The parasitic helminth infection neurocysticercosis (NCC) is the most common cause of adult-acquired epilepsy in the world. Despite the serious consequences of epilepsy due to this infection, an in-depth review of the distinct characteristics of epilepsy due to neurocysticercosis has never been conducted. In this review, we evaluate the relationship between NCC and epilepsy and the unique characteristics of epilepsy caused by NCC. We also discuss recent advances in our understanding of NCC-related epilepsy, including the importance of anti-inflammatory therapies, the association between NCC and temporal lobe epilepsy, and the recent discovery of biomarkers of severe epilepsy development in individuals with NCC and seizures. As generalized-onset seizures by definition involve networks on both sides of the brain at onset, they are always associated with impaired awareness. Both focal- and generalized-onset seizures are then classified by the type of symptoms experienced at the onset of the seizure, either motor symptoms  or non-motor symptoms .Seizures can be classified, according to the International League Against Epilepsy, as either focal- or generalized-onset seizures.14 In the case of NCC, the epileptic abnormality is typically caused by focal lesions,15 although other chronic changes resulting from NCC infection  may disturb normal neuronal activity.17 Additional factors that may also play a role in epilepsy development for some patients with NCC include a genetic predisposition toward a lowered seizure threshold and/or a strong host immune inflammatory response.20 Although NCC is a leading cause of epilepsy, little attention has been paid to the distinct characteristics of NCC-related epilepsy. In this review, we examine the unique characteristics of epilepsy development in NCC.Regardless of the seizure type, epilepsy of any etiology is believed to develop in the setting of an epileptic abnormality that impairs normal neuronal activity, an altered seizure threshold, and/or additional factors.21 These seizures may have multiple relapses associated with episodic lesion-associated inflammation. What distinguishes these acute seizures from epileptic seizures is that acute seizures do not continue in the absence of an active immune/inflammatory response to the parasite. It is only when this inflammatory response is also accompanied by the formation of synchronous firing patterns and epileptic networks where epileptogenesis (epilepsy development) occurs. Once these functionally connected epileptic networks form, it is unclear if they ever go away.When discussing seizures associated with NCC, it is important to distinguish early-onset seizures provoked by an active NCC infection and resulting inflammation (non-epileptic early-onset seizures) from epileptic seizures, which are unprovoked. Early-onset–provoked seizures are thought to be primarily incited by an immune inflammatory response to antigens released either from a naturally degenerating lesion or following treatment.23 or 2) transient release of NCC antigen during remodeling of calcified lesions causing an immune inflammatory response. An inflammatory response to NCC calcifications was demonstrated in a histologic examination of two calcified lesions.25 In subjects who have formed epileptic neural networks, an acute lowering of the seizure threshold can lead to additional epileptic seizures. Various studies have shown that up to 30% of NCC patients with epilepsy who were successfully treated for NCC can have persistent seizures despite being treated with both antiparasitic and anticonvulsant medications.21 These recurrent seizures are associated with worsened quality of life and increased mortality.35Factors associated with prior NCC infection can acutely lower the seizure threshold even in the absence of active infection. This can occur because of 1) neurotoxicity of calcium released from calcified lesionsEpilepsy due to NCC has many distinct characteristics that may lead to clinical differences and altered optimal treatment strategies compared with epilepsy of other etiologies. Because this syndrome is common, it is worth evaluating these distinct characteristics. In this review, we discuss the features of epilepsy in subjects with NCC and review new advances in our understanding of NCC-related epilepsy.Taenia solium, most commonly via the fecal–oral route. The infection passes through several stages that can be distinguished on imaging studies, from a living (viable) cyst, to an inflamed (degenerating) lesion, and finally culminates in the formation of a granuloma that either resolves (is no longer visible on imaging) or forms a calcified lesion  and secondarily generalized-focal seizures (15–61% of subjects).41Neurocysticercosis occurs when an individual is infected by the ova of d lesion .23,36–3844 In a retrospective cohort study, a strong relationship was found between the semiologic classification of the seizure and the location of the NCC lesion,45 but other studies have not found this association. One explanation for this discrepancy could be that different studies may have included distinct subpopulations of patients with NCC. Many of the studies which did not find an association between semiology and lesion characteristics included primarily calcified lesions, which represent subjects at a late stage of disease who thus may present differently.47 These conflicting results also further highlight the exceedingly heterogenous nature of NCC.Various studies have evaluated for an association between characteristics of NCC lesions and seizure semiology , with mixed results. In some studies, the frontal lobe lesions were more likely to present with focal seizures, whereas the temporal lesions and those in the degenerating stage were more likely to secondarily generalize.40 This finding is especially important as a separate randomized controlled study found a significant reduction in seizures in subjects treated for NCC; this reduction was primarily driven by the subgroup of subjects for whom the cyst resolved.48 However, the authors noted that the cyst resolution only partially reduced the likelihood of continued seizures, and therefore, additional methods capable of preventing recurrent epileptic seizures in NCC patients need to be identified.Several studies have demonstrated a benefit of antiparasitic treatment in reducing the frequency of seizures in subjects with NCC. In a double-blind, placebo-controlled trial of subjects with viable NCC lesions, treatment with albendazole and dexamethasone was shown to decrease the number of generalized seizures during 30 months of follow-up. The number of total seizures, however, was not decreased by treatment. Unsurprisingly, those who were treated with albendazole in this study were more likely to have resolved their cystic lesions on follow-up imaging than those randomized to placebo.49 Unfortunately, regardless of the underlying cause of seizures, currently available anticonvulsant drugs do not prevent epileptogenesis or cure epilepsy.50 Therefore, for early intervention to prevent epilepsy in NCC patients to be possible, researchers must first 1) develop an increased understanding of the processes involved in epileptogenesis and 2) identify biomarkers capable of identifying subjects early in their clinical course who will go on to develop epilepsy. Such biomarkers would also help avoid unnecessary interventions in patients with a low likelihood of seizure recurrence and could identify novel therapeutic targets to prevent epilepsy.Early intervention is thought to provide the best possibility for preventing epilepsy in subjects with an initial seizure, as each seizure may increase the brain’s ability to produce seizures.52 These imaging findings include 1) an increased number of total, degenerating, and calcified lesions at baseline18 (53 (54 on posttreatment magnetic resonance imaging (MRI) scans. One potential drawback of perilesional edema as a predictive factor for persistent seizures is that this finding could either be caused by vasogenic edema from a seizure or could be due to immune-associated inflammation. One strategy to address this could be to reimage with MRI after a period of at least 2 months following a known seizure to evaluate edema persistence. This would not completely rule out that perilesional edema is due to seizures, however, as the patient could have subclinical ictal activity. A recent study examining calcified NCC lesions with perilesional edema using PET scans found that 12 of 13 new NCC lesions  expressed immune-mediated inflammatory proteins, which remained present from 2 to 9 months following treatment.55 Future studies using this technology could, therefore, help better understand the pathophysiology of perilesional edema.To date, no biomarkers have been identified that can successfully predict which NCC patients with acute seizures will develop epilepsy. However, several imaging findings have shown promise in identifying those at increased risk for treatment-resistant epilepsy, as it occurs in up to 30% of subjects with NCC.seline18 , 2) persne18  56 This inflammatory response in NCC appears to play an important role in epileptogenesis, natural involution of lesions, and response to treatment. Inflammation is thought to lead to epileptogenesis either by causing gliosis and/or by causing persistent areas of blood–brain barrier dysfunction.39In both porcine models of NCC and PET studies in humans, lesions of NCC showed evidence of perilesional inflammation.58 Finally, a strong serologic response (≥ 4 bands)59 to parasite antigen on NCC EITB was associated with both epilepsy development and with a more severe course once epilepsy was established.60As evidence to support an association between the inflammatory response and epileptogenesis, subjects with active epilepsy due to NCC, compared with infected subjects without epilepsy, have 1) higher serum levels of blood–brain barrier breakdown molecules (MMP-9) and increase in pro-inflammatory cytokines , 2) increased expression of lymphocyte adhesion molecules, and 3) increased likelihood of mutations in the Toll-like receptor 4 that lead to an increased Th1 (pro-inflammatory) response .19,20,5762 and may be able to impact disease progression.63 Steroids have proven beneficial in reducing the frequency of adverse events among subjects with 1) viable intraparenchymal lesions who are treated with antiparasitic medications,64 2) single enhancing or multicystic lesions,65 3) subarachnoid or spinal disease, and 4) those who require ventriculoperitoneal shunt placement.66 However, the lengthy treatment courses and high doses of steroids often required to control inflammation associated with NCC can lead to significant side effects, including hyperglycemia, avascular necrosis of joints, and glaucoma.67 Newer anti-inflammatory medications such as methotrexate and TNF-α inhibitors likely merit further study to assess their ability to suppress inflammation with potentially fewer side effects than steroids. Although data are limited, one case series included four patients with episodes of perilesional edema and seizures, all of whom reported clinical improvement after treatment with 25 or 50 mg of etanercept weekly for a median of 400.5 days.68 Further studies should be carried out with a larger number of patients to confirm these findings.Given the importance of inflammation in epilepsy due to NCC, several studies have evaluated for a clinical utility of glucocorticoids such as prednisone and dexamethasone to reduce seizures or inhibit epileptogenesis. Steroids have been shown to reduce the incidence of acute seizures in NCC69) are common.70 