It is well established that mothers with above-normal pre-pregnancy BMI are at increased risk of breastfeeding cessation; however, the impact of pregnancy weight-gain (PWG) is less well-defined. Excess PWG may alter the hormonal preparation of breast tissue for lactation, increase the risk of complications that negatively impact breastfeeding , and may make effective latch more difficult to achieve.N = 1207). PWG category was interacted with pre-pregnancy BMI . Confounders of the relationship of interest were evaluated using directed acyclic graphs and bivariate analyses; variables not on the proposed causal pathway and associated with the exposure and outcome were included in multivariate models. Results: After adjustment, women of normal and obese pre-pregnancy BMI with greater-than-recommended PWG had 1.39 (1.03–1.86) and 1.48 (1.06–2.07) times the risk of any breastfeeding cessation within the first 3 months postpartum compared to women with normal pre-pregnancy BMI who gained within PWG recommendations. Overweight women with greater-than-recommended PWG were at increased risk of cessation, although not significantly (aHR [95% CI]: 1.29 [0.95–1.75]). No significant relationship was observed for exclusive breastfeeding cessation.Our objective was to determine the impact of PWG and pre-pregnancy BMI on the risk of breastfeeding cessation utilizing the Institute of Medicine’s 2009 recommendations. Cox proportional hazards models were utilized to estimate the risk of cessation of exclusive breastfeeding, and cessation of any breastfeeding among women who initiated exclusive and any breastfeeding, respectively, in a cross sectional sample of survey respondents from a New York county  and other issues that may lead to early breastfeeding cessation. As overweight and obesity continue to rise among women of reproductive age , a compr2), where the previous recommendations were limited to women with a BMI of 29 kg/m2 or less : 1.29 [0.95–1.75]). Women with pre-pregnancy obesity showed a trend toward increased risk cessation in both less than recommended and as recommended PWG categories . Women of normal pre-pregnancy BMI who gained less than recommended showed a trend towards increased risk of cessation compared to the reference group (aHR [95%CI]: 1.35 [0.92–1.99])  , future studies in large cohorts should evaluate whether a portion of the increased risk of poor breastfeeding outcomes among mothers who gain more than is recommended is due to these pregnancy and birth factors. Due to the small number of women with underweight pre-pregnancy BMIs, we were unable to evaluate the impact of pregnancy weight gain and weight status on breastfeeding outcomes in these women. Additionally, self-reported breastfeeding data were utilized for this study. As noted, the source of weight data on the birth certificate is consistent , however it is possible the some of the weights in these records were drawn from prior visit documentation rather than direct measurement.Additional factors that could not be evaluated in this analysis include gestational diabetes, hypertensive disorders of pregnancy, postpartum hemorrhage, pre-eclampsia and C-section. These conditions are more common among women who gain excess weight during pregnancy and increase the risk of maternal intensive care unit admission and longer hospital stays, however, given that this increased risk is conferred at least partially While studies have demonstrated that PRAMS self-report measures of breastfeeding initiation show a high degree of agreement with the birth certificate , similarDespite these limitations, this analysis has several key strengths. The sample of respondents included in this study are representative of the county from which they were sampled allowing for evaluation of this relationship in a sample that represents the communities with similar demographic characteristics. These results are likely generalizable to populations with similar characteristics, however, are not generalizable to women with underweight pre-pregnancy BMIs and should be replicated in other populations. Categorizing pre-pregnancy BMI status and pregnancy weight gain using birth certificate data  eliminates concerns about social desirability bias that is often present in self-report data regarding weight. Additionally, this association should be evaluated in regions with greater diversity in terms of cultural, ethnic and racial backgrounds, and with differing health care infrastructure around pregnancy and breastfeeding support in order to understand the importance of these factors in the association of interest. Finally, having data regarding breastfeeding outcomes extending to 4 months allowed us to evaluate the risk of cessation over a long follow up period, and provides detailed time to event breastfeeding data that captures the individual variation in breastfeeding outcomes in this population.Weight gain in excess of the Institute of Medicine’s 2009 guidelines leads to increased risk of poor breastfeeding outcomes. Efforts to reduce overweight and obesity in women before child bearing, in addition to increasing the number of mothers who gain weight during pregnancy within Institute of Medicine guidelines would be expected to improve breastfeeding outcomes. Given that these modifiable breastfeeding risk factors impact an increasingly large proportion of pregnant mothers in the United States, effective interventions aimed at not only lowering pre-pregnancy BMI, but also reducing excessive weight gain during pregnancy could significantly improve breastfeeding outcomes on a national scale.Additional file 1: Supplemental Table 1. Crude: Time to cessation of breastfeeding ~ Pregnancy weight gain (PWG)* BMI.Additional file 2: Supplemental Table 2. Adjusted: Time to cessation of breastfeeding ~ Pregnancy weight gain (PWG)*BMI.


Currently available treatments of diabetic kidney disease (DKD) remain limited despite improved understanding of DKD pathophysiology. The complement system is a central part of innate immunity, but its dysregulated activation is detrimental and results in systemic diseases with overt inflammation. Growing evidence suggests complement activation in DKD. With existent drugs and clinical success of treating other kidney diseases, complement inhibition has emerged as a potential novel therapy to halt the progression of DKD. This article will review DKD, the complement system's role in diabetic and non-diabetic disease, and the potential benefits of complement targeting therapies especially for DKD patients. Diabetic kidney disease (DKD) is a common microvascular complication of diabetes mellitus. It is a chronic, progressive disease characterized by kidney function decline through hyperfiltration with or without proteinuria not attributable to concomitant kidney diseases. DKD is the leading cause of both chronic and end-stage kidney disease  worldwide. In the United States, it affects 25–43% of diabetic patients and is the primary kidney diagnosis in more than 50% of subjects requiring kidney replacement therapy , 2. Of nThree classes of drugs are approved by the FDA to treat DKD: angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonists, and the sodium-glucose cotransporter 2 inhibitors (SGLT2i). Other therapeutic interventions include the use of glucagon-like peptide-1 receptor agonists, blood pressure and lipid control, dietetic interventions, and physical activity . HoweverThe complement system is the main soluble component of the innate immunity, primarily known to facilitate pathogen clearance by lysis and opsonization of target cells. Complement dysregulation is observed in most autoimmune disorders and is the central pathogenic mechanism of various systemic diseases . As for kidneys, complement activation and deposits are frequently seen in immune-complex mediated glomerular diseases. In the past decade, newly discovered genetic mutations associated with the alternative complement pathway activation have been implicated in several glomerular diseases, alluding to a novel classification of membranoproliferative glomerulonephritis (MPGN) and more importantly, therapeutic options . In partHerein, we will review the available evidence about the pathogenic role of complement activation in DKD progression. Moreover, we will discuss available and experimental drugs able to modulate complement activity to identify novel therapies for DKD.The definition of DKD embraces a variety of pathogenic mechanisms, histological lesions, and clinical manifestations. Generally, DKD is considered a glomerular disease and is classified as a microvascular complication of diabetes. Nevertheless, the whole kidney tissue is involved, and a considerable fraction of patients show significant tubular injury , 10. SinHyperglycemia is thought to play an important role in the pathogenesis of diabetic complications. Glycation is the bonding of glucose molecules to proteins or lipids without enzymatic regulation. It causes the accumulation of misfolded and pro-oxidant macromolecules called advanced glycation end-products (AGEs). As a consequence of hyperglycemia, AGEs accumulate in kidney tissues and cause intracellular oxidative stress and extracellular hyaline deposits. Intracellular AGE accumulation activates the transcription factor NFκB  in bloodThe complement system is constituted by soluble and membrane-bound proteins that respond to alarm signals and, through a proteolytic cascade, generate a plethora of immune effectors. Three main triggers that activate the complement cascade include : the binBesides these well-established functions, complement receptors are extensively expressed by immune and non-immune cells and exert a variety of pathophysiologic effects . Our groThe ability of the complement system to target specific surfaces whilst distinguishing foreign or altered surfaces from the host requires ample regulation as uncontrolled complement activation could lead to disease. Evidence of such complement dysregulation is noted in many kidney disease types and this changed understanding of disease pathogenesis can improve clinical management. For instance, improved understandings of the alternative pathway in complement significantly revised the classification of membranoproliferative glomerulonephritis (MPGN). Based on the positivity of immunoglobulin (Ig) staining, MGPN now are divided into Ig (+) C3 (+)-MPGN such as infection, autoimmune/rheumatological diseases, or monoclonal gammopathy-mediated GN, and Ig (−) but C3 (+)-MPGN including C3 glomerulonephritis (C3GN) and dense-deposit disease (DDD) . EvidencSystemic or locally produced complement activation has also been implicated in the disease pathogenesis of antibody-mediated glomerular diseases. Diseases such as membranous nephropathy and lupus nephritis are characterized by complement deposition which is both a biomarker and direct mediator of disease . In antiin vitro and in vivo (db/db) model  model , suggestEvidence of glycated complement components was noted in DKD patients over three decades ago ; glycateAnimal models of diabetes have illuminated a deeper understanding of the potential role for the complement system in disease pathogenesis [See  for an ePreclinical data have indicated the relevance of the lectin pathway in the pathogenesis of DKD. In the streptozotocin-induced diabetes model, MBL-knockout mice had reduced kidney weight, urinary albumin excretion, and kidney fibrosis when compared to wild-type diabetic animals . Other aWhile these models provide valuable insight for preclinical studies, there are limitations to these animal studies. DKD progression in humans requires many years, whereas many animal studies are limited to weeks or months. Therefore, it is necessary to validate these animal findings in human studies.While complement activity is noted in diabetes, complement activation is particularly evident when kidney disease occurs. In a gene-expression analysis of postmortem kidneys, complement was mostly altered in early DKD when only limited glomerulosclerosis was evident . Also, cMultiple clinical studies reported hyper-activation of the lectin pathway in DKD similar to the animal studies mentioned above. In 191 T1D patients from the Finnish Diabetic Nephropathy Study, MBL levels were 50% higher in those affected by proteinuria and significantly correlated with Hb1ac and kidney glucose disposal rate . In a coDownstream complement products have been associated with DKD activity as well. Urinary excretion of C3b, Bb, and MAC have been noted in DKD