A high incidence of thrombosis in hospitalised patients with COVID-19 was identified early during the pandemic. Accurately quantifying thrombotic risk may assist prognosis and guide appropriate thromboprophylaxis. Observational studies have estimated the rate of thrombosis in both hospitalised and non-hospitalised patients with COVID-19, and how this corresponds to the severity of illness. In this review, we provide an overview of the incidence and prevalence of arterial and venous thrombotic events in patients with COVID-19 and highlight the limitations in the studies to date. Asymptomatic individuals with COVID-19 and those with mild symptoms are at very low risk of thrombotic complications. However, rates of thrombosis are substantially increased in hospitalised patients, and are strikingly high in those patients who are critically-ill requiring treatment on the intensive care unit and especially those requiring extracorporeal membrane oxygenation. Clinicians managing such patients need to be aware of these risks and take appropriate steps with respect to thromboprophylaxis and heightened clinical vigilance. Large prospective observational studies will more accurately quantify thrombotic rate, and randomized controlled trials are currently investigating optimal thromboprophylactic strategies. The rates of arterial and venous thrombotic complications in COVID-19 are low in non-hospitalised individuals with asymptomatic or mild disease.Thrombotic risk increases with severity of COVID-19 illness, with those on intensive care being at greatest risk.Limitations in the available research include the reliance on retrospective observation studies, variable reporting of types of thrombosis, and the variable use of screening.Randomized controlled trials currently underway will guide us as to the optimal thromboprophylaxis strategy to improve outcomes in these patients.Early in the COVID-19 pandemic, it was identified that the rate of thrombosis in hospitalised patients with COVID-19 was relatively high, and attributed to a pro-thrombotic state . A numbeThe incidence of a thrombotic event in patients who test positive for COVID-19 but suffer from either no or mild symptoms or do not require hospitalisation appears to be very low. In a large dataset of 6153 patients by Hill et al., 3405 individuals were identified who had tested positive for COVID-19, but did not require hospitalisation [This suggests that in otherwise well patients who have tested positive for COVID-19 but do not require hospitalisation, the rate of thrombosis is low. However, there are a number of issues, particularly related to the inherent biases of observational studies when the outcomes are likely to be a low frequency. Firstly, patients may not report symptoms, or have treatment for thrombosis managed elsewhere. Secondly, patients may have only had mild symptoms of COVID-19, but then develop a thrombosis that requires hospitalisation, and as such may not be included in the “outpatient” cohorts described above. Finally, publication bias may have contributed to fewer publications that document a low prevalence of thrombotic complications than those reporting unexpectedly high rates of such events.Nonetheless, there are case reports and series of outpatients who have developed thrombotic events in the context of COVID-19. Uppuluri et al. report the case of a 32-year-old man who developed symptoms of pulmonary embolism (PE) 12 days following an outpatient diagnosis of COVID-19, subsequently confirmed on computed tomography pulmonary angiogram (CTPA) . Akel etThere are much more abundant data regarding the incidence of thrombotic complications in patients who have been hospitalised, with multiple prospective and retrospective studies from populations across the world Fig. . In a laDrawing upon data from multiple databases, comprising 28,173 patients with COVID-19 , one meta-analysis estimated an in-hospital prevalence of VTE (PE or DVT) of 14.1% . HoweverHospitalised patients can be subdivided into those managed on the ward, compared to those managed on intensive care. A recent large meta-analysis comprising 9350 ward (non-ICU) patients across 20 studies identified an overall VTE incidence of 7.1% (95% CI 4.8–9.8) . A furthArterial thromboses, including those in the cerebral, myocardial, mesenteric beds as well as limb ischaemia also warrant consideration, although in general the incidence of arterial thrombotic complications in patients with COVID-19 is less well established than that of VTE. In one large retrospective observational study of 3556 patients, 32 (0.9%) developed an ischaemic stroke, 4 occurring without any symptoms of COVID-19 prior to the event, and in the remainder of cases, stroke developed up to 27 days following COVID-19 symptom onset . Two furA meta-analysis of 4466 patients (5 articles) reports a 1.2% pooled incidence of ischaemic stroke in patients with COVID-19 . IncidenIt currently remains uncertain whether other prothrombotic comorbidities compound the coagulopathy observed with COVID-19. Patell et al. reported thrombosis in COVID-19 across a population of cancer and non-cancer patients in a single centre and identified similar overall incidence of thrombotic events between 45 cancer patients and 353 non-cancer patients .The incidence of thrombosis in patients with COVID-19 requiring care on the ICU appears to be higher than that in non-ICU hospitalised patients. Klok et al. reported on 184 ICU-treated patients with COVID-19, and based on clinical suspicion, identified a crude cumulative thrombosis incidence of 57% (95% CI 47–67%), comprising 65 patients with a PE, 3 with other venous thromboses, and 7 patients with arterial thromboses . FraisseIn one of the largest cohort studies, Bilaloglu et al. reported on 829 ICU patients with COVID-19, where thrombosis diagnosis was made based on clinical suspicion . They foThe incidence of thrombosis is particularly high in the ICU patients when systematic screening with ultrasonography is performed to identify DVT. Longchamp et al. performed lower limb venous ultrasound in 25 ICU patients, confirming DVT in 6 patients (24%) . Ren et A proportion of the most critically unwell patients with COVID-19 require advanced cardiac ICU support including extracorporeal membrane oxygenation (ECMO). Patients requiring ECMO require anticoagulation to prevent circuit thrombosis, and are known to exhibit high rates of both thrombotic and bleeding complications . SeveralThe added thrombotic complications related to ECMO include that of the extra-corporeal system: central lines, the centrifugal pump and the oxygenator. The presence of thrombus in the extra-corporeal system can cause complete system failure with a high risk of mortality. Beyls et al. describe one such event, where a patient with COVID-19 died of a major oxygenator thrombosis shortly after starting veno-venous ECMO .Bemtgen et al. describe a high incidence of circuit thrombosis in COVID-19 patients, despite continuous intravenous heparin infusion, adjusted to an APTT of 40–50 s . CompariThe risk of thrombosis is highest in patients with severe COVID-19, but the risk of thromboembolism does not cease at hospital discharge. Given that some patients will have ongoing symptoms for weeks following discharge, and during this time may be at a lower functional state with reduced mobility than prior to the illness, and given the potential ongoing, albeit reduced, inflammatory state post-discharge, there remains a risk of increased tendency to thrombosis. To date, there are limited studies investigating the risk of VTE in patients following hospital discharge. Patell et al. reviewed 163 patients with COVID-19 followed for a median 30 (IQR 17–46) days following hospital discharge . PatientAnother retrospective analysis compared a large cohort of 1877 patients discharged following admission with COVID-19, with a cohort of 18,159 patients discharged from 2019 following other medical admissions . AmongstThrombotic complications, in particular venous thrombosis and thromboembolism are highly prevalent in patients with COVID-19. From the studies available, it seems evident that as the severity of COVID-19 illness increases, requiring higher levels of care, so does the rate of thrombosis.Several guidelines have been produced, in part building upon these data to provide advice to clinicians as how to interpret this when implementing thromboprophylaxis strategy –39. HoweThere are a number of limitations pertaining to the data reported here on the prevalence of thrombotic complications. The vast majority of studies are retrospective observational studies. Secondly, identification of thrombosis is generally dependent on the clinical suspicion, any conventional clinical features of VTE are frequently absent or difficult to recognise especially in those on the ICU. There is also the logistical challenge of performing imaging tests in patients that may expose a technician to COVID-19, as well as the challenge of transporting critically unwell patients to CT scanners. Amongst individuals with COVID-19 in the community, who are either not admitted or discharged home without admission, VTE may be missed and under-reported.In patients with critical illness with COVID-19, especially towards the end of life, when identification of thrombosis may not trigger change in management, VTE may not be investigated with confirmatory imaging. Under-reporting of VTE in critically-ill patients is supported by autopsy data, with one autopsy study identifying DVT in 7 of 12 patients (58%) with PE being the direct cause of death in 4 patients (33%) .Another important limitation is the variable reporting of different types of thrombosis in different studies. Several studies only report DVT, whilst others only PE, and few reports on arterial thrombosis. Furthermore, given that the vast majority of studies in ICU-treated patients indicate that standard anticoagulant thromboprophylaxis may be insufficient, bleeding events have been documented in very few studies to date. A recent large meta-analysis of thrombotic complications in patients with COVID-19 identified just 5 articles documenting bleeding events, compared to 44 documenting thrombotic events .Lastly, studies employing routine screening for VTE report a higher incidence of thrombotic complications than those relying on clinical suspicion. However, whether identification and subsequent treatment of small and potentially asymptomatic thrombotic complications, for example subsegmental PEs, will improve outcome, is unclear and as such, the role of routine screening remains debatable .This paper demonstrates high rates of thrombosis in patients with severe illness related to COVID-19. An important hypothesis being considered is whether treating patients who are at the highest risk of thrombosis with a higher dose of prophylactic anticoagulation will reduce the thrombotic burden and improve outcomes. One of the first randomized controlled trials to report on this hypothesis is the INSPIRATION trial, which randomised 562 ICU inpatients to either an intermediate or standard dose of prophylactic anticoagulation . There wThe association of COVID-19 with venous thrombotic complications is undisputed, with the severity of COVID-19 being directly proportional to the risk of thrombotic complications. The prevalence of arterial thrombotic events is still not well understood. Large prospective observational studies are urgently needed to better define thrombosis risk, in varying cohorts including those with advancing age, diabetes, obesity and in Black, Asian and minority ethnic cohorts, including in the outpatient and post-discharge period.