Calcium is known to be neurotoxic, and other diseases with neurologic calcifications are also associated with epilepsy. In a select number of patients with cerebral calcifications , the bisphosphonate drug disodium etidronate was found to decrease seizure recurrences, presumably by chelating solid-phase calcium phosphates.22 However, this has not yet been studied in calcified NCC lesions.Epilepsy rates have been shown to be higher in areas where NCC calcifications  the NCC lesion could cause chronic interictal or ictal discharges that injure and kindle the hippocampus or 2) NCC-associated inflammation could directly damage the hippocampus.79As the neuroanatomic locations of calcified NCC lesions do not always correlate with areas of abnormality on electroencephalogram (EEG), some researchers have suggested that calcifications may be only an incidental finding in epilepsy patients.P-value < 0.05).80 In addition, in a case–control study, patients with NCC had decreased hippocampal volumes compared with control subjects. On a subgroup analysis , this decrease in hippocampal volumes among NCC patients was found to be primarily due to changes among untreated patients. Mean hippocampal volumes in this study were 3.44 cm3 on the right and 3.38 on the left for untreated NCC patients compared with 3.92 cm3 and 3.84, respectively, for control subjects (P < 0.001).81 Furthermore, in a cohort of patients surgically treated for mesial temporal lobe epilepsy, among patients with a single calcified NCC lesion, the lesion matched the side of the HS in 43 of 58 cases (74.1%), significantly more than would be expected because of chance .82As evidence for a causative association between calcifications and HS, among patients presenting for an evaluation for epilepsy surgery, patients with HS were less likely to have another precipitating injury of epilepsy identified if they had NCC calcifications  compared with those with HS alone , pathologic examination of a resected calcified NCC granuloma with recurrent episodes of perilesional edema showed a marked mononuclear infiltrate.25 Despite the immunologic causes that appear to underlie inflamed calcifications, the utility of corticosteroids or other immunosuppressive medications in this setting has not been established. Corticosteroid withdrawal has been shown to initiate (or exacerbate) pericalcific edema,86 and therefore, currently there is no sufficient evidence that the benefits of steroids outweigh the risks.Recent evidence suggests that calcifications may also lead to epilepsy through an immune inflammatory mechanism. NCC calcifications had long been thought of as immunologically inert, until Nash et al.88 16 subjects who had calcified NCC lesions plus either evidence that a specific calcified lesion was the seizure-provoking focus (based on seizure semiology and/or EEG findings) or association of the calcification with unilateral HS were treated surgically. Those without evidence of HS underwent resection of the causative lesion, whereas those with HS were treated with resection of both the calcified lesion and the anterior temporal lobe (ATL). Following surgery, 93.8% (15/16) of subjects experienced complete resolution of their seizures. However, resection of solely the NCC lesion or the ATL alone was not found to be successful in subjects with both HS and a causative lesion due to NCC . Although surgical resection has been promising in this small group of patients, which surgical technique is best and what subset of patients are most likely to benefit from such an intervention are not yet known.Because of the focal nature of NCC lesions, a promising option for a subset of patients with medically refractory epilepsy and NCC has been surgical resection. Although a minority of NCC patients would likely qualify for such treatment, patients may benefit from surgery if they have 1) intractable epilepsy with an NCC lesion within the boundaries of the epileptogenic zone or 2) developed HS in conjunction with NCC. In two small reviews,Recent studies have identified two primary processes, inflammation and calcification (in association with HS or not), which appear to be important for epilepsy development in patients with NCC. Factors associated with these processes  may be able to identify those at increased risk for severe seizure disorders early in their disease course. Both of these processes could potentially be treated with currently available medications that could be repurposed for use in NCC (either steroid-sparing anti-inflammatory medications such as methotrexate or etanercept or bisphosphonates). Studies of these treatments in NCC patients are needed as a large portion of those with NCC and epilepsy continue to seize despite treatment.


Aquaporin 4 (AQP4) is the most abundant water channel in the central nervous system (CNS). Its expression is confined to non-neuronal glial cells, predominantly to astrocytes that represent a heterogeneous glial cell type in the CNS. The membrane of astrocyte processes, which align brain capillaries and pia, is particularly rich in AQP4. Several isoforms of AQP4 have been described; however, only some (AQP4a (M1), AQP4 c (M23), AQP4e, and AQP4ex) have been identified in the plasma membrane assemblies of astrocytes termed orthogonal arrays of particles (OAPs). Intracellular splicing isoforms  have been documented, and most of them are postulated to have a role in the cell surface distribution of the plasma membrane isoforms and in the formation of OAPs in murine and human astrocytes. Although OAPs have been proposed to play various roles in the functioning of astrocytes and CNS tissue as a whole, many of these still need to be described. OAPs are studied primarily from the perspective of understanding water permeability regulation through the plasma membrane and of their involvement in cell adhesion and in the dynamics of astrocytic processes. This review describes the cellular distribution of various AQP4 isoforms and their implications in OAP assembly, which is regulated by several intracellular and extracellular proteins. Aquaporin 4 (AQP4) is one of the three types of aquaporins (AQPs) that have been described in astrocytes . Besides2  when recorded by direct stochastic optical reconstruction microscopy [OAPs or AQP4 supramolecular assemblies in the plasma membrane of astrocytes can be readily detected experimentally by neuromyelitis optica (NMO) IgG antibodies applied to impermeabilized cells . NMO IgGcroscopy . Recentlcroscopy . In the croscopy . The sizcroscopy  have notcroscopy .In addition to the predominant plasma membrane distribution, AQP4 isoforms are also distributed intracellularly, as shown by several studies on isolated murine astrocytes ,20,21,22OAPs are aggregations of several AQP4 isoforms in the plasma membrane distinctively observed in astrocytes . AQP4 moAltered composition of the extracellular milieu can also affect OAP structure. Time-dependent changes in the size of OAPs were demonstrated in rat astrocytes overexpressing AQP4e exposed to hypoosmotic conditions mimicking brain edema . In addiIn addition to plasma membrane movements, substantial rearrangements of AQP4 isoforms have been identified within the cytoplasm, with the potential to affect the arrangements of plasma membrane isoforms see also . For insWater permeability through AQP4 channels in astrocytes may be regulated by several mechanisms and the role of OAPs in this is still not entirely clear. Altered expression levels of plasma membrane isoforms, delivery of isoforms via intracellular vesicles, phosphorylation of the channel, and rearrangement of isoforms within OAPs have been described among the processes that affect water permeability . The rolTherefore, one of the most conspicuous findings in recent years is the discovery that intracellular isoforms may affect the assembly and abundance of OAPs and consequently water homeostasis of astrocytes. When the truncated intracellular AQP4-Δ4 isoform, which is not a functional water channel, was co-transfected with the full-length AQP4, cells showed diminished water transport activity due to a reduction in the plasma membrane expression of AQP4, triggered by a dominant-negative effect as a consequence of heterodimerization between AQP4 and AQP4-Δ4 in the endoplasmic reticulum . FurtherPositioning of individual AQP4 channels and OAPs in the plasma membrane of astrocytes has been receiving increasing attention in recent years, especially in connection with extracellular matrix proteins and proteins that directly or indirectly bridge the plasma membrane and AQP4 . While tAgrin, an extracellular matrix protein, is a heparan sulfate proteoglycan and is a component of the basal lamina of blood-brain barrier (BBB) microvessels. It has been shown to be implicated in mechanisms that define the plasma membrane distribution of AQP4. This role of agrin was first described in human glioblastoma samples ,39 and wThe presence of exogenous agrin has been described to affect changes in cell volume in hypoosmotic conditions . AstrocyAlthough agrin acts as a key molecule important for increased density of OAPs in astrocyte endfeet , it is oLaminin, another of the main structural proteins of the perivascular basal lamina, has also been demonstrated to induce plasma membrane clustering of not only AQP4 but also β-dystroglycan and Kir4.1 channels in hippocampal and cortical astrocytes ,43. CoatIn vivo studies supported the data obtained in cultured cells by showing that interaction between perivascular laminin and α-dystroglycan enables AQP4 positioning in perivascular astrocyte endfeet, which is also supported by a similar distribution of AQP4, α-syntrophin, and dystrophin–dystroglycan complex ,47. The Agrin and laminin interact with a plethora of transmembrane proteins and cytoplasmic proteins that dynamically interact with the plasma membrane protein pool. Among these proteins is dystrophin–dystroglycan complex, composed of α- and β-dystroglycan, which is crucially involved in the plasma membrane localization of OAPs in astrocytes. Whereas α-dystroglycan binds to agrin and laminin ,43,46,49One of the important steps in confirming the participation of α-dystroglycan in the localization of OAPs comes from experiments in which OAPs positively co-stained for α-dystroglycan and agrin ,41. DeleDystrophin, a component of the multi-protein dystrophin–dystroglycan complex, is a cytoplasmic protein connected directly to β-dystroglycan via its amino terminal domain ,55,56 F. In humaDystrophin interlinks (likely indirectly) with intracellular protein α-syntrophin , and thi−/− mouse model, the prominent localization of AQP4 in astrocyte endfeet was compromised simultaneously with decreasing expression and cortical mislocalization of α-syntrophin [Syn−/− mice does not affect the total levels of AQP4c (M23) and AQP4a (M1) expression in the brain, the polarization pattern of AQP4 in the astrocytic endfeet adjacent to the endothelial basal lamina in the cerebral cortex is significantly diminished in astrocytes devoid of α-syntrophin [In addition to AQP4c M23), which was primarily proposed to be the linking partner of α-syntrophin , AQP4ex , which wntrophin . Althougntrophin ,64,66. Ontrophin .The lack of α-syntrophin expression affects water regulation properties of astrocytes, which further substantiates the proposition that abundant localization of AQP4 in perivascular endfeet of astrocytes is essential for the regulation of water homeostasis in the brain. Namely, perivascular and subpial astrocyte endfeet have a swollen appearance in the brain of α-syntrophin-null mice, suggesting that clearance of water in the brain is reduced . FurtherThe