patients  and moreComplement activation is a primary pathogenic mechanism in numerous inflammatory disorders and substantial efforts have been made to develop complement-targeting drugs. In March 2007, Eculizumab became the first complement-specific drug being approved by the FDA for the treatment of paroxysmal nocturnal hemoglobinuria and, subsequently, aHUS . NowadayGrowing evidence has unveiled the primary role of complement receptors in DKD. Morigi et al.  showed tWhile animal models of DKD invite promise for the use of C3aR and C5aR inhibitors, the study of these inhibitors in humans is limited. A C5aR inhibitor CCX168) has been branded by ChemoCyntryx as Avacopan and released for experimental human use . In ANCA has beenC1-INH is the recombinant form of human C1 esterase inhibitor and is commercially available as Cinryze (Shire pharmaceutics). C1-INH is a complement regulating protein that halts the classical pathway and regulates the intrinsic coagulation cascade. It is currently FDA-approved for the treatment of hereditary angioedema  . A phaseEculizumab is a murine humanized monoclonal antibody that binds with high-affinity C5 and prevents its cleavage and the generation of C5a and MAC. It is commercialized by Alexion Pharmaceuticals as Soliris. Eculizumab was sequentially approved for the treatment of paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and neuromyelitis optica in adults who are anti-aquaporin-4 antibody positive.Eculizumab has also been used with promising results to treat C3 glomerulopathies , 75 and OMS721 is a monoclonal antibody targeting MBL-associated serine protease-2 (MASP-2), the protease that binds MBL and cleaves C4 and C2. OMS721 is currently under investigation for TMA, aHUS (NCT02222545), and IgA nephropathy (NCT02682407). Given the specificity for the lectin pathway, this agent could represent a valid option for DKD without inducing broad complement inhibition.in vitro that DPP-4 inhibitors block also complement-activating serine proteases and inhibit in particular the lectin pathway. The authors hypothesized that DPP-4 reduce complement injury in DKD and randomly assigned 137 patients to sitagliptin or placebo. Sitagliptin-treated patients had reduced circulating levels of MBL and soluble MAC after 12 weeks of treatment.Dipeptidyl peptidase-4 (DPP-4) is a serine protease that inactivates glucagon-like peptides and is targeted by a widely used anti-diabetic drug class (DPP-4 inhibitors). Hoffman-Petersen and colleagues  demonstrAs previously noted, hyperglycemia can cause glycation of CD59 which normally inhibits MAC complex formation . GlycatiDiabetic kidney disease is a frequent complication of diabetes and the leading cause of chronic kidney disease worldwide. In recent years, new drugs have considerably improved DKD management by targeting glucose metabolism; however, different disease mechanisms may promote DKD progression once the initial injury is established. The complement system is hyper-activated in DKD and has been recognized as the main mechanism of disease progression. In particular, AGEs trigger the lectin pathway, whose activation significantly associates with patient prognosis. Besides MAC generation, the complement cascade triggers kidney injury through complement receptors expressed on kidney cells. Pre-clinical studies demonstrated that complement inhibition prevents DKD progression and complement receptor targeting is a promising therapeutic strategy.KB, SD, and SY wrote the manuscript. PC supervised the manuscript. 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.


It is well established that protein biogenesis is tightly linked to various protein quality control processes, which monitor errors in protein folding, modification, and localization. However, little is known about how cells cope with translocation defective polypeptides that clog translocation channels (translocons) during protein translocation. This review summarizes recent studies, which collectively reveal a set of translocon-associated quality control strategies for eliminating polypeptides stuck in protein-conducting channels in the endoplasmic reticulum and mitochondria.Protein translocation across membranes is a critical facet of protein biogenesis in compartmentalized cells as proteins synthesized in the cytoplasm often need to traverse across lipid bilayers  These pvia an ER-localized SRP receptor. Nascent chains are handed over to the Sec61 translocon, which uses the energy from translating ribosomes to move polypeptides across the membrane. For membrane proteins, the translocation process is coupled to membrane integration of hydrophobic TMDs through a lateral gate of the translocon , which is recognized by signal recognition particle (SRP) in the cytosol. SRP then targets the ribosome-nascent chain complex to the ER membrane anslocon . For a sanslocon .A relatively small number of proteins such as those bearing a suboptimal signal sequence or the so-called tail-anchored (TA) proteins (carrying a single membrane targeting TMD at the carboxyl-terminus) use a posttranslational mechanism for ER targeting . In thisThe biogenesis of membrane and secretory proteins is monitored by various protein quality control (PQC) mechanisms, which safeguard defects in protein folding, assembly, and localization. Deficiencies in ER-associated PQC result in accumulation of malfunctional proteins and trigger ER stress, which contribute to the pathogenesis of many human diseases .via a process termed retrotranslocation, which requires a membrane complex and the associated ubiquitination machinery. Polypeptides are subsequently extracted from the membrane by an abundant ATPase complex named Cdc48 in yeast or p97 in mammals (The best studied ER PQC process is the ER-associated protein degradation (ERAD) pathway . In this mammals . This AT mammals . Genetic mammals , while r mammals .via interactions with SRP and an SRP receptor and reroutes them to an Hrd1-containing retrotranslocon for ubiquitination-mediated degradation  pathway , which aradation .via a mechanism similar to the well-established cytosolic PQC system . IAPP is a signal sequence-containing secretory protein that can clog the translocon by induced oligomerization prior to ER targeting. IAPP oligomerization-associated cytotoxicity in pancreatic β cells is considered a key contributor to type 2 diabetes. Overexpression of an oligomerization-prone IAAP variant in yeast also causes cytotoxicity, which was attributable to a translocation defect because the suppressor function of Ste24 depends on its declogging activity  (Translation of faulty mRNAs bearing no stop codon (NS mRNAs) represents a major trigger of ribosome stalling both in the cytosol and on the ER membrane . NS mRNAy (RIDD) . This prUnlike ribosome stalling in the cytosol, translation arrest during cotranslational translocation generates a unique proteostasis crisis because the arrested products are embedded in the Sec61 translocon: A significant portion of the substrate upstream of the stalling sequence is either in the ER lumen or has been integrated into the ER membrane and thus could have participated in cotranslational events such as protein folding, glycosylation, and disulfide bond formation. Consequently, translation arrests not only produce defective nascent polypeptides, but also generate clogged translocons and a blockade in ER import . Apparenvia the lateral gate, it often needs to wait in the hydrophobic lipid environment for assembly with downstream TMDs via non-hydrophobic interactions. Recent studies have identified the EMC as a potential TMD-specific chaperone  . In thisIn sharp contrast to the well-documented RQC mechanism, the limited information regarding ribosome stalling on mRNAs encoding secretory or membrane proteins fails to paint a consistent model. The aforementioned contradiction may be resolved if cotranslational TAQC mechanisms are substrate-tailored . A conceIn yeast mutants lacking a functional EMC, the biosynthesis of a mutant ABC transporter Yor1ΔF is compromised, which appears to cause ribosome stalling . LakshmiTo study how mammalian cells cope with ribosome stalling during cotranslational translocation, we recently generated an ER stalling reporter containing a polyadenine segment downstream of an ER targeting signal sequence. The reporter also contains a GFP-encoding sequence upstream of the stalling site and an RFP-coding sequence downstream. Translation stalling results in a translocon-associated translation product bearing GFP but no RFP. As expected, this translocon-clogging nascent chain is short-lived, but surprisingly, it is not degraded by the proteasome. Instead, it is transported out of the ER and disposed of by lysosomes. Intriguingly, this process requires the modification of the ribosome subunit RPL26 with a ubiquitin-like molecule named UFM1 (ubiquitin-like modifier 1)  Wang et.Protein modification by UFM1, a process dubbed as UFMylation is a conserved posttranslational modification found in most eukaryotes except in yeast and fungi . In analWe and other recently identified the 60S ribosomal subunit RPL26 (ul24) as the principal target of UFMylation . Biochemin vitro erythropoiesis model, RPL26 UFMylation is upregulated during erythroid differentiation, which coincides with an increase in the secretory flow. Compromising ribosome UFMylation impairs protein secretion, induces ER stress, and ultimately inhibits hemoglobin production .S. cerevisiae, which constantly monitors the TOM machinery under non-stress conditions to prevent channel clogging , which import mitochondrial proteins synthesized in the cytosol . The mitclogging . A key pclogging .via the Tom translocon (Both the MitoTAD and MitoCPR mechanisms appear to cope with polypeptides stalled during posttranslational translocation, which accounts for most translocation events at the mitochondria. However, recent studies also suggest the existence of localized protein translation in proximity to the MOM, which may result in cotranslational protein import into the mitochondria . Accordianslocon . This deanslocon . Intriguanslocon , 2017. Tanslocon .Translocation clogging at the MOM might also induce mitophagy, a form of macroautophagy that selectively removes damaged mitochondria to resolve translocon jamming. Protein translocation across membranes is critical for cell physiology because it is a fundamental protein biogenesis step for clients destined to secretion or the endomembrane system. While the translocation machineries in different organelles have been extensively studied, the quality control mechanisms dedicated to the surveillance of these translocation processes have just begun to surface. Although TAQC is now being characterized in the ER and mitochondria, similar quality control processes may exist in other organelles such as peroxisomes and chloroplasts.As a newly emerged field, there are many outstanding questions to be explored for TAQC. What are the physiological substrates of the each TAQC pathway? Are these TAQC mechanisms regulated by stress or under aging conditions in animals? How do cells sense clogged translocons in different TAQC pathways? What are the fates of the clogged substrates in each pathway? More importantly, as TAQC mechanisms are essential for the normal flux of proteins during biogenesis, the declogging function of TAQC may be particularly important for specialized cells with a high demand for protein translocation. Under these circumstances, failure in TAQC may contribute to the pathogenesis of human diseases. For instance, it would be interesting to investigate whether UFMylation-mediated TAQC plays a role in neuronal development and whether the pleiotropic neurological disorders associated with UFMylation deficiencies are caused by defects in TAQC. In short, future studies along these directions will surely reveal the full biological scope for these new PQC pathways, and also shed important insights on various pathophysiological TAQC conditions that are intimately associated with human diseases.LW and YY conceived the topic and wrote the manuscript together. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Gangliogliomas are predominantly low-grade primary brain tumors comprised of neuronal and glial components that are found in both pediatric and young adult populations. In the majority of cases, surgical resection of these tumors is curative. However, tumor location in eloquent centers of the brain can make surgical intervention inappropriate. Additionally, a subset of tumors progress to anaplastic ganglioglioma which carries a poor prognosis, despite resection. Activating mutations in the MAPK pathway, such as BRAF V600E, have been identified in many of these tumors. Tumors carrying such mutations have demonstrated susceptibility to MEK inhibition therapy. However, there remains a subset of ganglioglioma that do not contain a known mutation in the MAPK pathway and thus have not been targeted with MEK inhibition therapy. Here, we present a young adult ganglioglioma patient without identified MAPK pathway activation mutations who demonstrated a significant and sustained response to MEK inhibition with trametinib. Gangliogliomas are primary brain tumors most commonly arising in children and young adults less than 30 years old but are seen in the older adult population as well. Like pediatric patients, adult ganglioglioma are predominantly low-grade 91.4%) [.4% [1]. In recent years there has been extensive work describing the genetic landscape and potential therapeutic targets in these tumors. The BRAF V600E mutation makes up a significant amount of the genetic alterations found in ganglioglioma, but other MAPK and non-MAPK pathway mutations have been described . PekmezcGiven the prevalence of MAPK activation described in gangliogliomas, MEK inhibitors such as trametinib may provide a significant therapeutic option. MEK inhibition in conjunction with BRAF inhibition has shown promising results in BRAF V600E mutation in adult anaplastic ganglioglioma . AdditioA 32-year-old woman presented with two to three years of progressive right-sided arm greater than leg weakness. Her history consisted of a bifrontal brain mass at the age of 11 years. At that time, she underwent biopsy, which was said to be consistent with germinoma, however, the pathology was not able to be re-reviewed. Adjuvant treatment consisted of craniospinal radiotherapy with resolution of disease without recurrence. At the time of her current presentation, MRI imaging showed an enhancing left thalamic mass . Biopsy Due to the great majority of characterized mutations in ganglioglioma identified as players in the MAPK pathway , we hypoNCT03213691) and other trials . And while there are several studies including targeting the MAPK pathway in brain metastatic melanoma and other tumors, there are currently no primary adult CNS tumor studies evaluating the potential of MEK inhibitors for patients with verified or presumed MAPK pathway activation. This case and the success of MEK inhibition in primary pediatric low-grade gliomas would suggest a potential avenue of new research for adult low-grade and ganglioglioma patients [There are multiple ongoing studies of MEK inhibitors for central nervous system tumors in pediatric patients including the Pediatric MATCH (


Autoimmune encephalitis (AE) is a rapidly progressive inflammatory neurological disease. Underlying autoantibodies can bind to neuronal surfaces and synaptic proteins resulting in psychiatric symptoms, focal neurological signs, autonomic dysfunction and cognitive decline. Early and effective treatment is mandatory to reduce clinical symptoms and to achieve remission. Therapeutic apheresis, involving both plasma exchange (PE) and immunoadsorption (IA), can rapidly remove pathogenic antibodies from the circulation, thus representing an important first-line treatment in AE patients. We here review the most relevant studies regarding therapeutic apheresis in AE, summarizing the outcome for patients and the expanding clinical spectrum of treatment-responsive clinical conditions. For example, patients with slowly progressing cognitive impairment suggesting a neurodegenerative dementia can have underlying autoantibodies and improve with therapeutic apheresis. Findings are encouraging and have led to the first ongoing clinical studies assessing the therapeutic effect of IA in patients with anti-neuronal autoantibodies and the clinical presentation of dementia. Therapeutic apheresis is an established and well tolerated option for first-line therapy in AE and, potentially, other antibody-mediated central nervous system diseases. Autoimmune encephalitis (AE) is a rapidly progressive inflammatory neurological disease with subacute onset. Patients may present with behavioral changes and altered mental status as well as reduced levels of consciousness and new focal neurological signs or epileptic seizures . FurtherAE comprises both, antibody-mediated and paraneoplastic, i.e., cytotoxic T-cell-mediated, encephalitides. Clinical presentation is diverse and depends on the specific underlying antibody . As moreα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors), inhibitory transmitter receptors (GABAB (gamma-aminobutyric acid B), GABAA (gamma-aminobutyric acid A), glycine receptors), ion channel subunits and cell adhesion molecules (Caspr2 (contactin-associated protein 2), IgLON5) or soluble synaptic proteins .The most common and best-known form of antibody-mediated AE is NMDA (N-Methyl-D-Aspartat) receptor (NMDAR) encephalitis, defined by cerebrospinal fluid (CSF) IgG antibodies targeting the NMDA type glutamate receptor. Patients present with subacute onset of psychiatric symptoms, autonomic instability, focal neurological signs and behavioral changes as well as new-onset epileptic seizures and reduced levels of consciousness. Other AE-defining autoantibodies bind directly to excitatory transmitter receptors besides NMDAR  bind to intracellular antigens  and therefore do not cause the neurotoxicity directly; they rather serve as valuable biomarkers for an underlying tumor, often small cell lung cancer and gynecological tumors. The neuronal damage in these cases is, rather, caused by cytotoxic T-cells with oligoclonal T-cell receptor expansion and autoreactivity against neuronal structures. Among the antibodies targeting intracellular antigens, GAD (glutamic acid decarboxylase) and amphiphysin antibodies are an exception as they are not necessarily associated with a tumor and seem to be pathogenically relevant despite their intracellular antigen location .At this point, there is no clear evidence-based treatment standard for AE. Established treatment strategies for first-line therapy of AE include high-dose corticosteroids (three to five days course of 1000 mg intravenous methylprednisolone), intravenous immunoglobulins (IVIG) (2 g/kg body weight over three to five days), as well as therapeutic apheresis. Cyclophosphamide and the CD20-antibody rituximab  might be added in case of persisting or relapsing symptoms and as a long term maintenance therapy. Most centers favor a low threshold for rituximab initiation given its good safety profile and potential effect in preventing relapses. Many other treatments have been used with variable success, including mycophenolate mofetil, methotrexate or azathioprine. It is broadly agreed that immunotherapy needs to be started as early as possible after symptom onset to be most effective. Nevertheless, marked recovery can be seen in some patients with antibody-mediated AE in whom therapy is only started months after disease onset. The choice of adequate therapy depends on the clinical syndrome and the underlying antibody. However, comparative treatment studies in patients with AE are sparse and focus on the most common forms of AE, such as NMDAR encephalitis.In paraneoplastic AE with antibodies targeting intracellular proteins, rituximab, intravenous immunoglobulins and therapeutic apheresis often have only little effect as the antibodies are not directly pathogenic. Here, neuronal damage is caused by cytotoxic T-cells. Evidence of a tumor requires prompt and complete removal in order to withdraw the auto-antigen expressed by the tumor that triggers the production of autoantibodies. Nevertheless, despite advanced immunotherapy and tumor removal, in many cases neuronal damage in paraneoplastic AE progresses.Therapeutic apheresis and the removal of autoantibodies is a major therapeutic option in AE. The pathophysiological binding of antibodies to their antigens can thereby be reduced.To conduct the review, we followed the PRISMA  guidelines and screened the articles independently for their respective eligibility .We included all articles about patients with autoimmune encephalitis—antibody-mediated as well as paraneoplastic—treated with plasma exchange or immunoadsorption. Treatment regimen, such as concomitant immunotherapy, as well as details about the apheresis itself (plasma exchange (PE) or immunoadsorption (IA), number of courses) had to be specified in the article. Further, outcome measures, such as the modified Rankin Scale (mRS) or structured neuropsychological assessment had to be provided. The mRS ranges from zero (no symptoms) to six (death from the disease), and a change of ±1 mRS point is considered as clinically significant improvement or deterioration. Cut-off for independent living is at ≤2 mRS points.The following strategy was used to find previous literature and trials : MEDLINETherapeutic apheresis is an important treatment option in a range of inflammatory central nervous system diseases . It has Therapeutic apheresis has been shown to be safe and effective leading to measurable laboratory and clinical improvement in several inflammatory diseases of the central and peripheral nervous system, including myasthenia gravis, Guillain-Barré syndrome and multiple sclerosis. Apheresis is recommended by the German Society of Neurology as escalation treatment of severe courses of AE. Patients should be treated with apheresis at least five times every other day. In cases with predominant CSF antibodies seven to ten treatment courses are usually needed for relevant reduction of CSF antibody titers. Before receiving therapeutic apheresis, patients mostly show either severe clinical symptoms on hospital admission or an insufficient response to therapy with high-dose cortisone or IVIG.Much has been learned from acquired myasthenia gravis, which represents a “model disease” for the much later discovered forms of autoantibody-mediated AE. It could first be demonstrated that removal of the disease-defining acetylcholine receptor antibodies using plasma-exchange led to marked symptom improvement . AntibodTherapeutic apheresis has been shown to improve clinical symptoms in different antibody-mediated diseases. According to the American Society for apheresis (ASFA) guidelines PE and IA are strongly recommended for different antibody-mediated encephalitis forms ranging from low to moderate evidence. In contrast, the therapeutic role of apheresis is not yet established for paraneoplastic neurological syndromes and individual decision-making is necessary . In NMDATherapeutic apheresis offers two different procedures. On the one hand is plasma exchange (PE), where a defined plasma volume is removed and replaced by human albumin or fresh frozen plasma. On the other hand is immunoadsorption (IA), a procedure that more specifically removes immunoglobulins and immune complexes by passing the plasma over an adsorber column, allowing reinfusion of the patients’ own plasma. Two different IA procedures were used in the reviewed articles: either a regenerative double column system or a disposable tryptophan column. Tolerability and therapeutic effects do not show relevant differences between PE and IA in recent studies ,19,26. RUsually a minimum of five sessions of apheresis is performed. When patients show a CSF predominant antibody, more sessions are generally needed in order to eliminate the antibody in the central nervous system. Most studies included in this review describe a central venous catheter in an internal jugular vein as vascular access. Only Hempel et al. use a peripheral vein to perform IA and in order to treat patients as outpatients. However, they report significant patient drop-out due to a failure of repeatedly accessing the vein .The treated plasma volumes can be calculated using Sprenger’s formula . DependiThe specific mechanism of antibody removal has been shown to be a more beneficial treatment of NMDAR encephalitis than intravenous methylprednisolone alone. In a retrospective study 2/14 patients showed significant clinical improvement following steroids, whereas 9/14 patients who received additional PE improved in the mRS during the third and fifth cycle of apheresis . This isMany patients with AE treated with apheresis receive prior treatment with high-dose steroids or IVIG. The decision for treatment with apheresis is often only made after unsuccessful or incomplete recovery