To date, almost all solid malignancies have implicated insulin-like growth factor (IGF) signalling as a driver of tumour growth. However, the remarkable level of crosstalk between sex hormones, the IGF-1 receptor (IGF-1R) and its ligands IGF-1 and 2 in endocrine driven cancers is incompletely understood. Similar to the sex steroids, IGF signalling is essential in normal development as well as growth and tissue homoeostasis, and undergoes a steady decline with advancing age and increasing visceral adiposity. Interestingly, IGF-1 has been found to play a compensatory role for both estrogen receptor (ER) and androgen receptor (AR) by augmenting hormonal responses in the absence of, or where low levels of ligand are present. Furthermore, experimental, and epidemiological evidence supports a role for dysregulated IGF signalling in breast and prostate cancers. Insulin-like growth factor binding protein (IGFBP) molecules can regulate the bioavailability of IGF-1 and are frequently expressed in these hormonally regulated tissues. The link between age-related disease and the role of IGF-1 in the process of ageing and longevity has gained much attention over the last few decades, spurring the development of numerous IGF targeted therapies that have, to date, failed to deliver on their therapeutic potential. This review will provide an overview of the sexually dimorphic nature of IGF signalling in humans and how this is impacted by the reduction in sex steroids in mid-life. It will also explore the latest links with metabolic syndromes, hormonal imbalances associated with ageing and targeting of IGF signalling in endocrine-related tumour growth with an emphasis on post-menopausal breast cancer and the impact of the steroidal milieu. The hypothalamic-pituitary (HP) axis is the central regulator of endocrine action, controlling the function of a number of endocrine glands including thyroid, adrenal, gonadal and the growth hormone (GH)/insulin growth factor-1 (IGF-1) system, which modulate a myriad of physiological processes. The HP axis integrates various stimuli, including those generated internally such as energy levels, stress, inflammation and also in response to external stimuli (dark/light cycles and temperature)  and orchThe regulation of the GH/IGF-1 system is dependent on the integrity of the hypothalamus, pituitary and liver. The primary source of circulating GH is the somatotrophs of the anterior pituitary gland, however, it is also synthesised in other tissues including reproductive tissues, lymphoid tissues and the gastrointestinal tract . The pulThe GH/IGF-1 system changes over the human lifespan and these alterations are associated with, although not necessarily causative of, metabolic alterations and ageing related disease. GH pulsatile secretion is impacted by a vast array of factors including: gender, sex steroids, age, nutritional status, body composition, visceral adiposity, sleep, physical activity, and metabolic stress .During the period of increased growth associated with puberty, GH secretion rates increase resulting in a twofold to threefold increase in serum IGF-1 concentrations , 1996 wiThe effect of age on spontaneous GH secretion is less pronounced in pre-menopausal women  with secThe IGF signalling network consists of IGF-1R, IGF-2R, and the insulin receptor (IR). IGF-1R and the IR are classified as receptor tyrosine kinases and share a similar structure. Ligands involved in the IGF signalling pathway include IGF-1 and IGF-2. IGF-1 has highest affinity for the IGF-1R  with mucAs well as the hypothalamic pituitary GH axis, IGF-1 secretion is controlled by autocrine/paracrine signals in peripheral tissues. Interestingly, the paracrine versus endocrine sources of IGF-1 can impact its function. Paracrine IGF-1 has a greater effect on mammary gland branching than IGF-1 from endocrine sources . FurtherThe IGF signalling pathway is influenced by a number of different factors. In order to gain more insight into the activity of this pathway studies have also looked at the levels of its regulatory proteins, the IGFBP family . IGF-1 bIn adulthood, estrogen is the primary sex-steroid agonist of GH secretion in both women and men , 2006. EAlthough androgens are known to stimulate the GH/IGF-1 axis in men there is scant information in the literature about the impact of androgens on the GH/IGF-1 axis in women, which may be a significant factor especially during the post-menopausal period. This is an interesting point when you consider that in prostate cancer it has been shown that estrogen can substitute to upregulate IGF-1R when androgen levels are low . It is gIn men, testosterone concentrations positively correlate with the regularity of GH secretion, with concentrations of both hormones, diminishing with increasing age . It has Studies have shown that nutritional status is a strong determinant of IGF gene expression, not only in liver, but also in other tissues; with fasting shown to reduce serum and tissue IGF-1 levels , althougAs alluded to earlier, the importance of the HP axis in the regulation of somatotrophic growth and steroid production and associated feedback undergoes significant changes with age. An insightful study by Polycystic ovary syndrome may provide useful clinical insights into the relationship between androgen excess, insulin signalling and metabolic dysfunction. PCOS is a common chronic health condition affecting up to 10% of all women, and is defined by androgen excess, anovulation and often polycystic ovarian morphology on imaging . It has in vitro, androgen excess induces peripheral insulin resistance and pancreatic β-cell insulin hypersecretion in both female human and mouse cell culture models, predisposing to onset of β-cell failure and T2DM  cell lines compared to non-luminal cell types . It has 2 induced phosphorylation of the IGF-1R and subsequent activation of ERK  targeting IGF-1R such as dalotuzumab (MK-0646), ganitumab (AMG479), cixutumumab (IMC-A12), and figitumumab (CP-751871) were investigated. These did not show therapeutic benefit, some reasons postulated for this include crosstalk between the IGF-1R and other growth factor pathways such as the IR  and feedclinicaltrials.gov database. None of these have progressed any further and therefore have not contributed to the clinical management of the disease. That said, a significant point to note is that drugs targeting IGF did work well in some patients, but the limiting factor was the ability to enrich for these patients using validated biomarkers. Therefore, the important questions to be asked here are; why targeting IGF hasn’t been significantly beneficial in the clinical setting? What have we learnt from these clinical trials? And are we any closer to identifying a robust biomarker for IGF targeted therapies in cancer?The IGF-signalling system has long been an area of therapeutic interest in the treatment of breast cancer which was further supported by the findings of both the European Prospective Investigation into Cancer and Nutrition  and the In vitro and patient-derived xenograft (PDX) models of prostate cancer have since found evidence that PTEN and the abnormally spliced ligand independent androgen receptor variant 7 (AR-V7) could have potential use as biomarkers for response to this combination of therapy. In prostate cancer cell models xentuzumab failed to inhibit AKT phosphorylation in PTEN-null cells. Regarding AR-V7, its expression was increased in the xentuzumab + enzalutamide group compared with the enzalutamide-only group (A more recent approach being investigated is the use of therapies targeting the IGF ligands directly to reduce their bioavailability. Dusigtumab (MEDI-573) and Xentuzumab (BI-836845) are dual IGF-I/IGF-II neutralizing antibodies which have now entered clinical trial. Xentuzumab has a binding affinity for both IGF-1 and IGF-2. The advantage of this drug is that it does not target the isoform B of the IR which is involved in glucose metabolism and therefore does not induce the hyperglycaemia and metabolic toxicity observed with IGF-1R targeted therapies or TKIs . Xentuzuly group . Xentuzuly group . An innoly group .Of key importance to the advancement of IGF targeting therapies in cancer is a comprehensive understanding of its role under normal physiological conditions. In normal prostate cells IGF-1 maintained differentiated cellular characteristics, however in prostate cancer cells it induced a mesenchymal phenotype . While tThe interconnected IGF/IGFR and sex steroid signalling pathways play crucial roles in normal growth and development and their perturbation is often associated with diseases of metabolism. What remains to be understood is how age, estrogen and visceral adiposity jointly regulate the secretion of GH in ageing humans and how this may differ between genders. Both androgens and estrogens influence IGF-1 release, and their respective signalling pathways are intertwined. This has important implications for the many cancers that are linked to the development of metabolic syndrome in ageing adults such as tumours of the breast and prostate. Furthermore, as outlined previously IGF signalling is integrated with nutritional status and metabolism and now more than ever a focus on lifestyle interventions such as diet and exercise is necessary. With future studies in this area a greater understanding of IGF pathway activation will certainly lead to a resurrection of IGF targeted therapies in cancer but critically this time with an ability to identify the patients who will most benefit from it.RB and MM conceived and co-wrote the main manuscript. MO’R and MS contributed to the sections with particular clinical relevance to endocrine disorders. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The Covid-19 pandemic has led to substantial and unexpected increases in morbidity and mortality in France. Vulnerable populations housed in accommodation centres have a greater risk of infection because collective housing and their dependence on social support services mean it is more difficult to apply preventive measures. They are also at greater risk of developing severe forms of Covid-19 and waiting longer before seeking healthcare (for Covid-19 or other) treatment. We aimed to identify the factors associated with SARS-CoV2 infection in the most vulnerable populations in the city of Marseille.The study sample comprised users of various services provided by the association AAJT in Marseille, France, some presenting symptoms suggestive of Covid-19 and others not. All had routine health surveillance provided by AAJT’s dedicated healthcare team between March 2020 and May 2020. Using univariate and multivariate analyses, we studied the influence of several variables on morbidity associated with Covid-19.p < 0.005) – which included persons testing positive and those suspected of being infected – were younger. The study sample’s male/female ratio was seven. In our multivariate analyses, living in a shared apartment and poor adherence to social distancing measures were factors associated with Covid-19 infection. Furthermore, mental health problems - such as anxiety disorder - were very frequent in the study sample.The study included 64 participants, 29 of whom tested positive for Covid-19 and 35 control subjects. Median age was 21.16 years old. Individuals in the ‘Covid-19 case’ group (Allocating more and specific housing units to structures providing accommodation services to the most vulnerable people would seem to be a decisive factor in controlling the spread of SARS-CoV2, and deserves more attention from public authorities.The online version contains supplementary material available at 10.1186/s12889-021-11716-6. Corona Virus Disease 2019)  In this context, the present study highlights the need to allocate more individual housing units to social structures. The collective nature of bathrooms and showers in social shelters is also an important point to consider in terms of the spread of SARS-CoV2. Implementing a strategy to ensure that only one person at a time uses these facilities - followed by disinfection - could be beneficial. Furthermore, developing psychological supports often already offered in social structures is of paramount importance. The anxiety-inducing context of the current Covid-19 crisis and the associated restrictive measures are potential triggers or aggravators for states of psychiatric and psychological disorders [By focusing on patients affected by Covid-19, we aimed to identify the determinants of infection in socially vulnerable populations in Marseille, with a view to limiting the spread of Covid-19 while waiting for more generalized vaccination to begin.[8isorders . Given tisorders . NGOs shisorders .The fact that AAJT is a large, well-established association providing not only social support and accommodation, but also and healthcare follow-up services in Marseille on a daily basis, justified exclusively involving this organisation and its users in our study to understand emerging Covid-19 issues in the city’s vulnerable populations. Constraints linked to accommodation conditions and the very long waiting times before screening opportunities were a major concern for both the association and its users. Irrespective of how well a user implements Covid-19 protective measures, the efficacy of the latter depends on accommodation conditions. Similarly, some AAJT service users had serious and potentially deteriorating co-morbidities. Therefore, the risk inherent to collective housing justifies the attribution of individual housing for social structures. Moreover, diagnosing infected persons early means it is then possible to implement a truly effective isolation strategy and protect other users . FortunaAdditional file 1. Questionnaire used to collect data (English version). This is an english translation of the questionnaire developed specifically for this study.