involvement of AQP4 expression in the potential for astrocyte migration was first observed in cultured cortical astrocytes, where astrocytes devoid of AQP4 showed greatly impaired migration compared with their wild-type counterparts . At the −/−AQP4 cortical astrocytes and glioblastoma cells [+ channel Kir4.1 was detected in low- and high-grade astrocytomas and oligodendrogliomas [Processes that affect the migration potential of astrocytes are complex and related to interactions of AQP4 tetramers with the cytoskeleton, as demonstrated in astrocytoma cells . An impoma cells . For insma cells . Enrichema cells . The leama cells . On the ma cells . Togetheogliomas . This fiogliomas . If the ogliomas .Distinct localization patterns of AQP4 isoforms that are present in the human brain  imply thIn addition to extracellular matrix proteins and proteins interlinking with the dystrophin–dystroglycan complex that guide positioning of OAPs in the plasma membrane of astrocytes, external stimuli may also affect OAP arrangements. In addition to changes in osmolality and other multifaceted processes that modify AQP4 expression and hence the distribution and the formation of OAPs in traumatized brain, hormones are the most prominent factors for alterations in AQP4 expression  and possIncreased levels of dopamine have been demonstrated to reduce proliferation of striatal astrocytes and in downregulation of AQP4 expression . HoweverOver the last decades, sex hormones have been increasingly recognized as important agents that affect the functioning of several cell types and their functions in the CNS. In certain cases of acute neurological injuries, the gender issue is important, as was observed in experimental models and in humans . Of seveProgesterone is another sex hormone that can play an important role in several aspects of CNS insult, such as traumatic brain injury (TBI) and stroke, having the potential for treatment of TBI and other neural disorders in humans . ProgestNeuroprotective effects of estradiol have been reported in several studies. Protective actions of estradiol in neurodegenerative diseases and injuries are exerted by several complex mechanisms that regulate the proliferation and survival of cells and their anti-inflammatory responses . ProtectRegarding altered AQP4 expression in the development and reabsorption of brain edema, several hypotheses have been proposed. On the one hand, the upregulation of AQP4 channels has been proposed as beneficial in relieving the early phase of vasogenic edema  and on tIn addition to cell volume regulation and the development of brain edema, other roles of estradiol and other sex hormones of astrocyte function that are involved in recovery after neurotrauma  should be considered.The effect of AVP on swelling of astroglia cells was described three decades ago when it was documented that AVP causes gradual persistent astroglial swelling . Later, + and K+ that occur rapidly after focal brain injury may result in cellular edema [+ release from neurons [+ [Large extracellular ionic fluxes of Naar edema . Facilit neurons . Water f neurons . Thus, aurons [+ ,108.The increasing number of newly discovered AQP4 isoforms in astrocytes, combined with the additional roles proposed for those that have already been studied for an extended period of time, are advancing our knowledge about the various roles of AQP4 protein in the brain. Newly emerging data on AQP4 isoforms that have been identified to date are calling for reconsideration of some of the existing models of AQP4 functions. For example, in recent years, three additional plasma membrane isoforms have been discovered. One of them, AQP4e, was identified in rats and shown to localize in OAPs and to affect RVD in astrocytes. Therefore, an updated model of OAP structure in rat astrocytes, involving (at least) three plasma membrane isoforms  needs to be designed. Two AQP4ex plasma membrane isoforms that were recently identified in human astrocytes and revealed as crucial for binding NMO IgGs were also shown to build OAPs. Hence, updated modeling of OAP organization and dynamics is also required for human astrocytes. In addition to plasma membrane AQP4 isoforms, recently described intracellular isoforms AQP4b, AQP4d, and Δ4, are also being revealed as modulators of the abundance and size of OAPs. New discoveries about particular isoforms in OAP assembly and abundance, the role of isoforms that are detected as single tetramers in the plasma membrane of astrocytes, and the role and dynamics of intracellular isoforms extend our view of AQP4 channels as regulators of water homeostasis of the brain. Cell adhesion and migration processes intimately linked to AQP4 isoforms are yet to be unraveled in normal physiologic and pathologic conditions, such as acute neurotrauma and neurologic diseases where particular isoforms and assembly in OAPs in the plasma membrane of astrocytes might affect the development and the outcome of the particular pathologic conditions. Several intracellular and extracellular matrix proteins are emerging as important factors for positioning and polarization of AQP4 tetramers and OAPs at the plasma membrane of astrocytes. The role of these proteins and their interactions with APQ4 channels in normal and pathologic conditions are still emerging and require further attention to understand the larger picture of the changes that are taking place in traumatized brain. Additionally, the consequences of hormone imbalance and the composition of the extracellular milieu affecting ion homeostasis are crucial and need to be investigated in further studies.


Few studies have examined associations between socioeconomic, psychosocial and environmental characteristics with biological markers of aging among nationally representative older adult samples. This symposium will present results from four studies that examine the associations between 1) socioeconomic factors , 2) environmental characteristics  3) social isolation ,and 4) subjective well-being  as they relate to biomarkers of aging . Biomarker samples were obtained in 2017 via dried blood spots from 4,648 (88%) of the 5,265 self-responding participants of the National Health and Aging Trends Study (NHATS). NHATS is an ongoing study that conducts annual in-home interviews, which recruited a nationally representative cohort of Medicare beneficiaries aged 65+ residing in the contiguous United States in 2011 and replenished the sample in 2015. All analyses for the four studies presented in this symposium adjusted for demographic and socioeconomic characteristics and other potential confounders. Sampling weights were applied to account for study design and non-response so that inferences can be generalized to US adults aged ≥67 in 2017. Sessions of this symposium will highlight the socioeconomic, psychosocial and environmental characteristics that are associated with aging biomarkers. These results have clinical, policy and public health implications. These results can inform the development of interventions and policies aimed at improving biologic aging across the lifespan and reducing disparities in biologic aging.


More importantly, the dysregulation of tRFs has important clinical implications. They are potential diagnostic and prognostic biomarkers of gastric cancer, liver cancer, breast cancer, prostate cancer, and chronic lymphocytic leukemia. tRFs may become new therapeutic targets for the treatment of diseases such as hepatocellular carcinoma and respiratory syncytial virus infection. Finally, we point out the existing problems and future research directions associated with tRFs. In conclusion, the current progress in the research of tRFs reveals that they have important clinical implications and may constitute novel molecular therapeutic targets for modulating pathological processes.tRNA-derived fragments (tRFs) are a new category of regulatory noncoding RNAs with distinct biological functions in cancers and stress-induced diseases. Herein, we first summarize the classification and biogenesis of tRFs. tRFs are produced from pre-tRNAs or mature tRNAs. Based on the incision loci, tRFs are classified into several types: tRF-1, tRF-2, tRF-3, tRF-5, and i-tRF. Some tRFs participate in posttranscriptional regulation through microRNA-like actions or by displacing RNA binding proteins and regulating protein translation by promoting ribosome biogenesis or interfering with translation initiation. Other tRFs prevent cell apoptosis by binding to cytochrome Figure 1). The generation of tiRNAs and tRFs takes place in multiple biological processes, which implies that these fragments are not random cleavage products Recently, multiple studies have revealed that small noncoding RNAs (sncRNAs) exist extensively and have a diversity of functions in humans Figure 1). These tiRNAs are mainly generated by stress exposure, such as oxidative stress, hypoxia, and virus infection tiRNAs are generated by ribonuclease angiogenin (ANG) incision at the middle site of the anticodons of mature tRNAs, so tiRNAs are also called tRNA halves Figure 1), while 3′ SHOT-RNAs are characterized by a hydroxyl at the 5′ end and an amino acid at the 3′ end, as they are derived from aminoacylated tRNAs In addition, another type of tRNA half known as the sex hormone-dependent tRNA-derived RNA (SHOT-RNA) is induced by sex hormones and cleaved by ANG High-throughput sequencing technology has revealed that there is more diversity in tsRNAs than is reflected in the existing classifications. Among tsRNAs, tRFs, with a length of 14 nt to 30 nt, have a greater number of distinct classes and function as key players in the regulation of gene expression at transcriptional as well as posttranscriptional levels, indicating that they are not merely byproducts of the random cleavage of tRNAs but are regulatory sncRNAs involved in physiological and pathological processes Since tRFs are the major types of tsRNAs, in this review, we focused on tRFs. We present updated views focusing on the specific molecular mechanisms underlying tRF-mediated regulation of mRNA stability and translation, stress responses and viral infections and their potential roles as biomarkers or therapeutic targets in cancers and virus infections.Figure 1).tRFs are produced from pre-tRNAs or mature tRNAs Figure 1).There are three additional groups of tRFs: tRF-1, tRF-2, and inter tRF (i-tRF) Studies have revealed that some tRFs target genic mRNAs and participate in posttranscriptional regulation in humans Preparatory bioinformatic analysis suggests that tRFs have sufficient sequence complementarity with endogenous mRNAs and thus may play a potential role in posttranscriptional regulation Leu and pre-miRNA, inhibited colorectal cancer cell proliferation by inhibiting the Notch signaling pathway by directly interacting with the JAG2 mRNA 3′ untranslated region (UTR) Gly-GCC possessed an miRNA-like structure and functions by binding a complementary target site Figure ) Figure ) Huang et al. reported that tRF/miR-1280, which is derived from tRNAtRFs may bind to RBPs and posttranscriptionally regulate gene expression Asp, tRNAGlu, tRNAGly, and tRNATyr, can competitively bind to YBX1 and block its interaction with oncogenic mRNAs Figure ). This posttranscriptional suppression depends on sequence complementary because tRFs have a motif that binds the sequence that YBX1 can recognize In breast cancer cells, Goodarzi et al. reported that under hypoxic conditions, several tRFs were upregulated, which then suppressed oncogenic transcript stability by displacing the 3' UTR from the YBX1 protein Glu has been found to be able to bind and displace the RBP nucleolin in