after these other therapies. It has been shown that both patients who did and patients who did not receive prior treatment benefitted from apheresis . In all In the study by Onugoren et al. all patients except one out of 19 were treated with high-dose prednisolone (median dose 4.9 g) in parallel to IA . It is rApheresis in patients with AE is most established in the acute phase of the disease. However, repeated apheresis might also be applied in refractory disease with clinical signs of AE and constant detection of high antibody titers. Yet in one study, repeated IA after 4.5 months (median) in six patients with antibodies against NMDAR, LGI1, Caspr2 and GAD that responded insufficiently to a first series of IA, did not show any further clinical improvement measured by mRS .In case of an underlying malignancy, tumor removal is essential for improving further disease course.A better outcome in patients with NMDAR encephalitis was strongly associated with an early start of immunotherapy (less than 40 days after symptom onset) . ResponsAccording to a prospective study, symptoms that responded best to apheresis include apathy, aphasia, stupor, sleep disorders, agitation, myoclonus and dystonia, sensory neuropathy, apraxia and seizures . In anotTreatment efficacy is more pronounced in patients with antibodies against cell surface antibodies  or in patients with intracellular synaptic antibodies (GAD), whereas no positive treatment effect was observed in patients with paraneoplastic intracellular antigens (anti-Hu) ,22. MarkMarked and rapid effects of apheresis can be seen in patients with epileptic seizures concerning seizure frequency. This was seen for patients with LGI1 and Caspr2 antibodies, where five out of seven patients became seizure free immediately after initiation of therapy with IA . This trSeveral case reports point to the efficacy of PE in drug-resistant status epilepticus caused by AE in children and adults. Both convulsive and non-convulsive status have been described as being responsive to apheresis. In patients with abnormal electroencephalogram (EEG) prior to apheresis, EEG normalization was observed after 5 cycles of PE. EEG improvement correlated with decrease in antibody titers ,32.Treatment of severe AE complicated by status epilepticus or autonomic instability might need to take place on an intensive care unit. On the ICU, benefit from immunotherapy, including apheresis, strongly depends on medical complications associated with a prolonged ICU stay .In patients with predominantly psychiatric symptoms related to treatable autoimmunity, corticosteroids are often hesitantly used given the potential side effect of steroid-induced psychosis. Overall, notable neuropsychiatric side effects can occur in up to 6% of patients who receive steroids ; howeverIn paraneoplastic AE with antibodies targeting intracellular proteins, therapeutic effects of rituximab, IVIG and therapeutic apheresis are usually limited as the antibodies are not directly pathogenic, but neuronal damage is caused by cytotoxic T-cells ,26. FurtAfter therapeutic apheresis with both PE and IA, a transient spurious intrathecal immunoglobulin synthesis of all three subclasses  can be observed. The transient intrathecal Ig fractions and increased IgG index are due to dropped serum IgG levels following apheresis. This “intrathecal pseudo-synthesis” regularly occurs in the first two days after apheresis in a majority of patients . Thus, oClinical indications for therapeutic apheresis in AE might expand to less recognized antibody-mediated conditions in the near future. We could recently demonstrate that asymptomatic mothers of a child requiring psychiatric in-patient diagnostics carried low-level pathogenic human NR1 antibodies more frequently than control mothers having a healthy child . To bettThe use of therapeutic apheresis has been shown to be safe in pregnant women, both for mother and fetus. A recent Italian study evaluated the use of apheresis during pregnancy. Among 48 pregnant women receiving apheresis one had suspected autoimmune encephalitis. Adverse events occurred in 2.1% of all patients analysed, which is reported to be lower than the Italian average. Peripheral veins are preferred as a vascular access during pregnancy to avoid the risks associated with a central venous catheter .Children with autoimmune disorders such as antibody-mediated AE can also be treated with therapeutic apheresis. PE was retrospectively evaluated in 22 children. All children had been treated with IVIG and/or steroids before PE. Each patient received a median number of six PE sessions. No PE-related mortality was observed and adverse events occurred in 2.2%, which is the expected average. Adverse events consisted of hypotension and urticaria. In total, three pediatric patients with antibody-mediated encephalitis were treated with PE, two patients improved and one patient showed partial recovery with persistent neurological deficits after three-year follow-up. One patient with paraneoplastic encephalitis did not benefit from PE and was lost to follow-up . AnotherCompared to ‘classic’ AE with subacute onset of neuropsychiatric as well as behavioral symptoms, antibody-associated dementias are a more slowly progressing group of diseases where decline in working and short-term memory as well as visuo-spatial deficits are the most prominent features. Detection of high-level autoantibodies in patients with dementia is rare, but autoimmune dementias represent a form of cognitive decline that is potentially treatable. A Mayo clinic study reported improvement of cognition in 64% of patients with suspected autoimmune dementia after immunomodulatory treatment . An undeIn a retrospective study analyzing 286 CSF and serum samples of patients with different dementia forms, 16% of the serum samples had NMDAR IgA, IgM or IgG antibodies compared to 4.3% in a healthy control group . BesidesClinicalTrials.gov Identifier: NCT03132272) investigates the effects of IA in patients with Alzheimer’s disease positive for agAAB. The group aims to demonstrate discontinuation of the vascular remodeling and slowing of cognitive decline following IA treatment.Another recent target in dementia patients are autoantibodies against G protein-coupled receptors. In a small trial analyzing the effect of IA in patients with mild to moderate dementia and agonistic autoantibodies (agAAB) against α-adrenergic receptors, treatment with four cycles of IA not only caused disappearance of autoantibodies, but resulted in stabilization of the cognitive and mental condition during the follow-up period of 12–18 months . AnotherApheresis not only has an acute effect on disease activity but can be used for longer lasting immunomodulation. In case of uncertainty of the immunological findings, first-line therapy including steroids or apheresis may serve as a diagnostic test to support the autoimmune etiology . In our Based on the new developments in this field, the ongoing identification of dementia-associated autoantibodies and the concern about overlooking treatable etiologies, we now offer diagnostic antibody testing in serum and CSF to every patient with suspected dementia in our memory clinic at the department of neurology at Charité. In this way we increasingly identify patients with a working diagnosis of Alzheimer’s, frontotemporal dementia or atypical dementia who have new or established autoantibodies against neuronal and glial proteins, as exemplarily shown in the case vignette of an 81-year-old gentleman . Box 1An 81-year-old dementia patient presented to our outpatient memory clinic at the department of Neurology at Charité—Universitätsmedizin Berlin with deficits in working and short term memory as well as difficulties in concentration. Symptoms began nine months prior to presentation with increasing loss of orientation for place and time, confusion and reported visual hallucinations. Brain MRI at symptom onset was unremarkable apart from microangiopathic lesions in the left temporoparietal lobe A. Basic In our center, the patient showed a persisting dysexecutive syndrome with amnestic and visuo-constructive deficits and apraxia. No other neurological deficits were observed. Montreal cognitive assessment showed mild cognitive impairment with a score of 21 out of 30. A second CSF analysis revealed normal cell count, but still increased protein of 1288 mg/L . No infectious cause was found. Extensive search for anti-neuronal autoantibodies in serum and CSF including indirect immunofluorescence staining on rodent brain sections detected neurochondrin IgG antibodies in the CSF B.The diagnosis of AE with oligosymptomatic memory deficits was established. A three-day course of intravenous methylprednisolone with 1 g/day led to improved gait, but no impact on cognition was observed. Because of the persisting memory deficits, two months later five sessions of IA were administered every other day and resulted in reduction of CSF neurochondrin antibody titers C. The paThe patient’s autoantibodies targeted neurochondrin, a leucine-rich protein expressed not only in the brain, but also in bones and cartilage. As neurochondrin is located intracellularly, the pathogenicity of neurochondrin antibodies is unclear and they might only be a biomarker of autoimmunity including T-cell-mediated neurotoxicity . NeurochDetection of antibodies against neurochondrin  in this The patient participates in an ongoing clinical trial (DRKS00016017) analyzing the role of anti-neuronal and anti-glial surface antibodies in cognitive disorders and potential improvement following IA. The study aims to identify dementia patients who harbor autoantibodies against structures of the central nervous system using cell-based assays for detection of established autoantibodies as well as screening assays using indirect immunofluorescence on unfixed murine brain sections. Autoantibody-positive patients with cognitive decline can enroll in the study and receive therapeutic apheresis . TreatmePredictors for beneficial outcome after treatment with apheresis in patients with AE include start of the treatment early in the disease before substantial irreversible brain damage has occurred. Nevertheless, after longer periods from symptom onset to therapy initiation, apheresis can also result in symptom improvement. Immunotherapy with steroids or IVIG prior to apheresis does not seem to have an effect on the overall outcome. Especially in patients with severe disease courses apheresis is a major treatment option and should be initiated early, possibly together with other immune therapies.For selected patient groups such as children and pregnant women as well as patients requiring ICU treatment, the safety and efficacy of apheresis could also be shown.According to all studies reviewed, the ASFA guidelines and the recommendations given by the German Society of Neurology, no benefit of apheresis in patients with onco-neuronal antibodies could be shown. Here, results are inconsistent, with most patients showing no therapeutic effect after apheresis, or even further deterioration, but in single patients clinical improvement could sometimes be seen. A clear treatment response to apheresis, both, PE and IA, is established in patients with antibodies against surface antigens or synaptic antigens. In the articles screened for this review, there was no difference in outcome of patients treated with PE or IA. Therapeutic apheresis is a valuable option within the complex multimodal immune therapy of AE. The benefit of treating patients with antibody-mediated AE with apheresis by far outweighs the possible side effects that were not severe and were mainly associated with the central venous catheter. Furthermore treatment with apheresis may be complicated by the necessity of an ICU setting and poor patient cooperation.Although predominant humoral autoimmunity seems to be rare in dementia patients and requires further study, the search for autoantibodies in these patients allows the detection of potentially treatable dementia forms and holds the potential to prevent further cognitive decline in selected patients. Thus, therapeutic apheresis is not only an important first-line therapy in patients with AE but may be increasingly considered in further patients who are positive for autoantibodies against neuronal structures. This already includes patients with cognitive decline but may in the future expand to novel clinical indications ranging from antibody-associated psychosis to autoantibody-positive pregnant women.