A rare entity of a B-cell malignancy with precursor B-cell phenotype and concomitant translocation t or variant MYC translocation exists. These cases show clinical, pathological and molecular overlap between precursor B-lymphoblastic leukaemia or lymphoma and Burkitt leukaemia or lymphoma (BLL).We report a case from February 2019 at the Charlotte Maxeke Johannesburg Academic Hospital, South Africa, of a 9-month-old infant with a predominantly extracranial soft tissue mass showing extradural extension. There was no involvement of the peripheral blood or bone marrow. Fine needle aspiration and Tru-Cut biopsy of the soft tissue scalp mass showed the tumour to be of precursor B-cell phenotype. Contrastingly, an immunophenotypic assessment revealed a high S-phase fraction and raised concern for BLL. This prompted testing for the translocation t by fluorescence in-situ hybridisation analysis, which confirmed this aberration.Based on the published experience of other centres, the patient was initiated on a BLL protocol. Despite an excellent clinical response, the patient succumbed to neutropenic sepsis six months after diagnosis.Leukaemia or lymphoma with translocation t or variant MYC translocation and precursor B-cell phenotype is a rare entity with a varied clinical presentation. This poses a challenge for diagnosis and classification and a clinical dilemma for the choice of treatment. Molecular studies have confirmed the involvement of the MYC gene in a translocation t or variant translocation, which is the molecular hallmark of BLL.2 This genetic aberration has largely directed the choice of therapy towards a mature B-cell or Burkitt-like protocol in almost all reported cases. It is uncertain whether this represents the best therapeutic approach as there is very limited data on both the duration of remission in these patients and the exact underlying molecular behaviour of this tumour.This entity has overlapping clinical and pathological features with Burkitt leukaemia or lymphoma (BLL) and precursor B-lymphoblastic leukaemia or lymphoma (B-ALL). It has been described across a spectrum of ages ranging from 32 months to 64 years, in both male and female patients. Peripheral blood and bone marrow involvement have been reported in the majority of cases, with isolated extranodal disease reported in only a few.This case study highlights this rare entity, as well as its associated diagnostic and therapeutic challenges.Parental informed consent was provided for this case report. The ethical clearance was obtained from the University of the Witwatersrand Human Research Ethics Committee , Johannesburg, South Africa, under the approval number M190356. Patient results were de-identified and stored in a secure database to ensure patient confidentiality.A 9-month-old male patient from Angola presented to the Charlotte Maxeke Johannesburg Academic Hospital, South Africa, in February 2019, with a 3–4-week history of progressive bilateral proptosis and scalp masses. The computed tomography scan identified bilateral occipital and temporal scalp masses with extracranial extension into the orbital cavities and sinuses, and extradural extension into the anterior cranial fossa. There was no hepatosplenomegaly or lymphadenopathy. A fine needle aspiration sample of the soft tissue scalp mass was submitted for flow cytometry, and a Tru-Cut biopsy (manufacturer unknown) for histological assessment at the National Health Laboratory Service. As part of the staging work up, bone marrow aspiration and a trephine biopsy were performed to exclude infiltration of the marrow.Flow cytometry was performed on the fine needle aspiration sample from the scalp mass on a FACS Calibur instrument , using the PAINT-A-GATE  and MODFIT  software. The following antibodies were used: CD19 FITC , CD10 PE , CD45 PERCP , CD34 APC , CD117 PE , HLA-DR FITC , CD13 PE , Kappa FITC , Lambda PE  and cytoplasmic TdT FITC .Immunohistochemical work up was performed on the Tru-Cut biopsy from the scalp mass using the following stains: CD20 , PAX5 , CD10 , TdT , CD34 , EBER ISH , BCL2 , and Ki67 .Fluorescence in-situ hybridisation for the detection of translocation t was performed using a Vysis LSI MYC/IGH/CEP8 tri-colour dual fusion probe , with orange, green and aqua signals reflecting the MYC, IgH and centromere 8 regions.6 Given the latter, Fluorescence in-situ hybridisation analysis was requested and found to be positive for the translocation t  .7 There was complete resolution of the proptosis and the scalp masses were no longer clinically evident. Sadly, the patient succumbed to neutropenic sepsis six months after diagnosis.The patient was initiated on a BLL regimen as per the FAB LMB 95 protocol with excellent clinical response.4 These individuals were both female and had no laboratory evidence of either peripheral blood or bone marrow involvement at presentation. The youngest, a 5-year-old, presented with abdominal, orbital and mandibular masses.4 The second, a 64-year-old, had multiple extranodal lesions and a chronic disease course.3 Similarly, the clinical presentation of our patient was with isolated extranodal disease. This pattern of involvement is often seen in Burkitt lymphoma and thus formed part of the differential diagnosis in our patient.The co-existence of the translocation t or variant MYC translocation with a precursor B-cell phenotype in an extranodal malignancy is extremely uncommon and has been reported in only one paediatric and one adult patient in the literature.8 It is a marker of immaturity and its expression in mature neoplasms such as BLL would be aberrant. Terminal deoxynucleotidyl transferase was found to be positive in ~30% – 40% of the tumour on both the fine needle aspiration and Tru-Cut biopsy specimens. While these results pointed towards a tumour at a precursor cell stage of development, the S-phase fraction analysis found the tumour proliferative activity to be very high. This measurement is performed by flow cytometry and uses DNA content to determine the proportion of cells in each phase of the cell cycle. Studies performed at our centre have shown the S-phase fraction of B-ALL to be characteristically around 10, whereas a fraction of more than 30, as seen in our patient, would be more typical of BLL.6 Comparisons could not be made with the other cases in the literature as S-phase fraction analysis was not reported. The Ki67 index detects a protein associated with cellular proliferation, which is present in all active stages of the cell cycle. Given that the Ki67 index and S-phase analysis measure different aspects of proliferation, these parameters are not always comparable. Notably, the Ki67 index is unhelpful in distinguishing B-ALL and BLL as values of over 95% can be anticipated in both.6 Despite the conflicting laboratory findings in our patient, the high S-phase fraction in the context of extranodal disease prompted the testing for, and confirmation of, the translocation t. B-cell lymphoma 2 (BCL2) expression is typically seen in acute lymphoblastic leukaemia and the possibility of a double-hit lymphoma was excluded by the presence of TdT.2 S-phase fraction analysis may therefore serve as an early indicator of this disease entity, albeit further investigation of its clinical utility is required. In summary, a combination of diagnostic modalities with evaluation by a multidisciplinary team is required to confirm this rare entity. We propose a laboratory approach to the diagnosis of lymphomas with features of B-ALL and BLL  translates into an actual proliferation advantage. In an attempt to address these questions, one study analysed the molecular characteristics of neoplasms positive for IG-MYC rearrangement and with precursor B-cell phenotype in 12 patients. Aberrant variable diversity joining recombination was shown in five patients and activating N- and K-RAS mutations were detected in seven; all neoplasms had a DNA methylation profile that clustered with that of precursor B-cells. In light of these findings, the authors of the study posed the question as to whether or not cases with MYC rearrangement better fit a B-ALL profile.Leukaemia or lymphoma with translocation t or variant MYC translocation and precursor B-cell phenotype is a rare entity with varied clinical presentation. Awareness of this entity and a high index of suspicion are required by both clinicians  and pathologists to prevent a misdiagnosis. Further studies into the molecular and clinical behaviour of this tumour are required to optimise the therapeutic approach.