breast cancer In fact, YBX1 is not the only RBP that may be displaced by specific tRFs. In fact, a novel tRF derived from mature tRNAtRFs play additional biological roles by activating or inhibiting protein synthesis via different mechanisms LeuCAG enhanced translation by facilitating ribosome protein biogenesis LeuCAG bound to duplexed secondary target sites in RPS28 mRNA and unwound the hairpin secondary structure to increase translation in human cancer cells Figure ). Thus, 3′ tRFLeuCAG plays a vital function in regulating the numbers of ribosomes. The greater the number of ribosomes, the more potential protein synthesis, eventually increasing cell growth and proliferation An innovative study by Kim et al. proved that a 22 nt length tRF called 3′ tRFGln19 interacted with the human multisynthetase complex (MSC) and then promoted RBP translation . However, the detailed mechanism needs to be determined.Keam et al. also convincingly showed the translational activation of tRFs Figure ). They identified that PUS7 was enriched in embryonic and/or hematopoietic stem cells and then bound to diacritical tRNAs and modified uridine (U) into pseudouridine (Ψ) at the U8 position (Ψ8) Figure ). In this structural model, translational initiation factors  interact with cytoplasmic poly(A) binding protein-1 (PABPC1) 7G-capped mRNAs . PUS7 and mTOG-Ψ8 loss affects translation regulation, resulting in the increased biosynthesis of proteins and the impairment of hematopoietic stem cell commitment, potentially leading to myeloid malignancies Recently, Guzzi et al. showed that in stem cells, pseudouridylation synthase 7 (PUS7) mediated pseudouridine (Ψ) disposition of particular tRFs (mTOG-Ψ8) to suppress translation (5C) by the methyltransferase NSUN2 was an innovative mechanism to suppress global protein synthesis 5C; the dysregulation of m5C increases the affinity between tRNA and angiogenin, leading to the accumulation of tRF-5s, which then repress protein synthesis and promote squamous tumorigenesis  to suppress cell apoptosis 1A) and 3-methylcytidine (m3C) in tRNAs. Demethylated tRNAs are more sensitive to the cleavage of ANG and easily generate tRF-5Gly-GCCc that was released from the mitochondria and then eventually strengthened ribosome assembly and finally prevented apoptosis of cervical cancer cells .Under sodium arsenite stress, Chen et al. revealed that the demethylase α-KG-dependent alkB homolog 3 (ALKBH3) induced tRFs to interact with cytochrome Under oxidative stress, Gkatza et al. reported that the activity of the cytosine-5 RNA methyltransferase NSUN2 can be suppressed, leading to the reduction of tRNA methylation and then the intracellular biogenesis of tRFs related to the repression of protein synthesis GluCTC (tRF-5GluCTC) was induced by RSV infection GluCTC recognizes a target site in the 3′ UTR of apolipoprotein E receptor 2 (APOER2) mRNA . APOER2 is a host anti-RSV protein whose inhibition favors RSV replication GluCTC is important for gene targeting GlyCCC and tRF-5LysCTTFigure ).Recent studies have demonstrated that infecting host cells with respiratory syncytial virus (RSV) can initiate a stress response by mediating ANG digestion of tRNAs to generate increases in tRFs Table 1).Based on the above, we can see that tRFs play various roles in cancers and virus infection (Table 2).In recent years, tRFs have become rising stars in the regulation of biological processes, and their deregulation has important clinical implications GluCTC in plasma exosomes than healthy controls, indicating that exosomal tRFs in plasma can act as novel “liquid biopsy” biomarkers for the diagnosis of cancer GlyCCC levels were significantly lower in peripheral blood mononuclear cells (PBMCs) from chronic lymphocytic leukemia (CLL) patients and could be considered a screening biomarker GlyGCC and i-tRFPheGAA have been reported to have prognostic and diagnostic value in CLL, respectively As high-throughput sequencing technology has spread, an increasing number of studies have demonstrated that aberrant expression of tRFs contributes to carcinogenesis and could represent new biomarkers for diagnosis LeuCAG resolved the hairpin structure of RPS28 mRNA and facilitated ribosome protein biogenesis to promote hepatocellular carcinoma growth LeuCAG prevents it from binding RPS28 mRNA, resulting in diminished ribosome biogenesis and apoptosis of hepatocellular carcinoma cells GluCTC and eventually block RSV replication Aberrant expression of tRFs not only plays a diagnostic and prognostic role in cancers but also indicates their potential usage as therapy targets for the treatment of disease In this review, we mainly focused on the possible applications of tRFs in cancers and viral infections. However, tRFs also play roles in other types of diseases, including neurodegenerative and metabolic disorders First, how many additional classes of tRFs exist? For restriction sequencing technology, the current nomenclature of tRFs is based on the different cleavage loci of tRNAs or their origins in tRNAs that transfer a specific amino acid. The nomenclature and classification of tRFs are still rudimentary and cannot provide clear basic information about tRFs. Second, which mechanisms are crucial for tRF roles? The distribution of tRFs may contribute to their biological roles. A recent report suggested that tRF-5s are located mostly in the nucleus, whereas tRF-3s and tRF-1s are mostly cytoplasmic Taken together, the evidence suggests that the roles and applications of tRFs still require intensive study. We are confident that future in-depth efforts will contribute to elucidating the mechanisms of tRFs and developing them as novel diagnostic biomarkers and therapeutic targets.


Injury surveillance in professional sport categorises injuries as either “new” or “recurrent”. In an attempt to make categorisation more specific, subsequent injury categorisation models have been developed, but it is not known how often these models are used. The aim was to assess how recurrent and subsequent injuries are reported within professional and elite sport.Online databases were searched using a search strategy. Studies needed to prospectively report injury rates within professional or elite sports that have published consensus statements for injury surveillance.A total of 1322 titles and abstract were identified and screened. One hundred and ninety-nine studies were screened at full text resulting in 81 eligible studies. Thirty studies did not report recurrent injuries and were excluded from data extraction. Within the studies that reported recurrent injuries, 21 reported the number and percentage; 13 reported only the proportion within all injuries; three reported only the number; five reported the number, percentage and incidence; and two only reported the incidence. Seven studies used subsequent injury terminology, with three reporting subsequent injury following concussion, one using an amended subsequent injury model and three using specific subsequent injury categorisation models. The majority of subsequent injuries (ranging from 51 to 80%) were categorised as different and unrelated to the index injury. The proportion of recurrent injuries (exact same body area and nature related to index injury) ranged from 5 to 21%.Reporting recurrent or subsequent injuries remains inconsistent, and few studies have utilised subsequent injury models. There is limited understanding of subsequent injury risk, which may affect the development of injury prevention strategies.CRD42019119264 Reporting the recurrent or subsequent nature of an injury remains inconsistent within research.Thirty of the articles that achieved the criteria for data extraction did not report recurrent injuries.Only three studies utilised subsequent injury categorisation models to categorise subsequent injuries, with only two using the SIC 1.0 and one using the SIC 2.0.Injury risk associated with participation in sport can vary depending on the type of sport and level of play –8. One oThe primary purpose of injury surveillance is to identify priority injury problems; however, a limitation with the current reporting of injury rates is the lack of distinction between new and recurrent injuries. Within traditional injury surveillance, all recorded injuries are new injuries unless they are the exact same injury as a previous one, in which case they are referred to as a recurrent injury . Yet oftAlthough consensus statements have outlined a standard method of reporting recurrent injuries –21, the This article systematically reviews the reporting of recurrent and subsequent injuries in prospective injury surveillance research within professional and elite sports that have a published, peer-reviewed consensus statement on the definitions and procedures for reporting injuries.The Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement was followed to ensure accurate reporting throughout . The sysInjury terms: injuries, injury, recurrent, subsequentSport terms: football, rugby, athletics, swimming, cricket, diving, waterpolo, tennis, horseracing, professional, eliteThe online databases of SCOPUS, Embase (via Ovid) and Medline (via Ovid) were used to search for articles. The search strategy included articles published after 2005, the earliest publication date of the peer-reviewed consensus statement in the included sports , until 2The terms were entered into the search engine as follows: injury OR injuries AND recurrent OR subsequent AND football OR rugby OR athletics OR swimming OR cricket OR diving OR waterpolo OR tennis OR horseracing AND professional OR elite.n = 170) was screened by the primary (CLB) and second author (JV-C). As there was an almost perfect agreement between authors (Cohen’s K 0.97), the primary author screened all remaining titles and abstracts. All full-text articles outlined for each stage of review following the screening of titles and abstracts were reviewed by both authors independently (CLB and JV-C). If there were any discrepancies between the authors on the inclusion of an article, these were discussed, and if no agreement was made, a third author (ISM) was used as an adjudicator. There was an almost perfect agreement on the full-text articles screened (Cohen’s K 0.83). Discussion on inclusion resolved any disagreement between authors.All results returned by the online databases were exported to EndNote for the organisation of references and removal of duplicates before titles and abstracts were screened. Once duplicates had been removed, titles and abstracts of all remaining articles were initially screened for the inclusion of injury reporting in professional- or elite-level football , rugby , athletics, aquatic sports , cricket, tennis and horseracing (injuries to the jockey only). Conference abstracts, commentaries and systematic and literature reviews were excluded, and only full-text articles were eligible. The selection of articles identified for full-text screening were then screened to identify whether they included the following: (1) prospectively collected data, which provides a more accurate method of data collection when recording the exposure and injury details within sport ; (2) datData were extracted from the eligible full-text articles and recorded in an Excel spreadsheet. The extracted data contained information on 1) how the data were collected (i.e. prospective cohort); (2) injury definitions and procedures used, including how new and recurrent injuries were defined; (3) length of data collection period; (4) number of teams or players and level of play; (5) sex of the participants; (6) number of injuries sustained overall, and where relevant the match and training