regulation of renal blood flow, alteration of cell fate and regulation of gene expression. NOX-derived ROS can alter renal cell fate by enhancing EMT by (i) MAP kinase activation, (ii) mesangial cell apoptosis, and (iii) ERK1/2 activation promoting cellular hypertrophy; this was shown in renal tubular epithelial cells , 94. ROSduce EMT , 96.in vitro. In vitro, apela inhibited the expression of TNF-α, MCP-1, and intracellular adhesion molecule-1 in renal glomerular endothelial cells induced by angiotensin II. Apela also inhibited the promotion of angiotensin II on the adhesion of THP-1 cells  were quantified. In all in vitro experimental groups , the expression of the genes was noisy with no consistent pattern. However, in the UUO animal model counterpart, TGF-β pathway-related genes were overexpressed in the ureteral obstruction group compared with the sham controls. Results suggest in vitro findings should be interpreted cautiously  in combination with supporting cells  and cells of the immune system, the cellular components of complex in vitro models should include all cell types involved in the disease response. In self-organizing organoid cultures, relevant cell types are derived during organoid development or differentiation, often with some spatial arrangement reminiscent of the native organ. Such is the case for kidney organoids and several protocols for the generation of human (pluripotent and adult) stem cell-derived kidney organoids were described  mechanism of interest. Microfluidic devices and microfabrication may represent the technological implementations needed to achieve organ vascularization and organ-organ interaction, which are crucial in complex disorders involving multiple organs/tissues as CRS. Multi-OoC could redefine the way human health research is done  and CRS t organs . Moreovet organs , 147.The ability to measure physiologically relevant parameters is crucial in the development of OoCs in general. When looking at studying CRS, it will be essential to be able to detect changes in both the cardiac and renal functions as necessary, in their respective compartments. Within the cardiac field, chips already exist that integrate electrodes  and impedance (which is an indication of contraction). In addition, most of the chips are transparent, allowing optical analysis for example to measure calcium transients. For the kidney structures on-chip, measuring nephron functionality will be challenging. Differential clearance of albumin should ideally be achieved as well removal of uremic solutes, when combined with endothelial cells . Alternain vitro, capturing the complex interaction of the organs, allowing for the measurement of critical readouts and recapitulating the hallmarks of the disease.in vitro may help unraveling what is still unknown about primary and secondary events.Cardiorenal syndrome is a broad term used to define the combined dysfunction of heart and kidney. It has recently gained attention through the realization that there is considerable reciprocal influence between the two organs that may help explaining the pathology of the disease and its variants. Although the biological mechanisms underlying the disease are complex and dynamic, new advances in modeling diseases in vitro models will soon contribute to a deeper understanding and modeling of CRS in humans. For this, the convergence of human stem cell biology with engineering technologies will enable control of environmental cues, the development of tailored assays and the design of relevant read-outs that reflect what occurs in patients. Cardiorenal OoC technology has the potential to be the next “best-in-class” platform to precisely model human CRS, allowing drug screening and development of novel treatments.Whilst animal studies have clearly been (and still are) valuable tools in understanding (patho)physiological signaling pathways underlying heart and kidney communication, we have argued here that innovative All authors outlined, drafted, wrote, and approved the final version of the manuscript.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.