During the coronavirus disease 19 (COVID-19) pandemic, extracorporeal membrane oxygenation (ECMO) has been proposed as a possible therapy for COVID-19 patients with acute respiratory distress syndrome. This pictorial review is intended to provide radiologists with up-to-date information regarding different types of ECMO devices, correct placement of ECMO cannulae, and imaging features of potential complications and disease evolution in COVID-19 patients treated with ECMO, which is essential for a correct interpretation of diagnostic imaging, so as to guide proper patient management. In January 2020, several cases of interstitial pneumonia were reported in Wuhan, China. Since mid-January 2020, the first cases of the disease were also found outside of China, and on 11 March 2020 the World Health Organization declared the coronavirus disease 19 (COVID-19) outbreak a pandemic .The clinical spectrum of COVID-19 can range from no symptoms to critical illness, with an estimated incidence of acute respiratory distress syndrome (ARDS) among hospitalized COVID-19 patients hovering around 33% , 5. WhilIn patients with COVID-19-unrelated ARDS, extracorporeal membrane oxygenation (ECMO) can be resorted to as a rescue therapy, allowing for temporary replacement of lung and/or heart function with artificial analogs in cardiac or pulmonary failure refractory to conventional clinical treatment . The eviOur purpose is to overview the imaging appearance of the catheters of ECMO devices in patients with COVID-19-related ARDS, as well as of potential complications that may arise secondary to either ECMO device placement or COVID-19 infection.The ECMO circuit consists of a pump that propels the patient's blood to an oxygen membrane  and two access routes (one for drainage and one for return of the blood to the patient) , 23. ECMAccording to the Extracorporeal Life Support Organization (ELSO) recommendations, VV-ECMO may be utilized for patients with COVID-19 and severe respiratory failure with expected outcomes comparable to patients supported with VV-ECMO before the COVID-19 pandemic. On the other hand, VA-ECMO may be utilized for patients with COVID-19 and severe cardiac failure. However, the experience regarding VA-ECMO in COVID-19 patients is more limited .In VA-ECMO, the drainage cannula is generally inserted into the internal jugular vein or into the femoral vein. The return cannula is preferentially placed into the femoral artery.The classic VV-ECMO configuration consists of two cannulae: a) the drainage cannula inserted into the femoral vein, with the tip at the junction between the inferior vena cava (IVC) and the right atrium, and b) the return cannula inserted into the internal jugular vein, with the tip to the level of the superior vena cava (SVC)-right atrial junction Fig. a.Fig. 1CAnother configuration is femorofemoral ECMO , in which the drainage cannula is introduced into the femoral vein and advanced upwards until the tip reaches the distal IVC. The return cannula is inserted into the contralateral femoral vein with the tip in the right atrium Fig. b.The third configuration is the dual lumen VV-ECMO. It consists of a single cannula with a double lumen inserted from the right internal jugular vein with its tip in the IVC  Fig. 1cc1c.According to the aforementioned ELSO guidelines, in COVID-19 patients dual lumen cannulae should be avoided if possible, as they take relatively longer time to be inserted and are associated with a higher risk of thrombotic complications and malpositioning, requiring repeat echocardiography with associated greater resource utilization and staff exposure .After the placement of ECMO cannulae, an X-ray of the chest and abdomen should be performed to evaluate their position and the distance between them, and to detect potential complications such as pneumothorax or hemothorax. In VV-ECMO, a distance of 15 cm between the tips of the two cannulae is recommended to avoid the phenomenon of “recirculation”, by which oxygenated blood coming to the return cannula is withdrawn through the drainage cannula without passing through the systemic circulation Lung ultrasonography may represent an alternative to chest X-ray and CT . Møller-Complications may be related either to the patient or to mechanical factors. The latter may involve circulatory impairment, most commonly due to clots in the circuit, cannula migration/misplacement, and oxygenator failure.In the EOLIA international clinical trial, patient-related ECMO complications were pneumothorax, acute kidney failure, cardiac arrest, neurological  and infective (ventilator-associated pneumonia) complications, and bleeding . In patiWhile chest X-ray can reveal complications such as pneumothorax or hemothorax , multideECMO patients are usually also intubated and treated with high-flow ventilation, so they may be subject to barotrauma. Pneumothorax and pneumomediastinum are frequent complications in COVID-19 patients treated with high-flow ventilation Fig. , as baroIn COVID-19 patients, bacterial pneumonia is the most common respiratory complication Fig.  43]. As. As43]. In invasively ventilated patients, ventilator-associated pneumonia is a further complication that should be considered. It is defined as a type of pneumonia that occurs 48 to 72 h after intubation in invasively ventilated patients.Neurological complications are a broad spectrum, possibly due to viral dissemination in the central nervous system or to an inflammatory response or immune dysregulation . Those aDuring ECMO treatment, systemic anticoagulation is required to reduce circuit clotting. Consequently, one of the most common complications is extensive hemorrhage. Hemorrhagic stroke Fig.  is one oOne of the risks related to circulatory components is the presence of clots in the circuit. Hence, anticoagulant therapy is warranted to prevent ECMO circuit from clotting and consequently to minimize the risk of thromboembolic events, such as stroke. Unfortunately, anticoagulation therapy increases the risk of hemorrhage . When heThe pathogenesis of spontaneous bleeding in patients with COVID-19 is unclear. Two possible explanations include increased serum proinflammatory cytokines and endothelial cell damage . ProphylIschemia of one or more limbs is the most common vascular complication in patients with VA-ECMO, with a reported prevalence ranging from 10 to 70%. The risk of ischemia is greater with larger cannulas (> 20Fr), in women, young patients, and in the presence of peripheral arterial disease. Possible clinical scenarios include pain, pallor, poikilothermia, motor or sensory deficit, and gangrene. Infection of the catheter insertion site, retroperitoneal bleeding, vessel wall dissection, and pseudoaneurysm formation are other common vascular complications  receptors expressed in the gastrointestinal tract , 68. TraThe presence of ACE2 receptors on kidney podocytes and proximal convoluted tubule cells could also explain the role of SARS-CoV-2 infection in the pathophysiology of acute renal failure . Acute kWang et al.  found a COVID-19 is a potentially life-threatening systemic disease that may require ECMO as a salvage therapy in patients who develop acute respiratory distress syndrome. Radiologists should have a comprehensive knowledge of ECMO devices and of the potential complications that may be encountered in COVID-19 patients treated with ECMO, which is essential for their proper management.


N = 107; Cam-CAN: N = 50). We discovered that the rhythmic and arrhythmic spectral properties of intrinsic brain activity can be robustly estimated in most cortical regions when derived from relatively short segments of 30-s to 120-s of resting-state data, regardless of instrument technology and resting-state paradigm. Using an adapted leave-one-out approach and Bayesian analysis, we also provide evidence that the stability of spectral features over time is unaffected by age, sex, handedness, and general cognitive function. In summary, short MEG sessions are sufficient to yield robust estimates of frequency-defined brain activity during resting-state. This study may help guide future empirical designs in the field, particularly when recording times need to be minimized, such as with patient or special populations.The principle of resting-state paradigms is appealing and practical for collecting data from impaired patients and special populations, especially if data collection times can be minimized. To achieve this goal, researchers need to ensure estimated signal features of interest are robust. In electro- and magnetoencephalography  we are not aware of any studies of the minimal length of data required to yield a robust one-session snapshot of the frequency-spectrum derivatives that are typically used to characterize the complex dynamics of the brain’s resting-state. We aimed to fill this knowledge gap by studying the stability of common spectra measures of resting-state MEG source time series obtained from large samples of single-session recordings from shared data repositories featuring different recording conditions and instrument technologies (OMEGA:  Though the full functional relevance of the approach is still debated , the potIntra-session variability in resting-state activity has been of recent interest for “fingerprinting” , functiowithin a single recording session. Thus, we define a robust estimate as a signal derivative that exhibits stable, consistent values when computed from a sufficiently long duration of data recorded during a single session.Previous electrophysiological reliability studies have focused on the consistency of various signal metrics across sessions separated by days, months, or years . Here, wWe also emphasize the recent interest in discriminating between periodic  and aperiodic  components of the power spectrum of neurophysiological brain signals . StudiesN = 107; N = 50; Here we qualify an estimate obtained from a given data length as robust when its group-wise consistency exceeds pre-defined thresholds across measurements. We use intra-class coefficient (ICC) statistics, with thresholds of ICC > 0.90 for excellent, ICC > 0.75 for good, and ICC > 0.50 for moderate levels of estimate stability . We exam2.2.1.We used subsets of data from two shared repositories: the Open MEG Archive OMEGA;  and the From the OMEGA dataset, 107 participants  were included based on the following criteria: no current neurological or psychiatric disorder, no history of head trauma, and no MRI or MEG contraindications. Participants without MEG, MRI, or demographic data  were also excluded. Eyes-open resting-state MEG data were collected from each participant at a sampling rate of 2400 Hz and with an antialiasing filter with a 600 Hz cut-off. Noise-cancellation was applied using CTF’s software-based built-in third-order spatial gradient noise filters. Recordings lasted a minimum of 4 min and were conducted with participants in the seated position as they fixated on a centrally-presented crosshair.We also selected a validation sample of 50 healthy adult participants  from the Cam-CAN dataset. The sample size of 50 was determined quasi-empirically, by modelling the time-by-ICC stability relationship as a function of sample size in the OMEGA sample see –2. From The data collection and management protocols for the OMEGA and Cam-CAN repositories were approved by the research ethics boards at the Montreal Neurological Institute and the University of Cambridge, respectively. All participants in both studies provided written informed consent in accordance with the Declaration of Helsinki.2.2.MEG preprocessing procedures for both datasets were similar to those reported by 2.3.recon-all. Source imaging was performed on the full-bandwidth data using only gradiometer data with individually fitted overlapping-spheres forward models  and two widely-used source estimation techniques with Brainstorm default parameters: the linearly-constrained minimum variance (LCMV) beamformer and dynamic statistical parametric mapping (dSPM). dSPM source imaging was performed using noise covariance estimated from empty-room recordings taken as close in time as possible to each participant’s visit. To verify that the inclusion of additional data in the form of empty-room recordings did not bias the stability analysis of dSPM, we also ran a control analysis using the identity matrix in place of the noise covariance matrix. These source imaging procedures yielded continuous time series per each spatially-resolved location  on the cortical surface for each participant. Only gradiometer data were used for source imaging to facilitate comparison across the OMEGA and Cam-CAN samples, as the MEG instrument used in the OMEGA study comprises gradiometer sensors only.Using approximately 100 digitized head points, MEG data were co-registered to each participant’s T1-weighted MRI that was segmented and labelled with Freesurfer  using reSource-imaged data were segmented into contiguous, non-overlapping 6 s epochs, resulting in a total of 40 epochs  for OMEGA participants and 70 epochs  for Cam-CAN participants. Vertex-wise estimates of power spectral density (PSD) were obtained using Welch’s method  and averaged over canonical frequency bands using Brainstorm defaults , and ICCs were calculated for each region and frequency between sets of PSD estimates averaged over progressively larger numbers of epochs from one to nepochs/2 . Note that gamma-frequencies were not subjected to FOOOF parametrization, as the FOOOF algorithm struggles with fitting PSD data properly above 40 Hz due to the loss of linearity in log-log space and lack of clear peaks in gamma frequency ranges and above (2) per each relevant location, frequency, and epoch  and extracted residuals from these linear models. We then re-ran all FOOOF temporal stability analyses on the fit-corrected FOOOF feature residuals.We parameterized the power spectra of each brain region using the FOOOF algorithm  implemennd above . Thus, wnd above  and the 2.6.individual stability contribution score per participant in the Cam-CAN participant sample  and Cam-CAN (n = 50) in The minimal recording durations required to derive robust, stable estimates of MEG spectral features using LCMV beamforming are illustrated for OMEGA  across participants in the OMEGA sample markedly improved the robust estimation of periodic activity in the delta band , none of the effects were significant at 4.Despite a substantial literature examining the relationships between frequency-defined neural activity and various states of cognition and disease, little research exists regarding appropriate data durations to derive robust spectral estimates of human electrophysiology signals. We believe that evidence-based recommendations concerning the minimum data duration required for extracting meaningful spectral features of brain activity are required to guide the design of resting-state studies and inform decisions when growing shared data repositories in the field. This work partially addresses this knowledge gap by quantitatively estimating the intra-session stability over time of source-imaged spectral features of resting-state MEG data.Overall, we find that spectral estimates of task-free MEG brain activity stabilize remarkably quickly when derived from durations as short as 30 s, and typically 120 s, of data. These results align with recent publications: one reporting stability of EEG graph theory metrics in under 10 s  and anotThese stability estimates support and extend existing research on the effects of data duration on neuroimaging signal reliability. Studies of data stability in fMRI have often recommended data durations of more than 10 min , which iWe also found that the stability of spectral features was similar between both MEG instruments tested (CTF vs. MEGIN/Elekta), sampling rates used in data collection (2400 Hz vs. 1000 Hz), and resting-state paradigms . We acknowledge the present study was not exhaustive with regards to all possible experimental factors that might influence MEG signal stability, but the MEG instruments and paradigmatic approaches encompass those most employed in the field. This is in agreement with a recent study showing similar longitudinal test-retest reliability of MEG derivatives, regardless of instrument type . The simWe also examined the robustness across varying data lengths of estimates of aperiodic and periodic spectral components of neurophysiological resting-state time series. The estimates of periodic components stabilized (ICC > 0.90) when derived from less than two minutes of data in the alpha and beta bands across the brain, and within 3.5 min of data in the theta band. We note that the theta, alpha, and beta bands are the most susceptible to exhibiting separable peaks in the human neurophysiological spectrum . This coWe found that estimates of the aperiodic slope were robust when derived from at least two minutes of OMEGA and Cam-CAN data. In contrast, the stability of the aperiodic offset differed between the OMEGA and Cam-CAN samples. Whereas the estimation of the spectral offset was stable for OMEGA data durations of about two minutes, its estimation from Cam-CAN data required longer durations and offset estimates in several frontal and medial regions did not reach excellent stability. We are unable to empirically determine the origin of this discrepancy in this study, as many experimental parameters  were different between the OMEGA and Cam-CAN samples. However, we can speculate that the parameters that most likely influence the low-frequency offset are the noise-cancellation method  and the resting-state paradigm . The estimation of the aperiodic offset is highly dependent on the correct estimation of lower-frequency periodic components, namely from the delta band, in which stability was also challenged in these cortical regions. Future research is needed to explore the sources of this disagreement between the OMEGA and Cam-CAN samples.It should be noted that some of our recommendations are predicated on the premise of equivalent stability between the OMEGA and Cam-CAN samples, as the OMEGA recordings (~4 min) were substantially shorter in duration than those collected for Cam-CAN (~8 min). This is supported by our finding that the median 95% confidence intervals for the stability of these samples overlapped at every frequency and data duration; however, the high dimensionality of our data precluded us from performing this analysis at every brain region. Thus, it remains possible that specific combinations of region, frequency, and data duration might exhibit higher stability in one sample versus the other. Theta frequency derivatives from the right superior temporal sulcus, for example, appear to reach excellent stability before 120 s in the Cam-CAN sample, but did not reach an equivalent level of stability in the 120 s of the OMEGA data. In cases where planned research will focus on these regions of non-overlap in our findings, we conservatively recommend the use of longer recording durations to ensure intra-session data stability. We also found that the estimation of high-gamma spectral power was not stable over the recording durations available from OMEGA and Cam-CAN. We can extrapolate from our data that future studies will require MEG resting-state recordings of at least 10 min per participant to derive stable estimates of high-gamma power . This paConsidering these limitations, we look at our present findings as a normative benchmark for neuroscience studies of resting-state brain activity. We hope that our recommendations will prove particularly useful to researchers when designing their MEG resting-state studies and extracting spectral features. Amidst concerns about the reproducibility and replicability of findings across scientific fields , it may 1


Multispectral imaging captures spatial information across a set of discrete spectral channels and is widely utilized across diverse applications such as remote sensing, industrial inspection, and biomedical imaging. Multispectral filter arrays (MSFAs) are filter mosaics integrated atop image sensors that facilitate cost-effective, compact, snapshot multispectral imaging. MSFAs are pre-configured based on application—where filter channels are selected corresponding to targeted absorption spectra—making the design of optimal MSFAs vital for a given application. Despite the availability of many design and optimization approaches for spectral channel selection and spatial arrangement, major limitations remain. There are few robust approaches for joint spectral-spatial optimization, techniques are typically only applicable to limited datasets and most critically, are not available for general use and improvement by the wider community. Here, we reconcile current MSFA design techniques and present Opti-MSFA: a Python-based open-access toolbox for the centralized design and optimization of MSFAs. Opti-MSFA incorporates established spectral-spatial optimization algorithms, such as gradient descent and simulated annealing, multispectral-RGB image reconstruction, and is applicable to user-defined input of spatial-spectral datasets or imagery. We demonstrate the utility of the toolbox by comparing against other published MSFAs using the standard hyperspectral datasets Samson and Jasper Ridge, and further show application on experimentally acquired fluorescence imaging data. In conjunction with end-user input and collaboration, we foresee the continued development of Opti-MSFA for the benefit of the wider research community. HoweCode 1  to [RGB] by inverting the following matrix relationship: The choice of color space generally depends on how the color is being displayed. Opti-MSFA uses the sRGB color space, and we select the CIE Illuminant D65 as the white point . The xyzTo evaluate the visual performance of the MSFA - the representation in RGB for visualisation purposes - the demosaicked MSFA signal for each channel is represented spectrally as the filter response for each channel then converted to RGB space to yield the RGB representation of the MSFA signal as Note that the J channels in the above representation simply correspond to the red, green and blue channels. This additional feature can enable comparison of the visual performance of MSFAs for both quantitative and visual performance.2.8Two publicly available reference datasets were used. The Samson dataset is composed of 952 x 952 pixels, with 156 spectral channels ranging from 401nm to 889 nm with a spectral resolution of 3.13 nm. To reduce the computational cost, a sub image of 95 x 95 pixels is extracted from the image starting at the  pixel index. Three endmembers are present . The Jasper ridge dataset is composed of 512 x 614 pixels with 224 spectral channels between 380 nm to 2500 nm at a spectral resolution of 9.46 nm. A sub image of 100 x 100 pixels is extracted from the  pixel index to reduce the computational cost. Channels 1-3, 108-112,154-166, and 220-224 were removed due to the atmospheric effects as is commonly done. This results in 198 channels. Four endmembers are in this dataset . The ground truth abundance map was spatially truncated to the appropriate size and region in both cases, and the endmember content was also adjusted to remove the relevant channels for the Jasper ridge dataset.A further experimental fluorescence dataset was used as a test case, given the interest in the application of MSI in biomedical fluorescence imaging. The data was acquired with a benchtop spectral imaging system, using a multi-LED source for illumination, as previously published . Briefly3.3.11.A commercial 2x2 RGGB CFA featuring off-the-shelf RGB filters .2.An empirically-designed 2x2 MSFA, similar to a Bayer filter (RGGB) but with a yellow filter instead of an additional green (RGYB) .3.A five-channel MSFA with center wavelengths and bandwidths optimized for spectral recovery; the spatial