injury incidence; and (7) number of recurrent or subsequent injuries, including the subsequent injury category, the recurrent injury rate and the type or severity of recurrent injuries. As the search criteria included only professional and elite sports with consensus statements, all articles included followed the injury definitions and data collection procedures of the respective sports consensus statements. Definitions of injury and recurrent injury in each of the sports consensus statements are shown in Table  how the Extracted data were summarised for each article. The methods of reporting injuries, the number and incidence of recurrent injuries and the proportions and categories for subsequent injuries were collated in order to provide a narrative overview of results. When analysing and reporting injuries in the current review, combining new and recurrent injuries was defined as the total injury rate.The online database search returned 1708 articles  used a time-loss definition for injury, five used a medical attention definition, five used a medical attention and time-loss definition and four used an all-encompassing definition. The number of injuries sustained within the studies ranged from 15 injuries across 3 years within 552 athletes [The number of studies within each sport varied, with 31 studies in football/soccer , 37–64, athletes  to 22,94athletes .Table 2n = 34), (2) whether there was any attempt at blinding the participants (n = 50) and (3) whether there was any loss to follow-up (n = 46).Scores ranged from 8 to 16, with a maximum possible score of 23. Studies with a lower quality score typically failed to report the following: (1) the participant characteristics  of the studies published after 2011 in the current study failed to categorise subsequent injuries. Although the SIC 1.0 and 2.0 were utilised in three studies in the current review, the study by Williams and colleagues [In an attempt to reduce the recall bias and potential lack of awareness associated with diagnosing recurrent injuries, a data-driven categorisation that can be applied post hoc to the injury data that is not reliant on specific clinical knowledge during the data analysis has been suggested . Followilleagues  used a mlleagues , which alleagues  were catlleagues , 70.The accurate diagnosis of subsequent injuries can have a significant clinical impact, especially when considering the influence of previous injuries on sustaining new injuries , 83, 84.A limitation in the current study was that the search strategy was limited to English language, meaning that articles reporting recurrent or subsequent injuries in other languages would not have been included. A further limitation was the restriction of studies within professional- or elite-level sports. The restriction to professional or elite sport resulted in studies reporting injuries within amateur and collegiate athletes being excluded from the review, even if recurrent injuries had been reported. However, ensuring only one level of sport was included in the review provides consistency both with the accuracy of injury diagnosis by professional medical provision and data collection procedures following a consensus statement for accurate comparisons between studies. Further research could incorporate all levels of sports, making comparisons between injury data to demonstrate whether discrepancies exist between playing levels. In addition, the results from some of the studies should be interpreted with caution due to the low score in the Downs and Black  assessmeReporting the recurrent or subsequent nature of an injury remains inconsistent within research, even with the publication of consensus statements and the subsequent injury categorisation models. Furthermore, only a few studies have utilised subsequent injury categorisation models to accurately categorise subsequent injuries, meaning that risk of subsequent injury following an initial injury remains unclear. The lack of recurrent and subsequent injury reporting shows research is not providing an adequate understanding of the injury risk, meaning that injury prevention protocols to mitigate against recurrent and subsequent injuries may be insufficient. As injury prevention relies on accurate injury surveillance data, utilising the SIC model in future research will allow clinicians and researchers to distinguish between new and recurrent or subsequent injuries and improve our understanding of the role of inter-injury relationships in tertiary prevention strategies.


Recent progress in the field of electroanalysis with metal nanoparticle (NP)-based screen-printed electrodes (SPEs) is discussed, focusing on the methods employed to perform the electrode surface functionalization, and the final application achieved with different types of metallic NPs. The ink mixing approach, electrochemical deposition, and drop casting are the usual methodologies used for SPEs’ modification purposes to obtain nanoparticulated sensing phases with suitable tailor-made functionalities. Among these, applications on inorganic and organic molecule sensing with several NPs of transition metals, bimetallic alloys, and metal oxides should be highlighted. Screen-printed electrodes are well-known suitable platforms for sensing devices’ development. Technology involving its preparation implies the use of many different substrates, such as ceramic, plastic, paper, or glass. In addition, the possibility of producing patterns of cells or electrodes with different architectures, such as single electrodes or conventional three-or-more electrode cells, interdigitated electrodes, flow cells, etc., affords a wide range of different applications. Its easy preparation and cost-effective production allows mass production of customized electrode configurations with devices made of different substrates, geometries, shapes, and sizes.In addition to its principal advantage that is customable manufacturing, screen-printed electrodes (SPEs) also offer the possibility of producing tailor-made surfaces to achieve desirable applications for the detection of specific analytes in several fields, such as industry, clinical, or academic research.Modification of the electrode surface in these devices is usually achieved by three well-known methods represented in There is a vast literature on the use of metal NPs for the detection of heavy metal ions, and several reviews have been published ,3,4,5,6.The first approach used for screen-printed electrode modification with nanoparticles copies the usual methodology previously explored with modifiers onto carbon paste electrodes. Functionalization in these devices was achieved by mixing modifying material with the carbon-based paste that was subsequently pressed and polished.In this way, ink mixing SPE modification consists in the preparation of an ink where three main components are always presented: Conductive particles usually made of carbonous material, a solvent/binder mixture that allows to transfer particulated matter onto the substrate, and the modifying agent, metal NPs in this case. Depending on the final application, the main parameters that should be optimized are the recipe of the ink, its rheology, substrate selection, and thermal curing.Screen printing starts with the positioning of the printing media upon the mesh screen followed by the application of pressure with a squeegee that forces printing medium through the previously designed pattern. Finally, a curing temperature is applied to dry the ink. This procedure can be applied several times in order to obtain more layers of the material onto the substrate, either conductive ink or dielectric material. Further details of the procedure were reviewed elsewhere .Being the first approach explored, original papers appear in the early 1990s. These publications employed conductive material attached directly to metal particles, so the modification consists of metallized carbon with platinum , palladiLater, pristine metallic particles previously separated from carbon were mixed with conductive material and binder ,13. AlthRecently, an interesting paper deals with suitable ink mixing modification procedures to achieve better results regarding the catalytic performance . Due to Although this technique is more suitable for printing detailed patterns onto flexible substrates, inkjet printing affords thinner conductive substrates so more metallic particles are exposed to the analyte solution, diminishing the aggregation effect when compared to the ink mixing method. Moreover, inhibition is avoided due to the lack of paste additives used for ink mixing that can decrease the catalytic activity. The results obtained with this methodology with silver NPs show a greater catalytic response against hydrogen peroxide. One more improvement can be made by using a decapping method, where hydrochloric acid is used to dissolve the capped agent of silver NPs. Following this protocol, agglomerated particles can be interconnected, decreasing the resistance and affording better electron transfer and, consequently, improving the analytical performance . This isThis is the easiest method employed to modify screen-printed electrodes. Only one parameter should be optimized: The final amount drop casted onto the electrode. This can be modulated by changing the size of the drop and the concentration of the metal NPs dispersed in the solution.Sensors developed with this methodology are based in two main strategies: Direct modification of selected NPs onto the working electrode or ex-situ fabrication of composites made of NPs attached with carbonous nanomaterials.The first strategy is easier to carry out, as no functionalization step is necessary. So, many metal NP solutions were used, such as bismuth , platinuIn order to overcome this issue, the second strategy is more commonly used since more reproducible nano-sized metallic centers are obtained. NPs are synthesized in this way onto a conductive carbonous substrate, in a similar manner to the particulated metallized carbonous materials firstly employed with the ink mixing method. When compared to the successive casting in the two steps of carbon nanomaterial followed by NPs , these nThis is the most common method used to modify SPEs with metal NPs because it is the best method to control the morphology of NPs in an accurate way. This methodology is based on the reduction of oxidized species, typically metallic water-soluble salts, at a fixed potential or current to obtain tailor-made metal particles grown on conductive substrates.3 [4 [3 [3)3 [2 [2 [4 [3 [2 [4 [2 [2PtCl4 [2PtCl6 [2 [3 [The parameters usually optimized are separated in two main groups: The ones related to the precursor solution where the salt type and concentration are involved, and the conditions of electrochemical deposition. A list of usual salts employed are AgNO3 ,32,33,343 [4 ,35,36 an3 [4 [3  for gold [3 [3)3 ,38,39 fo [3)3 [2  for coba)3 [2 [2 , CuSO4 [[2 [2 [4 , and CuN[2 [4 [3  for copp[4 [3 [2 ,45 and N[3 [2 [4 ,47 for n[2 [4 [2 ,49,50 fo [2PtCl4 , H2PtCl6 [2PtCl6 ,54,55,56PtCl6 [2  for platl6 [2 [3  for rhodl6 [2 [3 . The firThese methods are based on the application of a fixed potential. When a specific potential to deposit NPs is applied, their size and shape can be modulated. As the potential becomes more negative, the nucleation rate increases, so more NPs are obtained with a smaller particle size, increasing the electroactive surface area. Focusing on the shape, negative potentials around −0.1 V (vs. a silver pseudo-reference electrode) give rise to more spherical NPs since growth is favored more than nucleation while a more negative potential affords an heterogeneous