Protein supplementation improves physiological adaptations to endurance training, but the impact on adaptive changes in the skeletal muscle transcriptome remains elusive. The present analysis was executed to determine the impact of protein supplementation on changes in the skeletal muscle transcriptome following 5-weeks of endurance training.vastus lateralis were taken before and after 5-weeks of endurance training to assess changes in the skeletal muscle transcriptome. One hundred and 63 genes were differentially expressed after 5-weeks of endurance training in both groups . In addition, the number of genes differentially expressed was higher in the protein group (PRO)  when compared with the control group (CON) , with no time-by-treatment interaction effect . Endurance training primarily affected expression levels of genes related to extracellular matrix and these changes tended to be greater in PRO than in CON.Skeletal muscle tissue samples from the clinicaltrials.gov as NCT03462381, March 12, 2018.Protein supplementation subtly impacts endurance training-induced changes in the skeletal muscle transcriptome. In addition, our transcriptomic analysis revealed that the extracellular matrix may be an important factor for skeletal muscle adaptation in response to endurance training. This trial was registered at clinicaltrials.gov as NCT03462381.This trial was registered at  Skeletal muscle is an extraordinary malleable tissue which is demonstrated by its rapid remodeling and adaptation to exercise training , 2. RepeProtein feeding following endurance exercise has shown to affect mRNA-specific pathways involved in extracellular matrix, myogenesis, immunogenic response, and energy metabolism , suggestn = 21 vs PRO: n = 19). Baseline characteristics were not different between groups and can be found in Table In total, four subjects dropped out during the conduction of the study for various reasons. Analysis was executed on the 40 subjects who completed the 5-weeks training program . Table Figure Based on all significant genes altered  in each group, gene ontology biological processes revealed extracellular matrix organization as the process with the highest change in gene expression profile in both the CON group and the PRO group Table . In the We have recently demonstrated that protein supplementation enhances physiological adaptations to endurance training. The greater physiological adaptations elicited by protein supplementation were mainly observed during the first 5 weeks of training of a 10 week endurance training intervention . LikewisFive weeks of endurance training increased maximal aerobic capacity. Adding protein supplementation elicited a greater increase in maximal aerobic capacity and stimulated lean mass gain. For a more detailed discussion on the changes in physiological outcome measures the reader is referred to our recently published paper . At the 2max and stimulation of lean mass accretion).In this study we demonstrated that the physiological adaptive response to endurance training was accompanied by significant changes in the skeletal muscle transcriptome. Gene set enrichment analysis showed that endurance training caused significant changes in gene expression transcripts involved in extracellular matrix, which is in line with previous reports that have investigated changes in skeletal muscle transcriptome following prolonged endurance training , 14. SevThe extracellular matrix is composed of collagen, glycoproteins and proteoglycans . MoreoveOur observation that protein supplementation may increases extracellular matrix remodeling to endurance training is new and further elaborates on previous work, which demonstrates that addition of protein to post-exercise carbohydrate-lipid nutrition differentially alters the transcriptome involved in tissue structure and remodeling through regulation of extracellular matrix . GeneralIn contrast to the observed effect of protein supplementation on physiological adaptation, we were unable to find a clear additional effect of protein supplementation on the skeletal muscle transcriptome besides the extracellular matrix. It is possible that the effects of protein supplementation already started to manifest during the early hours of recovery from exercise, when mRNA abundance generally peaks , 28. AltThus far, much attention has been given to the acute molecular responses to a single bout of exercise, and the current theory suggests that acute signals predict/drive phenotypic adaptation over time. For example, the AMP-activated protein kinase and peroxisome proliferator-activated receptor-y coactivator-1ɑ, have been proposed as primary regulators of muscle tissue adaptation in response to endurance training –32. Whetclinicaltrials.gov as NCT03462381 and adheres to CONSORT guidelines for clinical trials. A detailed description of subject participation, experimental design, endurance training program, supplemental strategy, whole-body physiological outcome measures can be found in our previous publication [The investigation was approved by the Medical Ethical Committee of Wageningen University, in accordance with the Declaration of Helsinki. This trial was registered at lication . A schemBaseline (week 0) and post-intervention fasted muscle biopsies were taken as described by Bergstrom (1974) , and thelimma R library [P-values were calculated using Intensity Based Moderated t-tests (IBMT) [p-value < 0.05. Unadjusted p-values below 0.01 for the contrasts were considered statistically significant within the genes that passed the F-test. Gene set enrichment analysis was done using pre-ranked lists ranked by the t-values from the limma contrasts [Statistical analysis of gene expression changes was performed using  library . Contrass (IBMT) . Signifiontrasts , 37. We ontrasts . An adjuontrasts  and Grapontrasts , 40. A dontrasts .