distribution of the filters on the mosaic was produced using the binary tree pattern approach .4.An application-independent 3x3 MSFA design that was optimized on three reference HSI datasets that are not used in this evaluation  ; all banTo evaluate Opti-MSFA performance, we designed optimal MSFAs for unmixing endmembers in two real-world reference datasets and compared the results against published MSFA designs for the same datasets. The two standardized datasets are the Samson and Jasper Ridge HSI datasets  and OptiThese four MSFA designs were input into the processing pipeline and used to estimate abundance maps and associated unmixing error for the Samson and Jasper Ridge datasets. The Opti-MSFA pipeline was then used to produce an optimal MSFA design for each dataset. The optimization was first conducted for the visible wavelength range (400–700 nm), for ease of comparison against the reference MSFA designs, and then repeated for the entire wavelength span of the datasets for completeness. The RMS accuracy of the demosaicked and unmixed result was compared to the ground-truth abundance map. The optimization pipeline was repeated for 3, 4, 6, and 9 MSFA channels for both Samson and Jasper Ridge datasets, for comparison to the prior MSFA designs . Note thThe Opti-MSFA output for the Samson dataset , Table 4In the optimization results with a higher number of spectral bands for both data sets, some of the resulting spectral bands are nearly overlapping , 7. This3.2To further demonstrate the utility of Opti-MSFA, we designed optimal MSFAs to unmix fluorescence spectra from mixtures of fluorescence dyes, based on an experimentally measured spectral imaging dataset. These data provide an important application scenario relevant to biomedical imaging—in particular fluorescent guided surgery—in which to test the accuracy and performance of Opti-MSFA. Input endmembers  includedApplying the Opti-MSFA optimization to design an MSFA for the experimentally acquired data shows that the results for the 4 and 6 channel designs show remarkable similarly, with essentially four shared filters . The 6-c4.MSFAs enable cost-effective, compact and snapshot MSI for a wide variety of applications. MSFAs are preconfigured with a specific set of spectral channels, hence the design of optimized MSFAs that accurately reconstruct the spectral information contained within a scene using the fewest possible channels is imperative. Prior work has targeted either spectral filter selection or spatial arrangement, with only a small number of joint spectral and spatial optimization approaches reported; the majority also use limited datasets and crucially, are not available for use by the wider community.In this manuscript, we present the first version of the open-source MSFA design and optimization toolbox Opti-MSFA. Our demonstration on Samson and Jackson Ridge datasets show that some application-independent MSFAs can perform well on specific datasets but not on others. For example, the 9 channel MSFA tested performs the best out of the four reference designs on the Samson dataset but not on the Jasper-Ridge dataset. In contrast, the 6 channel MSFA performs well on the Jasper-Ridge dataset, but not on the Samson dataset. Using our Opti-MSFA tailored designs increases the performance in both cases, showing that ultimately, the best accuracy can be achieved using a custom MSFA. Our application of Opti-MSFA to a raw experimental dataset shows that Opti-MSFA is robust and effective for different data classes by using fluorescence rather than reflectance data, and shows that both can be unmixed from one another using this process. The outputs from these tests demonstrate that Opti-MSFA enables rapid comparison between different MSFA designs, as well as built-in optimization that can produce effective solutions.The generalizable framework of Opti-MSFA was developed with future integration of additional optimization and reconstruction methods in mind. The first version is capable of optimizing MSFAs for arbitrary input hyperspectral datasets with a host of user-controlled features, however, there remains a number of strategic aims for future development. One clear area is introducing more advanced and varied methods for spectral optimization, merit functions, different unmixing methods, and demosaicking approaches –56. AlonFurther, because the success and utility of such a toolbox is dependent on community adoption, we foresee development driven in conjunction with the community. For example: incorporating different design and optimization algorithms developed by researchers; benchmarking against methods integrated into Opti-MSFA and posting of results for other end-users; and uploading additional established hyperspectral datasets for others to investigate. Moreover, Python-based Opti-MSFA can be adapted and modified in a relatively straightforward manner to incorporate other powerful Python toolboxes, such as Keras or Tensorflow ,58, for The expansion of the toolbox to incorporate optics and alternate filter functions, represents an important direction for its next iteration. Firstly, in this work, the optical filter functions underpinning the MSFAs are represented by a Gaussian—simply described by the parameters of bandwidth and center wavelength—and hence straightforward to incorporate within an optimization framework. However, the normalized filter response,  effects –62, whic effects ,64, sens effects , etc. FoLastly, manufacturing considerations, such as fabrication tolerances could also be included as boundary limits, while a penalty could be introduced to merit functions when challenging or costly filter designs are considered. Longer term, we could also foresee incorporating polarization-sensitive filter arrays, which are now becoming more prevalent due to the introduction of polarimetric pixel sensors . Additio5.To address the challenge of spatio-spectral optimization of MSFAs for snapshot MSI, we introduce here Opti-MSFA: an open-access Python toolbox for the community . Opti-MS


Arachis species subjected to drought (A-metaDEG) and the root-knot nematode Meloidogyne arenaria (B-metaDEG) via meta-analysis, to identify core-stress responsive genes to each individual and concurrent stresses in these species. Transcriptome analysis of a nematode/drought bioassay (cross-stress) showed that the set of stress responsive DEGs to concurrent stress is distinct from those resulting from overlapping A- and B-metaDEGs, indicating a specialized and unique response to combined stresses in wild Arachis. Whilst individual biotic and abiotic stresses elicit hormone-responsive genes, most notably in the jasmonic and abscisic acid pathways, combined stresses seem to trigger mainly the ethylene hormone pathway. The overexpression of a cross-stress tolerance candidate gene identified here, an endochitinase-encoding gene (AsECHI) from Arachis stenosperma, reduced up to 30% of M. incognita infection and increased post-drought recovery in Arabidopsis plants submitted to both stresses. The elucidation of the network of cross-stress responsive genes in Arachis contributes to better understanding the complex regulation of biotic and abiotic responses in plants facilitating more adequate crop breeding for combined stress tolerance.Nematodes and drought are major constraints in tropical agriculture and often occur simultaneously. Plant responses to these stresses are complex and require crosstalk between biotic and abiotic signaling pathways. In this study, we explored the transcriptome data of wild  In addition, the foreseen impacts of climate change on crop productivity, including water shortage and disease emergence, stand a great challenge for global food security, propelling the need for the development of more adapted cultivars3. Over the course of evolution, plants developed complex networks to overcome stresses that rely on their ability to perceive external signals and activate an appropriate spectrum of molecular responses in a timely manner5. This include different signaling pathways mobilized to respond to abiotic and biotic stresses8, that in the case of simultaneous occurrence, lead to synergism or the prioritization of one stress response over another, often causing a decrease in plant tolerance to other hazards and losses in productivity8. The elucidation of key elements in plant multi-layered defense to simultaneous stresses provides new opportunities for enhancing plant resistance and can largely contribute to the improvement of crop yield11.Plants are constantly exposed to concurrent environmental stresses, such as drought and diseases which cause significant yield losses16, with the majority reporting increased resistance to fungi and abiotic stresses due to enhanced levels of annexins17, osmotin-like proteins18, polyamines20 or transcription factors23. Nonetheless, no studies on concurrent improvement for nematode and abiotic resistances have yet been reported.To date, few studies on plant responses to simultaneous abiotic and biotic stresses have been conducted26. In addition, SA and JA/ET defense pathways often act as mutually antagonistic28, with ABA appearing as a central modulator of the regulatory crosstalk between these phytohormones, directly impacting SA and JA biosynthesis29. The crosstalk between ABA/JA-ET30, ABA/SA31 and JA/SA32, that often causes antagonistic effects, is important to guarantee that those hormone cascades with low or no significant effects on the pathogen control are suppressed to avoid the waste of valuable physiological resources33. Nonetheless, a universal phytohormonal interplay does not apply to all cases, with the outcome of ABA and SA-JA/ET interaction being largely dependent upon the pathosystem, plant developmental stage, as well as on the timing of infection35.The role of phytohormones in activating the defense responses against biotic and abiotic stresses is well- known, with salicylic acid (SA) mediating responses mostly to biotrophic pathogens, jasmonic acid (JA) and ethylene (ET) to chewing insects and necrotrophic pathogens, and abscisic acid (ABA) to abiotic stressesArachis spp.) have evolved and adapted to a wide range of environments and harbor high levels of resistance to many pathogens and abiotic constraints39, constituting useful sources of resistance alleles against these stresses. Previous studies showed that Arachis stenosperma is highly resistant to the Root-knot nematode (RKN) Meloidogyne arenaria, triggering the Hypersensitive Response (HR) at the nematode feeding site41, whilst A. duranensis is more tolerant to water deficit by displaying a more conservative plant transpiration behavior42. While A. stenosperma HR response against M. arenaria has been linked to the activation of the JA pathway, the ABA-independent signaling pathway seems to be the main activator of the adaptive physiological changes in the drought-tolerant A. duranensis43.Peanut wild relatives , has enabled the comparison among transcriptomes from the most diverse conditionsArachis RNA-Seq data, and evaluated the use of this approach as a predictive tool for the discovery of genes responsive to combined stresses. We also evaluated the effect of the endochitinase (AsECHI) overexpression, a cross-stress gene identified here, on the improvement of Arabidopsis thaliana plants’ tolerance to combined nematode and drought stresses. Understanding the complex regulation of biotic and abiotic responses and their interactions is crucial to avoid trade-off effects that limit plant growth and yield and will facilitate a more efficient crop breeding for combined stress tolerance.In the present study, we investigated the transcriptional dynamics elicited by RKN infection in combination with drought, via the meta-analysis of our previously produced wild Arachis species under biotic and abiotic stresses were explored to identify unique and commonly stress-regulated genes. The overall expression trend and number