morphology . GeneralAlthough morphology is important, “best photo” is not often the goal but improved analytical performance, so the optimization has to be carried out to achieve a better response depending on the application goal. For example, more negative potentials (−0.6 V vs. Ag/AgCl) and shorter deposition times (190 s) than the ones employed to achieve nanoflowers are employed for the deposition of platinum onto ultramicroelectrodes . Mild reDeposition is usually performed in one potentiostatic reduction step by applying a potential negative enough to afford a reduction of the metal species to the zero-valence state, but sometimes, two steps  or more Examples of a well-controlled electrodeposition process are bimetallic alloys. These systems are based on particles that combine two different metals in one single particle as a core shell  approachElectrodeposition protocol is not the only parameter that should be taken into account when bimetallic alloys are developed. The selection of metals deposited, and the order of deposition are also crucial to achieve the best results. For example, a hydrazine sensor prepared with core-shell Cu-Pd NPs shows better results when copper is deposited first .Cyclic voltammetry is also employed for electrodeposition, but control is more difficult because more parameters need to be optimized, such as the scan rate, scan cycles, and potential window. Published works that use this technique with noble metals are mainly focused on the novelty of the substrates employed like graphite nanosheets , carbon Bimetallic alloys were also obtained when using cyclic voltammetry as the electrochemical technique. Gold and silver have been deposited together onto the carbonous working electrode, improving the sensitivity by slightly giving up the dynamic range . Silver Cyclic voltammetry is a useful technique when copper as a metal is employed, since passivated copper with an oxide/hydroxide layer is the species responsible for the catalysis of sugars and amino acids. Since the catalytic properties of CuNPs towards these analytes depend on the size, shape, and nature of CuNPs, several studies have been carried out to clarify the complex mechanism of electrocatalysis ,62. The Amino acids can also be detected with copper NPs because their carboxylic and amine terminals act like a chelating agent in a bidentate ligand. Their complexation with oxidized species of copper is capable of decreasing the detection potential at 0 V, while electrocatalysis increases this potential from the +0.4 to +0.8 V range. Interestingly, this phenomenon only occurs when 100-nm-sized copper NPs are electrodeposited onto SPEs, so accurate control of the particle size needs to be controlled. For this reason, a photo-irradiated electrodeposition method was developed based on potentiostatic electrodeposition applied under a xenon light source, because the modulation intensity of light is capable of controlling the size growth of copper NPs .These methods apply a constant negative current capable of reducing precursor metallic salt. The more negative the current applied, the higher the nucleation rate achieved, similar to potentiostatic methods. The deposition time is also a crucial parameter.−2) afford homogeneous nanoflower-like structures when nickel is reduced [−2) affords a more heterogeneous particle size distribution when copper is deposited [Although this technique is not extensively used, the employment of current instead of potentials is more convenient when using SPEs . Due to  reduced  while a eposited . This pheposited .−2) to the electrode system, provoking a quick reduction of metal ions at the working electrode of the SPE combined with the arising of numerous bubbles of hydrogen, hindering normal diffusion of the remaining ions of the precursor salt. Since no ion can occupy the space of hydrogen bubbles, the deposition is only achieved in the inner space among these bubbles so the final result is the formation of highly porous architectures where pores of hundreds of nanometers are easily obtained. This method has been employed to achieve 3D porous nickel structures with an extremely large electroactive area [The galvanostatic technique also offers the possibility of synthesizing porous metallic substrates with a high surface area via hydrogen evolution-assisted electrodeposition. This methodology consists in the application of a very large current density  cycles under atmospheric conditions, homogeneously distributed bismuth NPs of 2–5 nm separated and covered with a carbonous layer are produced in the surface of the working electrode. With this approach, it is possible to detect nanomolar concentrations of riboflavin without deaeration of the sample due to the presence of the carbon shell layer formed around the bismuth oxide NPs provoked by the own nature of the sparking process. The presence of this layer restricts oxygen interaction with bismuth NPs during voltammetric detection, affording lower blanks that allow measurements at lower concentrations .In this section, the main roles of the SPEs modified with NPs are revised. As mentioned above, heavy metal detection is the main application of metallic particles, but the extensive bibliography exceeds the scope of this review. Despite this, a brief mention is made of bismuth NPs that are the most commonly sensing phase employed in the detection of heavy metals since they are considered the most popular “green” substitutes for the classical mercury drop or film electrodes. Due to its wide cathodic range and its low toxicity, this allows the detection of heavy metal ions in a similar manner as mercury, but without the environmental risk of mercury waste disposal, such as bioaccumulation and acute toxicity by vapor inhalation ,69. ElecIn this section, the applications achieved with other metal NPS will be discussed, focusing on the specific role developed in the sensing device. In this way, three main different roles are considered. Metal NPs can act as a catalyst in enzymatic and non-enzymatic devices, as sensing phases for direct detection of several analytes, or as anchorage substrates for sensing platforms. In addition, two tables summarizing the real applications of SPEs based on metallic NPs see  and bimeMetallic NPs are capable of catalyzin relevant processes, such as peroxide reduction or carbohydrates oxidation, making the detection of enzymatic products, sugars, and amino acids in real samples possible. The following discussion is focused on the catalytical effect of several NPs against these and other analytes.Hydrogen peroxide monitoring is essential because it participates in classical enzymatic reactions extensively used in the biosensing field and it is an additive in many food, pharmaceutical, and environmental goods. Among enzymatic sensing devices, the most commonly used ones are based on peroxidases and oxidases due to their high selectivity and sensitivity. The reaction pathways of these enzymes involve hydrogen peroxide as the reagent or product, respectively, so it is the target analyte when detecting substrates with this two kind of enzymes. The monitoring is achieved directly in first generation biosensors, by the use of mediators in second generation biosensors or by the catalytic center of the enzyme in third generation biosensors. Although these successive improvements increase the use of enzymatic devices, they usually have poor chemical and long-term stability that limits their fabrication and increases the cost of the final device.Platinum group metallic NPs are a well-known electrocatalyst towards the oxidation or reduction of hydrogen peroxide . The mecSimilar to platinum, palladium shows high catalytic activity towards several electrochemical processes, making it possible to perform sensors based on screen-printing technologies . AlthougIridium NP-modified SPEs by the ink mixing approach were also used as transducers for enzymatic reactions. By means of peroxide detection, uric acid was detected via uricase reaction .As noble metal NPs are capable of detecting peroxide at low overpotential when compared to bare classical electrodes, they were also employed in enzyme-free devices. There are many advantages in the electrochemical detection of peroxide without the presence of an enzyme, such as the improvement in stability and reproducibility and the possibility of obtaining simple and inexpensive devices . MoreovePlatinum is the most common noble metal employed for non-enzymatic sensors. Due to its good electrocatalytic activity towards peroxide, platinum can be used to detect this molecule in several real samples, such as cosmetics , househoLike platinum, rhodium NPs were also used for peroxide detection. By a simple drop casting method, SPEs modified with rhodium NPs were used to detect hydrogen peroxide produced by autoxidation of polyphenols in tea extracts .In a similar manner to platinum-like metals, other noble metals, such as gold, silver, and copper, are employed in sensing devices. Sensors developed with these NPs show less catalytic activity towards hydrogen peroxide and they are very dependent on the particle size and preparation steps but offer cheaper devices compared to platinum-modified SPEs.Silver NP-based SPEs offer better catalytic properties towards peroxide detection than bulk silver electrodes. Unfortunately, sensors are only stable for a week because the main drawback of silver-based nanomaterials relies on its inherent and fast oxidation, so the stability is compromised for mass production purposes . Drop caFocusing on the field of biosensing and organic molecules, only a few works employing bismuth NPs were published  because Bismuth can act as a catalyst for hydrogen peroxide detection but only when it is combined with silver. Electrodeposition of bismuth NPs onto silver electrodes forms an alloy capable of catalyzing the reduction of hydrogen peroxide, including in real cosmetic samples. The existence of these alloys can open the door to a new metallic catalyst for sensing devices .Metal oxide nanoparticles were extensively used for sensing gases , but the2 [2 [2 [2, PdO, or IrO2 [Acting as a catalyst for peroxide oxidation, metallic oxides can be classified depending on their nature. The first group corresponds to common oxides, such as CuO ,79,80 an2  as the m2 [2 , RhO2 [82 [2 [2  and PtO2 or IrO2 . The lat or IrO2 , the ele or IrO2 , or ex-s or IrO2  and food or IrO2 .Metal alloys prepared with several elements, such as Pt-Ag  and Pt-PSeveral metallic elements are capable of catalyzing the hydrolysis of carbohydrates, making the detection of these analytes in real samples possible. Copper-modified SPEs were evaluated for sugar detection. The proposed mechanism for sugars’ oxidation  onto copGold surfaces are also capable of catalyzing sugar oxidation due to the presence of chemisorpted hydroxyl anions forming hydrous gold oxides, which are believed to be the catalytic component of gold electrodes. The main applications of this behavior relies on the development of novel non-enzymatic sensing in the health science field. In this way, glycated hemoglobin can be monitored in serum with electrodeposited gold nano-flowers . GlucoseAlthough metal oxides are capable of detecting hydroxide  directlyBimetallic alloys based on NPs capable of catalyzing carbohydrate oxidation were prepared. Cu-Ti  and Ni-CMetal NPs are also capable of monitoring other analytes, opening the path for new applications. Platinum NPs are capable of catalyzing the oxidation of small organic molecules, such as ethanol and formaldehyde, making the development of an alcohol sensor for wine and beer  and a gaPalladium