As the global COVID-19 pandemic continues, unabated and clinical trials demonstrate limited effective pharmaceutical interventions, there is a pressing need to accelerate treatment evaluations. Among options for accelerated development is the evaluation of drug combinations in the absence of prior monotherapy data. This approach is appealing for a number of reasons. First, combining two or more drugs with related or complementary therapeutic effects permits a multipronged approach addressing the variable pathways of the disease. Second, if an individual component of a combination offers a therapeutic effect, then in the absence of antagonism, a trial of combination therapy should still detect individual efficacy. Third, this strategy is time saving. Rather than taking a stepwise approach to evaluating monotherapies, this strategy begins with testing all relevant therapeutic options. Finally, given the severity of the current pandemic and the absence of treatment options, the likelihood of detecting a treatment effect with combination therapy maintains scientific enthusiasm for evaluating repurposed treatments. Antiviral combination selection can be facilitated by insights regarding SARS-CoV-2 pathophysiology and cell cycle dynamics, supported by infectious disease and clinical pharmacology expert advice. We describe a clinical evaluation strategy using adaptive combination platform trials to rapidly test combination therapies to treat COVID-19. Each category is complex, and of the armament of potential repurposed drugs for COVID-19, multitudes of mechanisms and pathways are in consideration. Selectively combining agents to complement each other by variable mechanisms of action across these categories may yield effective treatments for select phases of SARS-CoV-2 infection. Analogous to the evolution of antiretroviral therapies for HIV infection, it is important to formulate and test combination therapy regimens. Instead of sequentially testing monotherapies that will likely have modest clinical effects on their own, additive or synergistic effects can potentially be gained by combining antiviral drugs exploiting pharmacology throughout the spectrum of COVID-19 illness.4To illustrate, a recent large-scale compound repurposing effort identified more than 20 antivirals that should be further investigated for application for COVID-19.1 These trials cover all antiviral mechanisms noted earlier and applications from prophylaxis to treatment to reduction in the sequelae of the host inflammatory response. However, to date, only dexamethasone and remdesivir have been identified as effective therapies for severely ill patients.6 This low success rate might be due to the fact that the majority of COVID-19 clinical trials (87%) are evaluating repurposed drugs as monotherapy.1There are more than 750 clinical trials evaluating repurposed therapies for COVID-19.7 In comparison to the control group that received LPV/r monotherapy, the triple combination arm showed faster viral clearance and alleviation of symptoms, and shorter hospital stays.7 Although LPV/r monotherapy was no better than placebo alone in another hospitalized trial (RECOVERY),8 it is unclear whether both interferon beta-1b and ribavirin or triple combination therapy drove clinical benefits for hospitalized patients.Combination therapies for COVID-19 represent an attractive approach to drug development. Among the first trials published of effective interventions for COVID-19 was a combination of interferon beta-1b, lopinavir–ritonavir (LPV/r), and ribavirin for hospitalized patients.9Under normal circumstances, development of combination treatment strategies entails a stepwise evaluation process whereby first the individual components of a potential combination regimen are tested for clinical efficacy in isolation or as individual arms within a trial evaluating both single and combination regimens. This strategy allows sequential compilation of evidence for each drug before studying combinations, as outlined by the U.S. Food and Drug Administration in describing the so-called combination rule.This process is based on reducing exposure to ineffective or toxic drugs among participants until individual components have demonstrated treatment effects. Although this process is both scientifically rigorous and safe, it is slow, and may delay identification of unexpected synergistic effects, resulting in failure to evaluate potentially potent treatment cocktails.As the global pandemic continues, unabated and therapeutic options evaluated to date demonstrate limited effectiveness, and there is a pressing need to accelerate treatment evaluations. Among the options for accelerated development is the evaluation of combination strategies in the absence of prior monotherapy data. This approach is appealing for a number of reasons. First, combining two or more drugs with complementary antiviral or therapeutic effects permits a multipronged approach addressing the variable pathways of the disease. Second, if an individual component of a combination strategy offers a therapeutic effect, then the clinical trial should still detect treatment effects unless antagonism between components is present. Third, this strategy saves time, as it accelerates evaluation of prioritized combination regimens much earlier than would occur with a stepwise approach evaluating monotherapies before combinations. Finally, given the severity of the current pandemic, and the current absence of verified treatment options, the likelihood of detecting a treatment effect with combination therapy maintains scientific and patient enthusiasm for repurposed treatments.1 only 258 (13.1%) trials are evaluating combination therapies. Of these, 120 trials are taking a stepwise approach, and 138 trials are evaluating combination strategies without associated individual monotherapies. Determining whether these trials are implementing a forward  or backward stepwise approach is not possible from clinical trial registries.Despite these advantages, there has been limited research activity investigating combination therapy for COVID-19, in clear contrast to other disease areas. Among the 1971 registered clinical trials evaluating all therapeutic interventions  for COVID-19,11 Although this concept is not new, it has historically been controversial and limited to industry-run clinical trials, especially in oncology. In adaptive clinical trial designs, prespecified modifications are permitted with decision rules based on accumulated interim data.12 Adaptive designs have been controversial because interim data are often assessed multiple times, leading to a fear of inflated type I error rates. However, statistical measures can be implemented to control the type I error rate at the usual 5%. Important examples of adaptive platform trials directed toward COVID-19 include the RECOVERY,5 SOLIDARITY,13 and REMAP-CAP14 trials.Among the many scientific lessons of the COVID-19 pandemic has been the widespread embrace of adaptive clinical trials, particularly platform trials in which multiple interventions are compared simultaneously against a common control arm, and in which interventions may be added over time.Adaptive platform trials provide capacity in combination trials to add or remove ineffective treatment arms, allowing for sequential comparisons and a perpetual design wherein treatments can be prioritized or de-prioritized as evidence accumulates. We propose a platform trial to evaluate combination therapies in the absence of monotherapy evaluations . If the 16 Thus, combination strategies may miss efficacy of individual components undermined by antagonism. Furthermore, drug interactions may result in adverse events and toxicity not observed with monotherapy. Therefore, it is critical to engage clinical pharmacologists and infectious disease experts in guiding the selection and refinement of regimens to ensure that insights on SARS-CoV-2 pathophysiology, cell cycle dynamics, mechanisms of action, drug–drug interactions, safety signals, and clinical utility are appropriately incorporated in the selection of combinations for study.Our proposed approach is not without limitations. Evaluating combinations assumes that individual components are nonantagonistic and likely either additive or synergistic. There are important examples of drug therapies that exhibit antagonism.Ultimately, we argue that the best approach to quickly establish efficacy of potential therapies for COVID-19 is through studying combination therapies, ideally with demonstrated in vitro and in vivo activities and strong preclinical properties, in an adaptive framework to maximize the probability of clinical efficacy. This strategy challenges the traditional drug development dogma, but we believe that it will accelerate the development of repurposed drugs as combination therapies to treat COVID-19.


Glioblastoma (GBM) remains the most devastating primary central nervous system malignancy with a median survival of around 15 months. The past decades of research have not yielded significant advancements in the treatment of GBM. In that same time, a novel class of molecules, long non-coding RNAs (lncRNAs), has been found to play a multitude of roles in cancer and normal biology. The increased accessibility of next generation sequencing technologies and the advent of lncRNA-specific microarrays have facilitated the study of lncRNA etiology. Molecular and computational methods can be applied to predict lncRNA function. LncRNAs can serve as molecular decoys, scaffolds, super-enhancers, or repressors. These molecules can serve as phenotypic switches for GBM cells at the expression and/or epigenetic levels. LncRNAs can affect stemness/differentiation, proliferation, invasion, survival, DNA damage response, and chromatin dynamics. Aberrant expression of these transcripts may facilitate therapy resistance, leading to tumor recurrence. LncRNAs could serve as novel theragnostic or prognostic biomarkers in GBM and other cancers. RNA-based therapeutics may also be employed to target lncRNAs as a novel route of treatment for primary or recurrent GBM. In this review, we explore the roles of lncRNAs in GBM pathophysiology and posit their novel therapeutic potential for GBM. The vast majority of the human genome is transcribed, however, less than 3% of all transcribed genes are protein-coding. For decades, speculation over the roles of non-coding RNA (ncRNA) has ranged from junk transcripts to master epigenetic regulators. The majority of prior ncRNA research has emphasized the roles of small ncRNAs such as micro RNAs (miRNA). A particular class of ncRNAs, long non-coding RNAs (lncRNAs), have been increasingly recognized as playing large regulatory roles in normal function and in disease etiology ,2,3,4. LGliomas are tumors originating from glial cells in the central nervous system (CNS). The most aggressive form of gliomas is glioblastoma (GBM), the most common primary CNS malignancy in adults. GBMs can either be primary or arise from low grade glioma (LGG) with drastic differences in etiology dependent on primary or secondary malignancy. Secondary GBM tend to be IDH1 mutants with or without 1p/19q co-deletion . PrimaryLncRNAs are characterized primarily by length (>200 nucleotides) and lack of coding potential. In some cases, however, lncRNAs can contain short open reading frames for small peptides . LncRNAsThe diversity of lncRNAs in form lead to a vast variety of molecular functional roles in normal and aberrant biology. Owing to the size of the non-coding transcriptome, a complex regulatory system governing lncRNA expression has evolved. Developmental regulation of lncRNAs mean that lncRNA expression is cell-specific and temporally regulated. Tissue-specific expression of lncRNAs can contribute to the maintenance of differentiation and phenotype. Many EZH2-associated lncRNAs have been shown to have tissue-specific binding potential suggesting a tissue-specific role for lncRNAs in chromatin remodeling and cellular imprinting . This caThe versatility and persistence of lncRNAs lead to dramatic phenotypic control over cells. When applied in the context of malignancy, lncRNAs can begin to appear as master regulators of tumorigenic potential. Many lncRNAs have regulatory potential encompassing multiple intracellular or phenotypic processes. LncRNA activity can be seen across the spectrum of canonical cancer hallmarks . HoweverSustained proliferation and avoidance of growth suppressors is one of the key hallmarks of malignant cells. The lncRNA CASC11 has been shown to promote proliferation in bladder cancer through binding of miR-150 . The lncCancer cells frequently develop immune evasion mechanisms or even hijack normal immune cells. Tumors can induce pro-tumor inflammatory responses. LncRNAs have been shown to play a role in cancer cell programming related to immunity. The lncRNA SATB2-AS1 is normally expressed in colorectal tissues and is downregulated in CRC leading to an inverse relationship between expression and prognosis. SATB2-AS1 mediates H3 lysine 4 tri-methylation (H3K4me3) deposition and promoter methylation of SATB2, regulating TH1-type chemokine expression and immune cell density . A novelMany cancers possess metastatic potential, typically developed in a subpopulation of perivascular cells. Metastatic cells must migrate into the blood stream of lymphatic system, exit, and establish a new tumor at a distal site. There is evidence that lncRNAs may confer metastatic potential in these highly specialized cells. The novel lncRNA XLOC_006390 has been shown to facilitate tumorigenesis and metastasis in cervical cancer cells by serving as molecular sponges for miR-331-30 and miR-338-3p . LncRNA Cancer cells characteristically undergo uncontrolled cell division. A hallmark of many cancer cells is their ability to overcome replicative senescence and acquire replicative immortality via enhanced stability of chromosomes often through telomere lengthening . The priRelated to but separate from metastasis, cancer cell migration and stromal invasion have major implications in the progression and treatment of disease. GBM for example, is considered a whole organ disease as tumor cells are highly invasive/migratory and yet, GBM metastasis outside of the CNS is exceedingly rare . Highly Solid tumors have increased need for gas and nutrient exchange owing to their rapid growth. Tumor-associated blood vessels are also important in metastasis. Cancer cells frequently have the capacity to promote angiogenesis or can even differentiate into endothelial cells to form new vessels . A novelCancer cells classically acquire very high mutation loads owing to their numerous cellular divisions and to genomic instability. This instability can lead to the deletion of tumor suppressors or the amplification of oncogenes. One example of this is PTEN loss in GBM. LncRNA transcripts are emerging as regulators of genomic instability. We have already mentioned how the lncRNA TERRA is associated with telomeres and replicative immortality, but through its roles in chromosome binding, TERRA also plays a role in genomic stability . AnotherA hallmark of cancer cells related to replicative immortality, is their ability to avoid regulated, programmed cell death mechanisms. Several lncRNAs have been associated with this hallmark both positively and negatively regulating apoptosis. The well-characterized HOTAIR lncRNA has been found to suppress apoptosis in breast cancer cells through the miR-20a-5p/HMGA2 axis . SimilarAberrant metabolism in cancer cells has long been an attractive therapeutic target. Cancer cells frequently undergo the Warburg effect which describes increased energy production through aerobic glycolysis . LINC005Cancer stem cells (CSCs) are believed to be responsible for tumorigenesis and therapy resistance. CSCs have the capacity for self-renewal, multipotency, and survival. The replication and differentiation of CSCs create a hierarchical cell model within tumors, contributing to their malignancy and resilience. Niches within the tumor microenvironment (TME) are thought to support CSCs, but it has been discovered that lncRNAs contribute to the maintenance of stemness and of cellular fate programs. A usual suspect, MALAT1 has been shown to promote stemness in gastric cancer cells by enhancing SOX2 mRNA stability . In a poThe capacity of cancer cells to overexpress DNA repair machinery is an essential component of their resistance to conventional therapies including ionizing radiation or alkylating chemotherapy. Some lncRNAs regulate DNA damage response (DDR) transcriptional programs. NEAT1 induces expression of multiple DDR pathways including homologous recombination (HR) in multiple myeloma (MM) . NEAT1 aTumors, especially highly infiltrative tumors like gliomas, often encompass normal, non-malignant cells in their mass. Cancer cells are often able to co-opt normal cells to support their TME and facilitate growth/spread. Immune cells, particularly TAMs, can not only contribute to immune evasion, but also to growth. HISLA is released in extracellular vesicles from TAMs resulting in enhanced aerobic glycolysis and apoptotic resistance in nearby breast cancer cells . CarcinoThe roles of lncRNAs in glioma etiology is an emerging field. Many of the hallmark characteristics of glioma tumors have been found to be regulated by lncRNAs. The lncRNA regulatory story in glioma starts with tumor initiation and progression. A set of p53-regulated lncRNAs initially identified in colon cancer, were found to be expressed inversely with glioma tumor grade with lowest expression in GBM samples . These lThe roles of lncRNAs and their mechanisms of action are diverse. Many lncRNAs act on pathways in GBM via regulation of miRNAs either to promote or suppress glioma progression. LINC01446 promotes disease progression via the miR-489-3p/TPT1 axis . SimilarOne of the primary obstacles in the treatment of glioma are the extent of intra and intertumoral heterogeneity. LncRNA expression has been found to be dynamic across multiple single cells in GBM tumors and cell lines ,114. TheGliomas and GBM are highly infiltrative of normal stroma and are considered a whole organ disease. The invasive and migratory potential of glioma cells contributes to the difficulties in treating this disease and the eventual recurrence of these tumors. Glioma invasion is promoted in a HIF1α-dependent manner through the expression of lncRNAs H19 and AWPPH ,116. MigAnother hallmark of glioma and GBM is acquired resistance to standard therapies. The canonical mechanism of TMZ resistance in GBM is the expression and activity of DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). Lnc-TALC promotes MGMT expression by positively regulating the c-Met pathway . TreatmeMuch research goes into the discovery and classification of ever elusive disease biomarkers. Biomarkers come in three not always exclusive flavors: diagnostic, prognostic, and theragnostic. Diagnostic biomarkers are useful in distinguishing between normal tissue and cancer. Prognostic indicators exhibit a correlation of expression with prognosis/disease progression independent of therapy. Theragnostic biomarkers have detectable changes in expression which are predictive of therapeutic response. Cancer-related lncRNAs are sometimes detectable in serum and have also been found in circulating exosomes . Serum lThe multitude of functions of lncRNAs makes these transcripts attractive potential therapeutic targets as they are often low in abundance yet have broad and significant phenotypic effects. LncRNAs are versatile and prolific transcripts in normal biological activity as well as in aberrant disease states. They can exist in many forms with a wide variety of functional capacities. Expression is regulated in a cell-specific manner, as well as spatially and temporally. Despite their low abundance levels, lncRNAs can have major impacts on cellular programs and phenotypes. LncRNAs exhibit roles in all of the canonical hallmarks of cancer with potentials for conserved, pan-cancer activities that may be targetable. These transcripts are particularly expressed in the CNS environment and naturally play a multitude of roles in the most common primary brain malignancy, GBM. GBM remains an intractable, incurable disease for which standard therapies have little effect. These molecules could represent a novel class of biomarkers in GBM or even attractive therapeutic targets. A number of lncRNAs are associated with tumorigeneses, stemness, invasion, and therapy resistance in glioma. We have just begun to scratch the surface of the wide variety of roles of lncRNAs in glioma pathology, particularly in therapy resistance. Further exploration of lncRNAs in glioma may revolutionize our understanding of the disease and may result in novel therapeutic advances in what has up until now been a losing battle.


Rusophycus with an oval outline that possesses a distinctly three lobed axis with an elevated central ridge and regularly spaced transverse furrows that convey the appearance of discrete body segments. The anterior part is the most irregular region of the structure, and it is not always recorded. Morphotype 2 displays more elongated, tubular morphology. Careful observation, however, reveals that it comprises up to three successive morphotype 1 specimens organised in a linear fashion and partially truncating each other. Trilobate morphology and effaced transverse furrows are locally evident, but the predominant morphological feature is the continuous, elevated ridge. The detailed morphology of morphotype 1 and well-preserved, discrete segments of morphotype 2 closely resemble the dorsal exoskeleton of the enigmatic late Carboniferous euarthropod Camptophyllia, suggesting the possible affinities of the producer. Comparisons with patterns of Devonian phacopid trilobite exuviation suggest that the Naranco Formation trace fossils may have been produced by the infaunal activities of an euarthropod that anchored its dorsal exoskeleton in the firm sediment during the body inversion moult procedure. Our findings expand the phylogenetic and environmental occurrence of infaunal moulting in Palaeozoic euarthropods, and suggest a defensive strategy against predation, previously only known from trilobites preserved in open-marine deposits.Trace fossils represent the primary source of information on the evolution of animal behaviour through deep time, and provide exceptional insights into complex life strategies that would be otherwise impossible to infer from the study of body parts alone. Here, we describe unusual trace fossils found in marginal-marine, storm- and river-flood deposits from the Middle Devonian Naranco Formation of Asturias (northern Spain) that constitute the first evidence for infaunal moulting in a non-trilobite euarthropod. The trace fossils are preserved in convex hyporelief, and include two main morphological variants that reflect a behavioural continuum. Morphotype 1 consists of a structure that superficially resembles a  The abundance of euarthropod body fossils in the rock record has led to a comprehensive understanding of the morphological adaptations required for ecdysis, namely the distribution and variability of suture lines on the dorsal exoskeleton of major groups including trilobites and eurypterids5. These investigations have also produced insights on euarthropod palaeoecology through deep time, such as environmental triggers for mass moulting events7, adaptive advantages of particular moulting strategies5, and behavioural tradeoffs in response to predation9. However, the fossil record of moulting is strongly biased in favour of biomineralizing groups inhabiting open-marine environments, particularly during the Palaeozoic1, and thus the current understanding of this critical aspect of euarthropod evolution is drastically underrepresented for soft-bodied organisms.The process of ecdysis – in which the exoskeleton is discarded and substituted with a new one in order to accommodate changes in size and/or shape during growth – represents a fundamental biological constraint that has shaped the evolution of euarthropods for more than half a billion years10 that provide evidence of shallow infaunal moulting in an euarthropod taking place in a marginal-marine depositional environment. Our findings represent one of the earliest unequivocal trace fossils produced by moulting (ecdysichnia)11, and expand the record of infaunal moulting to include non-trilobite euarthropods9. The infaunal strategy revealed by these peculiar trace fossils may reflect a response to predation pressure on the behaviour of organisms inhabiting shallow- to marginal-marine environments during the mid-Palaeozoic8.Here, we describe unusual trace fossils from the Middle Devonian Naranco Formation of northern Spain12) Naranco Formation of Asturias in northern Spain : El Tranqueru and Punta La Vaca Fig. . Both loIn particular, the trace fossils documented in this study occur at the bases of 0.3–5.0 cm thick, erosionally based, very fine- to fine-grained sandstone locally showing parallel lamination, and starved, linguoid and combined flow ripples. Sole marks (e.g. groove casts) are common. These sandstone beds are typically separated by mudstone layers. Some sandstone beds and mudstone intervals display soft-sediment deformation structures, such as convolute lamination, load casts, and slumps of various scales. Overall these deposits are sparsely bioturbated. Interbedded, relatively thick and normally graded mudstone units tend to be unbioturbated.Conostichus, Planolites and Teichichnus, among others14. Ichnodiversity is low to moderate, reflecting the depauperate or stressed Cruziana Ichnofacies. The low ichnodiversity and the sparse bioturbation, coupled with the sedimentologic evidence, suggest various stressors, such as freshwater discharge, high rates of sedimentation, and erosion and by-pass. The ichnofossil-bearing intervals are interpreted as representing river- and storm-flood deposits, characterised by the interplay of hyperpycnal flows and wave action.Characteristic ichnotaxa commonly associated to the studied biogenic structures include Two different morphotypes have been identified based on the study of 65 collected specimens and 33 additional ones studied in the field. Of the ones collected, 46 correspond to morphotype 1 and 19 to morphotype 2. The new ichnofossils from the Naranco Formation consist of bilaterally symmetrical structures with clear antero-posterior orientation, and a distinctive trilobate morphology preserved in convex hyporelief Fig. .Figure 3Morphotype 1 consists of convex trilobated structures with an oval outline characterised by the presence of an axial region marked by two longitudinal furrows that define three lobes along the length of the trace fossil, the axial of which is wider and deeper than the lateral ones. Lobes display regularly spaced transverse furrows that evoke the appearance of body segments that gently taper in width posteriorly Figs.  and 4a. Morphotype 2 is similar to morphotype 1 in overall appearance, including the presence of a well-defined axial lobe with an elevated, central ridge, but differs in having a much more elongate shape, defining almost a tubular structure Figs.  and S2a.2  and combined locomotion and feeding activity (Cruziana) of benthic deposit feeding euarthropods with homonymous limbs15. Morphotype 1 implies the docking of the dorsal part of the exoskeleton in the firm mud, producing an oval symmetrical structure superficially resembling Rusophycus, whereas morphotype 2 involves concatenated morphotype 1 specimens resulting in an elongated structure recording linear movement through the sediment akin to Cruziana  to create linear structures recording locomotion (Cruziana-like). Contrary to Rusophycus and Cruziana, however, the Naranco Formation trace fossils are not the result of the interaction of walking legs or ventral anatomy against the sediment; they lack leg striations (i.e. “scratch imprints”) or any kind of appendage impressions  found in legitimate euarthropod trackways and burrows. Instead, these trace fossils are best interpreted as the product of the dorsal exoskeleton of an euarthropod pressed infaunally against the cohesive muddy sediment. This resulted in the distinctive trilobate morphology with a deeper axial lobe and segmented appearance expressed in morphotype 1, and the effaced surface of elongated morphotype 2 with the axial ridge. Initial anchoring of the dorsal exoskeleton (morphotype 1) was occasionally followed by subsequent dragging and re-anchoring in an adjacent consecutive position generating partially overlapping replicas of the dorsal anatomy in a continuous structure (morphotype 2). Successive animal body re-adjustments are recorded by subtle angle changes defining nested curve segments in morphotype-2 cross-sectional profile  British Coal Measures18. In fact, the enigmatic Naranco trace fossils closely resemble the dorsal trunk exoskeleton of Camptophyllia in overall shape, size range, the presence of paired longitudinal furrows forming three lobes, wide axial lobe with an elevated central keel, the presence of nine segments that taper in width posteriorly, and even a pointed back end , but been entrained in the hyperpycnal flows and transported seaward (as observed in the Naranco Formation). Second, the producers of the Naranco trace fossils may have actually lived in these shallow marginal-marine settings. If this is the case, these Camptophyllia-like euarthropods with a similar functional morphology (and likely ecology) to that of oniscid isopods could have originated in fully marine environments during the Devonian and subsequently migrated landwards during the Carboniferous. These environmental shifts through time are not unusual, and have been detected in various arthropod groups19, including those taxa more directly comparable with Camptophyllia 18. Further exploration of Devonian and Carboniferous strata may reveal similar biogenic structures to those herein described, providing crucial evidence to evaluate these competing hypotheses.Camptophyllia-like euarthropods living in shallow- to marginal-marine environments begs the question: what particular behaviour do these unusual fossils reflect? The convex hyporelief preservation , sedimentological evidence indicates frequent erosional (e.g. groove casts) and exhumation processes providing optimal conditions for firm substrates. The preferred orientations of specimens preserved on the large surface is consistent with relatively high-energy conditions under the action of currents. In this context, morphotype 1 records an upside-down resting stance anchored in a firm mudstone, whereas morphotype 2 resulted from a more prolonged process involving successive attempts of anchoring and release of the exoskeleton. This latter process resulted in repeated, consecutive, partially overlapped structures defining a linear structure (morphotype 2) spanning several centimeters. In our interpretation, the anterior region reflects a moulting strategy involving the disarticulation of the cephalic shield , substrate penetration may have helped to mitigate the relatively high-energy conditions at the sediment-water interface.The Naranco Formation trace fossils represent the first record of infaunal mass moulting in a soft-bodied euarthropod within a marginal-marine depositional setting, and significantly expand the known phylogenetic and environmental occurrence of this complex behaviour during the Palaeozoic. Previous trace fossil evidence for Palaeozoic moulting is based on the ichnogenus 22, and indicate that infaunal moulting evolved multiple times in different euarthropod lineages as a result of these fundamental selective pressures. This study illustrates the significance of trace fossils for illuminating the evolution of adaptive complex behavioural strategies through deep time and for expanding our knowledge of ecdysis commonly recorded in the form of body fossils23.In this scenario, the Naranco Formation trace fossils reveal that marginal-marine soft-bodied euarthropods may have been as susceptible to predation as mid-Palaeozoic trilobites living in fully marine environmentsThe two stratigraphic sections were measured and their component sedimentary facies described, taken into consideration lithology, physical sedimentary structures, bed boundaries, bed geometry, and fossil content. They were subsequently interpreted in terms of depositional processes and sedimentary environment. Information on the associated trace fossils was integrated with the sedimentary facies analysis. Sixty-five specimens of the moulting trace fossils were collected, and are housed at the Museo del Jurásico de Asturias (MUJA) and Museo de Geología (University of Oviedo) collections. Thirty-three additional specimens were studied in the field at El Tranqueru section Fig. . The horSupplementary information.


Papillomaviridae, Poxviridae and Herpesviridae which cause skin diseases in cattle. Two specimens were collected from cases clinically diagnosed as papillomatosis and pseudo-LSD. Both skin lesions were shown to harbor more than one viral species. This case report shows, for the first time, co-infection of zoonotic parapoxvirus with bovine papillomavirus and herpesvirus in skin lesions of cattle. In particular, the simultaneous presence of virions morphologically referable to parapoxvirus and papillomavirus confirms that the replication of both viruses in the same lesion can happen and the so-called papillomatosis can bear zoonotic viruses.We describe two cases of skin co-infections with epitheliotropic viruses, detected in two cattle during lumpy skin disease (LSD) surveillance in northern Italy. A diagnostic protocol including different molecular methods as well as negative staining electron microscopy was applied to detect the most common viral agents belonging to the family  Papillomaviridae family. Despite the benign characteristics of the lesions, in some cases, bovine papillomatosis can dramatically reduce production performance when lesions are spread throughout the body, involving teats and udders. We have previously shown that the so-called “papillomatosis” can be the result of multiple infections with epitheliotropic viruses, including zoonotic poxviruses [Skin represents the main barrier between external, potentially aggressive agents and inner, more vulnerable structures and represents a complex system harboring various populations of micro-organisms known as skin microbiota. Hyperproliferative skin lesions affecting the tissue and mucosa of cattle can be caused by bovine papillomavirus (BPVs), non-enveloped double stranded DNA viruses belonging to the xviruses ,2.Poxvirus and papillomavirus have been simultaneously detected in the skin lesions of cattle ,4, birdsHere we describe two further cases of skin co-infections with epitheliotropic viruses that have been detected during a surveillance program for the viral cutaneous diseases of cattle implemented in Italy following the onset of lumpy skin disease (LSD) in some eastern European countries.A diagnostic protocol, specifically set up to detect the most common viral agents causing skin diseases in cattle, was applied to cutaneous lesions collected from cattle involved in clinical cases reported in northern Italy. Briefly, skin lesions were tested for the presence of BPVs, bovine herpesviruses, parapoxviruses, orthopoxviruses and capripoxviruses using different techniques, combining molecular methods ,12,13,14The first clinical case (#7) was identified on February 2018 at the slaughterhouse in a 20 months-old Charolaise cow in which cutaneous proliferative lesions were observed  and pathThe second clinical case (#1616) was identified during an outbreak of pseudo-LSD in a Fresian dairy farm during summer 2018. The 100-day-old calf presented lumps, scattered circular areas of alopecia and surface crust formation on the back, neck, shoulders and head .In both cases, nsEM, RCA isothermal amplification, PCRs and sequencing of the amplification products were performed on pathological skin samples in order to identify the most common pathogens associated with cattle skin lesions, as well as bovine papillomavirus’ unknown circular genomes.In the samples from case (#7), nsEM identified the simultaneous presence of parapoxvirus mature virions and non-enveloped icosahedral particles morphologically referable to papillomavirus A.RCA followed by digestion with BamHI, HindIII and KpnI revealed the presence of BPV-12 while sequencing of the PCR products showed a co-infection with a zoonotic parapoxvirus and another bovine papillomavirus. The sequence obtained by the PCR specific for parapoxvirus (GenBank accession number: MT68213) showed a 99.81% identity to bovine papular stomatitis virus (BPSV) (GenBank accession number: MK285566). The presence of a second papillomavirus co-infecting the skin lesion was confirmed by the sequences of the E5 open reading frame (ORF) (GenBank accession number: MT682132) and partial L1 ORF (GenBank accession number: MT682133), which showed 100% identity to BPV-1 (GenBank accession number: MF384289.1).Previous reports have identified the co-existence of parapox and orthopoxvirus particles in skin lesions of cattle by EM . In humaIn samples from case (#1616), nsEM showed the presence of herpesviral particles B subsequHowever, the protocol used for diagnosis also included a pan-parapoxvirus PCR followed by Sanger sequencing that allowed us to detect the zoonotic pseudocowpoxvirus (PCPV) (Genbank accession number: MT682135). Genomic amplification did not allow us to identify any bovine papillomavirus (BPVs). This finding further proves that virus co-infections are also possible between herpes and parapoxviruses. The definitive PLSD diagnosis for sample (#1616) was based on nsEM and sequencing of BoHV2, but the detection of PCPV DNA cannot be ignored since parapoxvirus should always be included in differential diagnosis. The impossibility of identifying PCPV by electron microscopy can be the result of a low viral load. On the other hand, the presence of PCPV DNA in the pathological samples led us to speculate that the virus may use such lesions as reservoirs to spread and persist in the environment through the scab material produced by other epitheliotropic viruses.Our study confirms that cattle skin lesions may host diverse viral agents including zoonotic poxviruses. This is particularly important considering that all the parapoxviruses are responsible for common occupational zoonoses and humans can be infected after direct contact with affected cattle or indirectly through a contaminated environment as viruses survive for long periods when protected by organic material.