of DEGs varied for each species/treatment combination, with the dry-down treatment (abiotic stress) prompting the highest number of responsive DEGs in both species  + A. duranensis dry-down (DDD)), whilst the RKN-resistant A. stenosperma displayed the greatest number of DEGs in response to biotic stress  and biotic (B) libraries. Genes with the same expression trend among the abiotic and/or biotic were considered as A- and B-metaDEGs. By following these criteria, we assume that a better representation of the overall expression trend of the stress-responsive genes during the time of the stress imposition was obtainedA. duranensis and A. stenosperma). A total of 1,224 B-metaDEGs were identified, being 736 DEGs (60.08%) downregulated and 488 upregulated (39.83%) in both species  with the majority downregulated  and 530 upregulated (24.09%) Fig.  in both ies Fig. .Arachis core-metaDEGs, were genes responsive to stress regardless of plant genotype, type or timing of the stress imposed, constituting a valuable tool for better understanding the nature of stress resistance/tolerance in the genus, and to be further exploited as novel wild alleles involved in adaptability to stressful environments.In sum, only 7% (243) of all A- and B-metaDEGs were common between biotic and abiotic stresses, with the great majority 213) showing downregulation . In total, we found 3,345 DEGs in the cross-stress library (crossDEGs)  modulated in response to combined stress and validated by the meta-analysis, which are highly valuable for genetic breeding and biotechnological purposes.The comparison between the 3,345 de facto crossDEGs and the 3,424 A- and B-metaDEGs intersection, identified 86 DEGs in common  and de facto  analyses could be assigned to 33 different functional categories  genes forecast as A-metaDEGs, 7.3% (244) as B-metaDEGs and only 2.6% (86) as core-metaDEGs  into the main hormone pathways involved in plant defense Fig. A. Over 1Arachis, we demonstrated that the majority of the drought responsive genes involved in hormonal signaling were related to the ABA pathway39, while most genes responsive to RKN infection belonged to the JA pathway49. Nonetheless, the analysis of the de facto crossDEGs showed that another important hormone alternative defense pathway, the ET pathway, was more activated, with 32 genes modulated and classified as crossDEGs, whilst the JA and ABA pathways showed only 11 and eight crossDEGs, respectively. This indicates a shift on the main responsive hormone pathways normally triggered during each of these stresses alone, (e.g.) ABA for abiotic and JA for biotic stresses , which integrates signals from both JA and ethylene pathwaysA. stenosperma marker genes involved in ET, JA and ABA pathways corroborated this assumption. The expression of ERF2, a regulator of ET signaling, was strongly induced, while PDF1.2, a downstream ET‐responsive gene, was exclusively detected in the cross-stress treatment   Fig. . One of A. stenosperma (AsECHI), was successfully cloned in the pPZP_BAR binary vector and used for A. thaliana transformation. The overexpression of AsECHI transgene was confirmed in four overexpressing (OE) lines  at T2 generation by qRT-PCR analysis, with expression levels varying according to the transgenic line  were further submitted to the combined stress experiment.The 795 bp consensus coding sequence of ECHI from A. thaliana AsECHI-OE lines submitted to dry-down treatment displayed a slightly curled phenotype at the 9th day of drought imposition, while WT plants were severely affected  , biotic (B-metaDEGs) and the combination of both stresses (core-metaDEGs). Many of the metaDEGs here identified encode for defense-related proteins (PR), TFs, and genes involved in plant defense hormone pathways, and were differentially expressed according to the duration and type of the stress imposed. As different Arachis species (A. stenosperma and A. duranensis) with contrasting levels of stress tolerance were used in this meta-analysis, we assume that the sets of genes identified (metaDEGs) are not genotype-dependent and are well-conserved in the genus, thus constituting interesting core genes involved in the high adaptability of these wild species to their native environments under stressful conditions.In this study, we explored our previously produced wild 59, the co-occurrence of two or more different stresses make the prediction of the genes that play a major role in this concerted defense response rather difficult, due to the existence of additive, subtractive and combinatorial effects and also the activation of specific genes and pathways that are triggered solely during stress combination8. This seems to be the case in Arachis, as our meta-analysis was able to predict only 7.2% of the genes observed in our de facto cross-stress transcriptome. This low predictability for genes responsive to combined stresses suggests the existence of dedicated pathways and genes that have evolved to mitigate the effects of stress combination, which, by being different, would not have been picked up by the meta-analysis. These findings also corroborate the need to conduct de facto combinatorial stress experiments to fully identify the genes exclusively prompted by simultaneous stresses.As seen for different speciesA. thaliana30 showed that not only the set of responsive genes between single and combined stresses are different, but they rely on different signaling hormonal pathways, which seem to interact and inhibit each other. Likewise, based on our DEGs functional categorization, allocation to different hormonal pathways, and drawing on our previous works49, we predicate that individual and combined abiotic and biotic stresses trigger different phytohormone pathways and hold different modulation and cross-talk points in Arachis. This assumption is reinforced in this study by the qRT-PCR expression analysis of marker genes from hormone signaling pathways which showed that whilst JA and ABA were the prevalent hormone pathways activated in response to RKN and drought stresses respectively, ET also appears as one of the main regulators in Arachis plants submitted to both stresses simultaneously.Studies on hormonal signaling pathways and defense genes expression conducted in tomato and 60, the ET function as a regulator affecting both plant basal and systemic acquired resistance (SAR) in response to simultaneous stresses is less studied and seems to vary according to the plant-pathogen interaction62. Some reports suggest that stress-activated protein kinases  are responsible for integrating multiple environmental stresses via ET biosynthesis and stress perception63, while others point out that ET and JA cooperate through transcriptional induction of ERF  during pathogen attack, whilst ABA might show an antagonistic effect on JA/ET pathogen defense signaling30 , or positively affect JA biosynthesis in the activation of defense responses64. The latter seems to be the case here, as the ET pathway marker ERF2 and the ET-responsive PDF1.2 genes were highly or exclusively induced in Arachis plants submitted to cross-stress treatment, in synchronism with the induction of JA-responsive LOX3 and VSP2 genes, whilst the ABA-dependent regulator bZIP and RD29B were only slightly induced or downregulated in this condition.Although the role of JA/ET in the defense network against necrotrophic pathogens has been described in different plant speciesA. stenosperma defense responses against M. arenaria observed here and in our previous studies49, has also been reported in sweet potato infected with M. incognita, in which the ET-dependent ERF and JA-dependent MYC TF genes were induced65, and in Arabidopsis66.This protagonist role of the JA/ET pathways during 15. In this direction, genes and regulators identified in this study which include those responsive to both biotic (RKN infection), abiotic (drought) and the combination of these stresses, constitute a valuable gene pool to provide new insights into the transcriptional regulatory networks trigged by isolated and combined stresses and help us to better understand the genetic sources controlling the natural ability of wild Arachis species to resist to a number of environmental challenges. Moreover, this core stress-responsive genes constitute precise targets for biotechnological purposes to develop multiple stress resistance in crops.As plants need to balance the costs and benefits to initiate tolerance/resistance responses against a number of environmental constraints, they evolved a series of flexible signaling networks to optimize its adaptative responses. Cross-talk between these defense signaling hormonal pathways allows plants to tailor the appropriate defensive strategy to multiple stresses in a cost-effective mannerA. stenosperma (AsECHI), was identified as one of the few genes commonly induced in response to drought, RKN infection, and combined stresses in the de facto cross-stress experiments. Although there are many studies using chitinases on the enhancement of plant resistance against fungi67, only a few reports on transgenic plants overexpressing chitinase genes to increase resistance against sedentary phytonematodes69 or tolerance to abiotic stresses, such as cold and drought71 have been conducted. To our knowledge, this is the first time that the overexpression of a plant endochitinase gene is proved to confer resistance to isolated and combined RKN infection and drought stresses.In this study, an endochitinase from AsECHI, which increases tolerance to drought concomitant with a reduction of up to 30% in RKN infection, might be due to an increased accumulation of peroxidase activity in plants, as previously observed in transgenic tobacco72. In fact, peroxidases have a plural role in biotic and abiotic stress responses, as they act as signal molecules in the defense of plants against pathogen attacks and also contribute to increasing abiotic stress tolerance by counteracting the oxidative stress and also by favoring water retention in the cell walls, as suggested by73. Here, we demonstrated that transgenic AsECHI-OE plants exhibited higher RWC and reduced EL values than WT that suggest a greater ability for water retention and a low degree of membrane injury, with better protection against oxidative damage caused by isolated and combined stresses. In addition to peroxidase accumulation, the action of endochitinases can cause the liberation of glycoprotein oligomers in the plant cell wall or apoplast, which initiate defense signaling pathways such as those prompted by SA, also contributing to this increase in tolerance to biotic and abiotic stresses72. Therefore, the use of endochitinase genes such as AsECHI alone, or in combination with other plant defense genes, is an interesting sustainable strategy to improve crop tolerance and production.This dual effect observed in plants overexpressing Arachis. The further understanding of the complex regulation of biotic and abiotic responses and their interactions in order to avoid trade-off effects that limit plant growth and yield will enable a more efficient biotechnological crop improvement.With the onset of climate change, an increasing shift on the search for genes to be used for plant genetic engineering from individual to multiple stresses is occurring, as more sustainable crop management and disease control strategies are required. In the present study, we found common and specialized genes and pathways that coordinate plant responses to combined stresses in wild A. duranensis and A. stenosperma plants submitted to abiotic and biotic stresses were used for the meta-analysis (Supplementary Table 44. RNA-Seq libraries from abiotic (dry-down and dehydration) and biotic (nematode inoculation) treatments  for all the comparisons were considered as differentially expressed metaDEGs for Abiotic (A-metaDEGs) or Biotic (B-metaDEGs) stresses. The overlapping of A- and B-metaDEGs was conducted using the