nanoparticles are also capable of detecting formaldehyde and other analytes, such as hydrazine and sulphuric acid, with the classical approach of ink mixing. In addition, hydrogen was also tested in a proof-of-concept assay, making possible the application of palladium-based SPE sensors as gas detectors . AlthougIridium NPs are also capable of monitoring NADH produced in the dehydrogenase oxidation of glycerol. With this approach, a triglyceride biosensor was developed with a good correlation in bovine and human serum . In a siThe affinity of sulphur against gold offers the possibility of performing direct detection of sulphur species, obtaining devices capable of detecting sulfite in beverages  and freeSilver affinity towards sulfur and halide species was also explored for sensor development purposes. Sulfite was detected amperometrically with silver NPs in drinking water, pickle juice, and vinegar , and broDirect detection with bismuth NPs was only achieved when using deposition onto SPEs by sparking discharge. With this approach, it is possible to detect riboflavin in a nanomolar concentration in multivitamin real samples .Copper NPs are capable of a complex amino acid as previously mentioned, so α-,β-  and γ- aAlloys of Cu-Pd  and Au-AMetal oxides were successfully tested for direct sensing of pharmaceuticals in real samples by using other metal-transition oxides not previously mentioned like ZrO alone  or dopedMetallic NPs can also serve as carriers for sensing phases in other applications not mentioned before.Gold NPs were extensively used as an anchoring platform. Easy functionalization and tailor-made size, shape, and nature are advantages that offer a wide range of applications in the electrochemical sensors field . A recenBismuth NPs were also explored as a biosensing platform in mediator-free enzymatic SPE devices. These NPs are capable of complex oxidase activity tissues, affording a substrate capable of detecting phenolic compounds, thus direct monitoring the product of the enzymatic reaction instead of hydrogen peroxide was achieved. Deposition of cationic bismuth and negatively charged mushroom tissue together offers immobilization of polyphenol oxidase enzyme onto carbon SPEs, making the determination of phenol possible . With a Only a few more metallic NPs were studied as platforms for sensing devices with a rich related bibliography. Iron and cobalt NPs are one of the most assayed ones due to its low-cost production, tailor-made magnetic properties, easy functionalization processes, and biocompatibility. Iron NPs are often used as a magnetic carrier more than a catalyst  while coBimetallic clusters can also serve for biotin labelling detection in multivitamin tablets with Cd-Ti functionalized with neutravidin  and SERSAfter practically 25 years from the first publication, metal NPs are still nowadays employed as modifying agents in sensing devices based on screen printing technology. Ink mixing was the first approach explored capable of offering metallized SPE for sensing development. The main drawbacks of this method is the agglomeration of NPs, complex ink recipes, and bad reproducibility among batches. Drop casting methodologies offer a suitable approach to modify SPEs since the modification is carried out after ink preparation. NPs casted onto working electrodes offer a high active surface against analyte but agglomeration is also achieved. Nanoparticulated material with accurate tailor-made sizes and shapes is only obtained when using electrodeposition. Potentiostactic techniques are employed more due to their use for years in the classical three-electrode cell with a well-controlled reference electrode. However, galvanostatic techniques are more appropriate when working with SPEs. Deposition under a controlled current is not affected by potential variations from the pseudo-reference electrode, offering similar capabilities to control the nucleation and growth of metallic NPs as potentiostatic techniques. Large-scale synthesis is the main drawback of this method due to electrodeposition having to be done with each sensor separately, and the deposition step can be a large time-consuming process when thinking in large batch preparation.Recent scientific publications dealing with these sensing phases are focused on the development of novel strategies capable of affording a better analytical performance based on an increasing surface area or novel modifying agent synthesis. Newly developed electrochemical techniques that can increase the electroactive area in a great amount are based on previous knowledge not applied before with SPEs like hydrogen-evolution-assisted galvanostatic electrodeposition and spark discharge, or the use of carbonous nanomaterials. On the other hand, bimetallic clusters as modifying agents can be electrodeposited with an accurate growth control, affording novel surfaces with advantages from several metals in one particle, offering unknown metal synergies suitable for future screen printing devices based on metal NPs.


The adult liver has excellent regenerative potential following injury. In contrast to other organs of the body that have high cellular turnover during homeostasis , the adult liver is a slowly self-renewing organ and does not contain a defined stem-cell compartment that maintains homeostasis. However, tissue damage induces significant proliferation across the liver and can trigger cell-fate changes, such as trans-differentiation and de-differentiation into liver progenitors, which contribute to efficient tissue regeneration and restoration of liver functions. Epigenetic mechanisms have been shown to regulate cell-fate decisions in both embryonic and adult tissues in response to environmental cues. Underlying their relevance in liver biology, expression levels and epigenetic activity of chromatin modifiers are often altered in chronic liver disease and liver cancer. In this review, I examine the role of several chromatin modifiers in the regulation of cell-fate changes that determine efficient adult liver epithelial regeneration in response to tissue injury in mouse models. Specifically, I focus on epigenetic mechanisms such as chromatin remodelling, DNA methylation and hydroxymethylation, and histone methylation and deacetylation. Finally, I address how altered epigenetic mechanisms and the interplay between epigenetics and metabolism may contribute to the initiation and progression of liver disease and cancer. The adult liver has extraordinary regenerative potential. This knowledge dates back to the myth of Prometheus, who was condemned to an eternal punishment caused by an eagle eating his liver, which would replenish itself every day. Indeed, after partial hepatectomy, which leads to removal of up to 70% of the liver, liver mass and functions become significantly restored within a few days. Mechanisms underlying restoration of liver mass and function in response to hepatectomy have been elegantly reviewed elsewhere . In thisThe adult liver is a slowly self-renewing organ and does not exhibit a defined stem-cell compartment that maintains homeostasis, opposite to other organs that have excellent regenerative capacity, including intestine, stomach, and skin. A great number of experimental models indicate that cellular plasticity, intended as the capacity of liver cells to adopt alternative fates and functions in response to environmental changes, determines efficient adult liver regeneration following damage . A key qTwo epithelial cell types are present in the adult liver: (i) hepatocytes, which represent >60% of total liver cells and perform most of the metabolic and detoxification functions of the organ and (ii) cholangiocytes, which represent 3–5% of total liver cells and form biliary ducts that collect the bile produced by the hepatocytes and export it to the intestine for digestive purposes . Both hein vivo into bipotent liver progenitors that give rise to both hepatocytes and cholangiocytes  or conditional deletion of Arid1a in hepatocytes (mediated by infection with an AAV-Cre in Arid1a flx/flx mice) . Depletion of Arid1a following damage, in the recovering liver after a 12-week treatment with CCl4 or a 4-week treatment with DDC, reduced fibrosis and accelerated regain of normal weight. Thus, these data indicated that Arid1a deletion facilitated liver recovery from damage. At the molecular level, Arid1a deletion resulted in a significant reduction of Hnf4a+/Sox9+ bipotent liver progenitors in response to DDC treatment (2 weeks). At the molecular level, they found that Arid1a determined increased chromatin accessibility in the homeostatic liver, which facilitates YAP genomic binding after liver injury. YAP transcriptional activity was previously shown to promote hepatocyte de-differentiation into liver progenitors  . Sun et genitors . Consistrecovery . The difCdkn2a and Cdkn2b, which became up-regulated in Ezh1/2 knock-out (KO) livers. Consistent with the importance of hepatocyte proliferation and division in liver regeneration, epigenetic regulation mediated by the maintenance DNA methyltransferase DNMT1 and its regulator UHRF1 has been shown to play an important role in hepatocyte viability and regenerative capacity  and resulted in liver fibrosis after prolonged DDC-mediated liver injury   and can differentiate into hepatocytes  . Lineagetervals) , using as) Lgr5;  and adulatocytes . Moreoveatocytes  in intraatocytes . Togetheormation .Tet1 levels after liver injury in vivo (day 3 following DDC treatment) was only transient, since Tet1 expression decreased at the peak of DDC-mediated liver damage (day 5). In line with this, we found that 5hmC levels were transiently increased in cholangiocytes in vivo at day 3 at the transcriptional start site of >3000 genes, which are mainly involved in biological processes such as development, chromatin modifications, and cell-cycle (in vivo allows cholangiocytes to both respond to liver injury and return to the homeostatic state to avoid the detrimental effects of an aberrant progenitor response. Supporting this hypothesis, Lgr5+ cells have been identified as tumour initiating cells in mouse liver cancer (Of note, the increase in ll-cycle . Therefor cancer  and YAP r cancer .What epigenetic mechanisms regulate cholangiocyte differentiation into hepatocytes? Following experiments in zebrafish that led to the identification of a regenerative role for Hdac1 by repressing the expression of Sox9 and modulating the Notch signalling, These findings highlight the relevance of dynamic epigenetic regulation of cholangiocyte plasticity for liver regeneration in response to severe or persistent liver injury that compromises the hepatocytes. Of note, ductular reaction is often observed in human chronic liver disease, concomitantly with massive hepatocyte alterations and necrosis . TherefoHdac3 deletion in mouse resulted in hepatic steatosis (Epigenetic mechanisms are often regulated by metabolic inputs . For exateatosis . In additeatosis .Together, this suggests that investigating the complex interplay between epigenetics and metabolism will represent an important step to understand how the liver achieves efficient regeneration and identify the molecular mechanisms that influence initiation and progression of chronic liver disease.In this review, I have examined the role of several chromatin modifiers that regulate epithelial cell-fate changes to achieve efficient liver regeneration after injury in mouse models . It is wARID1A have been reported in >16% of human hepatocellular carcinoma (The expression levels of chromatin modifiers and genomic distribution of their epigenetic marks are often altered in human chronic liver disease and liver cancer . For exaarcinoma . Arid1a arcinoma . UHRF1 iarcinoma , as obsearcinoma . FurtherPharmacological interventions based on drugs targeting epigenetic activity have a huge potential for the development of therapeutic strategies in human disease and cancer . TherefoThe author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: With the increase in aging population, the rising prevalence of osteoporosis (OP) has become an important medical issue. Accumulating evidence showed a close relationship between OP and hematopoiesis and emerging proofs revealed that platelets (PLTs), unique blood elements, rich in growth factors (GFs), play a critical role in bone remodeling. The aim of this review was to evaluate how PLT features, size, volume, bioactive GFs released, existing GFs in PLTs and PLT derivatives change and behave during OP. Methods: A systematic search was carried out in PubMed, Scopus, Web of Science Core Collection and Cochrane Central Register of Controlled Trials databases to identify preclinical and clinical studies in the last 10 years on PLT function/features and growth factor in PLTs and on PLT derivatives during OP. The methodological quality of included studies was assessed by QUIPS tool for assessing risk of bias in the clinical studies and by the SYRCLE tool for assessing risk of bias in animal studies. Results: In the initial search, 2761 studies were obtained, only 47 articles were submitted to complete reading, and 23 articles were selected for the analysis, 13 on PLT function/features and growth factor in PLTs and 10 on PLT derivatives. Risk of bias of almost all animal studies was high, while the in the clinical studies risk of bias was prevalently moderate/low for the most of the studies. The majority of the evaluated studies highlighted a positive correlation between PLT size/volume and bone mineralization and an improvement in bone regeneration ability by using PLTs bioactive GFs and PLT derivatives. Conclusions: The application of PLT features as OP markers and of PLT-derived compounds as therapeutic approach to promote bone healing during OP need to be further confirmed to provide clear evidence for the real efficacy of these interventions and to contribute to the clinical translation. PLT. PLT9]. These GFs play a central role in the healing process and tissue regeneration, being used as messengers to regulate various processes . Tissue It has been observed that PLT derivatives  improved proliferation and osteogenic activity of bone marrow mesenchymal stem cells (BMSCs) and osteoblasts ,21,22. ATo date, many key questions remain unanswered and controversial, in particular concerning PLT function, size, volume, role of bioactive GFs released and use of PLT derivatives during OP pathogenesis. Thus, we carried out a systematic review in which we wondered: How do PLTs work and what changes occur in their function, features and/or structure  during OP? How do GFs released by PLTs or GFs existing in PLTs and PLT derivatives “work” during OP? Which are the main derivatives used in OP? and How are they used? In the present systematic review we tried to highlight and answer to these points, attempting to give an up-to-date tool for researchers and clinicians involved in PLT-mediated bone tissue regenerative applications in OP condition.The PICOS model was used to formulate the questions for this study: (1) studies that considered cells, animals and patients with OP (Population), (2) studies where one of the primary aims were to evaluate PLTs and PLTs derivatives during OP (Interventions), (3) studies that presented a control interventions (Comparisons), (3) studies that reported the effects/functions/roles of PLTs and PLTs derivatives during OP (Outcomes) and (4) preclinical (in vitro and in vivo) and clinical studies (Study design). Studies from 27 July 2009 to 27 July 2019 were included in this review if they met the PICOS criteria.We excluded studies investigating (1) PLTs functions and/or PLTs derivatives in pathological conditions different from OP, (2) pathological conditions where OP is a bone manifestation of another disease , (3) osteonecrosis of the jaw due to OP therapy, (4) PLT functions and/or PLT derivatives during the administration of drugs active on bone metabolism , (5) drug -induced osteoporosis. Additionally, we excluded case reports, abstracts, editorials, letters, comment to Editor, reviews, meta-analysis, book chapters and articles not written in English.Our literature review involved a systematic search conducted on 27 July 2019. We performed our review according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement . The seawww.mendeley.com”) to eliminate duplicates. Subsequently, the remaining full text articles were retrieved and examined by two reviewers . Any disagreement was resolved through discussion until a consensus was reached, or with the involvement of a third reviewer (MF).Possible relevant articles were screened using title and abstract by one reviewer (FS) and articles that did not meet the inclusion criteria were excluded. After screening the title and abstract, articles were submitted to a public reference manager  independently assessed the methodological quality of selected studies . In caseThe initial literature search retrieved 2928 studies. Of those, 1169 studies were identified using PubMed/MEDLINE, 167 using ProQuest, 134 using Scopus, 1141 were found in Web of Science Core Collection and 317 using Cochrane Central Register of Controlled Clinical Trials. After screening the title and abstract107 articles were run through Mendeley to eliminate duplicate articles. The resulting 49 complete articles were then reviewed to establish whether the publication met the inclusion criteria and 23 were considered eligible for this review. From the reference lists of the selected articles no additional publications were found. Search strategy and study inclusion and exclusion criteria are detailed in n = 13) (n = 10)   and stud(n = 10) . Each tan = 8; 75%), with the exception of two studies that have high risk due the lack of information [Risks of bias assessments for each clinical study were summarized in ormation ,45, for ormation  and outcn = 7) of the studies showed that groups were similar concerning baseline characteristics  and two studies showed that allocation was adequately concealed [n = 10), reported and detailed the primary outcome (n = 12) and were apparently free of other problems that could result in high risk of bias (n = 8)  .n = 9), two were in vivo studies, one was both in vivo and clinical and another was exclusively in vitro  value considering different cohorts of woman, i.e., healthy, osteopenic and OP ,46,47. T2, hemostatic sponge) to incorporate the PLTs derivatives and subsequently evaluated bone regeneration in calvaria [2 nanoporous implant associated with PRP [2 not only promoted the osteogenesis but also increased the expression of RUNX2 and COL1 genes and suppressed osteoclastogenesis with increased expression of OPG and decreased levels of RANKL [All 10 articles on PLT derivatives in OP were about vivo or both in vivo and in vitro studies. With the exception of the study by Rocha et al.  that usecalvaria ,58, tibicalvaria ,56 and icalvaria ,57. Clafcalvaria  evaluaticalvaria . Differewith PRP . Additioof RANKL . Despiteof RANKL ,57. Two of RANKL  and β-TCof RANKL  and lumbof RANKL  and incrDifferently from the above mentioned studies, four studies (40%) used PRP without scaffold/biomaterial to analyze the balance between adipogenesis and osteogenesis in bone regeneration , to induTo date OP management still remains a difficult task for clinicians and based on the progressively increase of aging population the global implications of OP and impaired bone healing are considerable. In the past decade, an increasing number of studies explored the use of new and advanced markers as well as of bioactive factors able to promote bone formation/regeneration during OP ,60. AlthIn this review, preclinical studies on PLTs function/features and growth factor in PLTs during OP mainly deal with PDGF, a naturally molecule released from the α-granules of PLTs, as part of the clotting process that occurs in response to injury . HomodimAnother key question concerning PLTs regards the role and use of their derivatives during OP. The rationale for PLT derivatives use in bone healing process is due to the abundance and accessibility of key GFs and other signaling molecules in PLTs ,66,67. Tscenario.Given the fundamental role of PLT features  and PLT derivatives in musculoskeletal repair and regeneration, their future role in OP is expected to expand. Additional researches are under way to further improve our understanding on PLT as markers for OP and on PLT derivatives as therapeutic treatment to enhance bone healing and control inflammation during OP. These future investigations will hopefully continue to shed more light on how PLTs could best used to further improve the outcomes of OP patients in the clinical


Quantitative skills are becoming central to the undergraduate and graduate curriculum in ecology and evolutionary biology. While previous studies acknowledge that students perceive their quantitative training to be inadequate, there is little guidance on best practices. Moreover, with the recent COVID‐19 sudden transition to online learning, there is even less guidance on how to effectively teach quantitative ecology online. Here, I synthesize a prescription of pedagogical best practices for teaching quantitative ecology online based on a broad review of the literature on multiple quantitative disciplines. These best practices include the following: (1) design and implement the class to meet learning goals using online strategies specifically; (2) create an open, inclusive, and welcoming online environment that promotes a sense of learning community; (3) acknowledge the diversity of talents and learning strategies; (4) use real‐world examples and assessments; (5) account for gaps in knowledge; (6) emphasize the modeling cycle process; (7) focus on developing ideas rather than tools or procedures; (8) if needed, introduce computational tools thoroughly before combining them with mathematical or statistical concepts; (9) evaluate the course constantly; and (10) put your heart and soul into the class. I hope these practices help fellow instructors of quantitative ecology facing similar challenges in providing our students with the knowledge and skills needed to meet the challenges of the future. Quantitative skills are becoming central to the undergraduate and graduate curriculum in ecology and evolutionary biology. While previous studies acknowledge that students perceive their quantitative training to be inadequate, there is little guidance on best practices. I synthesized a prescription of pedagogical best practices for teaching quantitative ecology online based on a broad review of the literature on multiple quantitative disciplines. In 2012, more than 70% of studies published in popular ecology journals applied some type of advanced statistical model . Therefore, this list of best practices emphasizes the quantitative/modeling aspect of the online course. For best practices for teaching computational skills, I encourage the reader to look at the extensive literature in the pedagogy of computer science  Feedback should be quick, personalized, and constructive. This constructive feedback addresses the learner by name, is balanced, specific, uses a positive tone, and asks questions promoting thinking ; Funding acquisition ; Investigation ; Project administration ; Writing—original draft ; Writing—review & editing .