graphical representation of the common genes by the Eulerr on-line software (http://eulerr.co) and genes found in common between A- and B-metaDEGs were considered as core-metaDEGs, regardless of their expression trend . Heatmaps showing the expression profile of DEGs were made in gplots package81 using average expression values for genes in the biotic and abiotic treatments11. For dotplot, we used the expression values from the core-metaDEG and crossDEG as input for ggplot package80.Genes identified as DEGs were functionally annotated by MercatorA. stenosperma (accession V10309) plants grown at 70% of field capacity (FC) under greenhouse conditions were submitted to four treatments , with each treatment comprising three sets  of 20 plants. At the end of treatments, plant roots were collected in liquid nitrogen and stored at − 80 ºC for RNA extraction.Six-week-old M. arenaria (race 1) juveniles (J2) were extracted from tomato roots as previously described40. Plants were inoculated with 10,000 J2 and kept well-watered under greenhouse conditions for 7 days after inoculation (DAI). The susceptible peanut cultivar (A. hypogaea cv. Runner) was also inoculated as a positive control.For nematode infection, 82.For the dry-down assay, plants were submitted to a gradual decrease of soil moisture (dry-down) by the withholding of irrigation for 7 days until 25% FC as described beforeM. arenaria inoculation and drought stresses in greenhouse conditions, as six-week-old plants kept at 70% FC were inoculated with 10,000 J2 of M. arenaria and submitted to water withdrawal for seven days. At the 7th day plants were collected and stored as above.For the cross-stress assay, plants were submitted to combined 83 and further purified with Invisorb Plant RNA Mini Kit . RNA was reverse transcribed and analyzed according to Morgante et al., (2013). In total, 12 paired-end cDNA libraries were constructed using the TruSeq™ Stranded Total RNA LT Sample Prep Kit from Illumima  and sequenced by Macrogen  using HiSeq-4000, 150 bp . Illumina raw reads were analyzed and quantified as before43 (PRJNA284674).Total RNA was extracted from roots of the three biological replicates from each treatment  using a modified lithium chloride protocolA. stenosperma (AsECHI) was synthesized and cloned under the control of the A. thaliana actin 2 promoter in the binary vector pPZP_BAR by Epoch Life Science Inc. . pPZP-BAR is derived from pPZP_201BK_EGFP85 in which we replaced the hygromycin resistance gene by the bar gene from Streptomyces hygroscopicus86 for glufosinate ammonium herbicide resistance. Wild-type (WT) A. thaliana plants (ecotype Col-0) were transformed using the pPZP-AsECHI vector as described by87. The eGFP-positive and glufosinate-resistant transformants were grown in a controlled growth chamber  to obtain transgenic AsECHI overexpressing (OE) lines, as described by88. All biotic and abiotic stress treatments were conducted using homozygous AsECHI-OE lines at the T2 generation.The complete coding sequence (795 bp) of an endochitinase-encoding gene from AsECHI transgene in A. thaliana OE lines and WT plants and of the hormone pathways marker genes in A. stenosperma were analyzed by qRT-PCR, using total RNA extracted with the RNeasy Plant Mini Kit  according to84. qRT-PCR reactions were performed in three biological replicates on the StepOne Plus Real-Time PCR System  and the relative quantification (RQ) of mRNA levels estimated using the SATqPCR web tool89. The specific primers of the target and reference genes are described in the Supplementary Table The expression levels of A. thaliana plants grown on sand:substrate mixture  were submitted to isolated and coupled biotic and abiotic stress assays. For the nematode assays, ten plants were inoculated with M. incognita J2  as described by Morgante et al. (2013) and kept at the growth chamber conditions. At 60 DAI, the number of nematode females on roots was assessed and statistically compared using the t-test (p < 0.05)88.Four-weeks-old 2) were collected per individual for the determination of the Relative Water Content (RWC) and electrolyte leakage (EE), according to Vinson et al. (2020)90.For the dry-down assay, irrigation of 20 plants was suspended for nine days (when plants showed wilting symptoms) in the stressed group, whilst the control group of individuals was kept under 70% FC. At the end of the treatment, three leaf discs  for four additional days, after which all plants were rewatered. The non-inoculated plants were maintained at 70% FC. The number of M. incognita females in the OE lines was assessed at 60 DAI as described above.For the cross-stress assay, nine plants per OE line and WT were first submitted to water withdrawal for seven days and then inoculated with Supplementary Information.Supplementary Table S2.Supplementary Table S3.Supplementary Table S4.Supplementary Table S6.


The COVID-19 pandemic caused by the novel SARS-CoV-2 coronavirus is an ongoing pandemic causing severe health crisis worldwide. Recovered COVID-19 patients go through several long-term side effects such as fatigue, headaches, dizziness, weight loss, and muscle loss among others. Our study sought to determine the molecular mechanisms behind muscle loss in COVID-19 patients. We hypothesized that multiple factors such as cytokine storm and therapeutic drugs  might be involved in muscle loss. Using the Gene Expression Omnibus database, we identified several studies that performed RNA sequencing on skeletal muscles with the treatment of cytokine, glucocorticoid, and antiviral drugs. Our study identified cytokines, such as IL-1b, and IL-6, associated with altered regulation of several genes involved in the myogenic processes, including Ttn, Cxxc5, Malat1, and Foxo1. We also observed that glucocorticoid altered the expression of Foxo1, Lcn2, Slc39a14, and Cdkn1a. Finally, we found out that the antiviral (RNA-dependent RNA polymerase inhibitor) drug regulates the expression of some of the muscle-related genes . Based on our findings, we hypothesize that the cytokine storm, glucocorticoids, and antiviral drugs might be synergistically involved in COVID-19-dependent muscle loss. The Coronavirus disease 2019 (COVID-19) is an infectious disease caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). As an airborne virus transmissible through droplets, the spread of COVID-19 has resulted in a global pandemic that continues to put a massive strain on healthcare around the world. While the initial focus was put on the respiratory effects of COVID-19, time wide variety of side effects were noted to be associated with this novel virus across several organs. Acute infection with the virus has shown to present with a wide range of severity, and common symptoms including fever, cough, shortness of breath, and myalgias [As we continue to manage patients who have recovered from acute COVID-19, observing long-term effects from infection is a major cause for concern. It is estimated that 10 to 20% of patients with symptomatic acute COVID-19 will experience persistent effects lasting greater than one month , 4. TermMuscle loss, or sarcopenia, has also been demonstrated as a consequence in the COVID-19 patients , 7-12, bIn this study, we sought to identify possible molecular mechanisms by which muscle loss might be induced in COVID-19 patients using Gene Expression Omnibus (GEO) database and published literature. We hypothesized that a combination of several factors , glucocorticoid, and antiviral drugs) is responsible for inducing muscle loss in COVID-19. In this study, we identified several genes differentially regulated by the factors mentioned above involved in muscle loss.Through the Gene Expression Omnibus (GEO) database, we searched the published literature for studies that carried out experiments with the three elements of our hypothesis. To evaluate the effect of the cytokine storm, we searched for studies treating cells with major cytokines involved, such as IL-6 and IL-1b. We first used search terms "IL-6" and "Il-1b" along with terms like "skeletal muscle." If no studies were found on the GEO database with these criteria, we broadened the search to find studies that treated other types of cells with these molecules. A similar search strategy was employed with "dexamethasone" and "remdesivir." We included human  mice model studies  and cell culture studies . Details of the study design of GEO dataset are provided in The overall effects of the COVID-19 pandemic have been immense, yet we still do not completely understand the issues that have and will continue to afflict patients in the long term. Sarcopenia is one such issue that can have a significant negative impact on patients, especially elderly populations. It has been well-documented that patients infected with COVID-19 are prone to developing significant weight loss and clinical cachexia -15. MuscThe cytokine storm associated with many viral illnesses, including COVID-19, is associated with a massive release of inflammatory cytokines . Levels of these pro-inflammatory molecules have been shown to be increased in frail and sarcopenic patients across several studies -19. We sIL-1b is another major player involved in the cytokine storm. In a study by Braun et al., IL-1b was injected into the mouse cerebral ventricles to determine its role in signaling in the central nervous system and the effect on skeletal muscle catabolism . SignifiNext, we analyzed studies involving glucocorticoid agents, as the anti-inflammatory effect of these medications was thought to prevent progression to respiratory failure and death in COVID-19 . In a stLastly, we searched for evidence that antiviral agents may play a role in muscle loss, as the drug remdesivir has been used across the world in the treatment of patients with COVID-19. Due to the lack of studies found on the Gene Expression Omnibus analyzing remdesivir's effect on cellular gene expression, we looked for other antiviral drugs with a similar mechanism (RNA-dependent RNA polymerase inhibitor). Ribavirin is another RNA-dependent RNA polymerase inhibitor and is currently used as a therapeutic for the hepatitis C virus. This drug works as a nucleoside analog and binds the RNA polymerase enzyme at the same binding site as remdesivir . Thomas Cyclin D1 (CCND1) is an important member of the cyclin family of genes which was downregulated with ribavirin treatment . SkeletaOur study does have some inherent limitations. We identified only a selected number of genes affected by the various treatments that play a role in muscle biology. These are likely only a fraction of the total number of genes implicated in sarcopenia as a whole. Secondly, we could not study gene expression changes in human skeletal muscle cells across all of these studies. Another important factor to consider is our inability to determine gene expression that COVID-19 infection itself has on skeletal muscle in combination with glucocorticoids and antiviral medications. In the future, further gene sequencing of human skeletal muscle cells infected by the coronavirus in combination with dexamethasone and remdesivir treatment is needed. This would likely provide a more accurate idea of the genetic mechanisms behind sarcopenia in COVID-19 patients. In summary, we found several differentially regulated gene expression that occurs when cells are treated with cytokines, glucocorticoids, and antivirals. These changes present the possibility that muscle atrophy may be induced by these conditions separately or synergistically. In patients infected with severe COVID-19 with long-term symptoms, it is likely they have experienced the cytokine storm as well as treatment with glucocorticoids like dexamethasone and the antiviral drug. Based on our findings, we speculate that an additive effect of these three factors might involved in muscle loss . Due to www.aginganddisease.org/EN/10.14336/AD.2021.0817.The Supplementary data can be found online at: