We investigated these discrepancies that question the sufficiency and robustness of hippocampal theta generation using a biophysical spiking network model of the CA3 region of the hippocampus that included an interconnected network of pyramidal cells, inhibitory basket cells (BC) and oriens-lacunosum moleculare (OLM) cells. The model was developed by matching biological data characterizing neuronal firing patterns, synaptic dynamics, short-term synaptic plasticity, neuromodulatory inputs, and the three-dimensional organization of the hippocampus. The model generated theta power robustly through five cooperating generators: spiking oscillations of pyramidal cells, recurrent connections between them, slow-firing interneurons and pyramidal cells subnetwork, the fast-spiking interneurons and pyramidal cells subnetwork, and non-rhythmic structured external input from entorhinal cortex to CA3. We used the modeling framework to quantify the relative contributions of each of these generators to theta power, across different cholinergic states. The largest contribution to theta power was that of the divergent input from the entorhinal cortex to CA3, despite being constrained to random Poisson activity. We found that the low cholinergic states engaged the recurrent connections in generating theta activity, whereas high cholinergic states utilized the OLM-pyramidal subnetwork. These findings revealed that theta might be generated differently across cholinergic states, and demonstrated a direct link between specific theta generators and neuromodulatory states.Hippocampal theta oscillations (4–12 Hz) are consistently recorded during memory tasks and spatial navigation. Despite several known circuits and structures that generate hippocampal theta locally  Howev[in vivo , and the[in vivo . As a se[in vivo –13, or t[in vivo –17. Spik[in vivo ,19. A th[in vivo ,20–23; e[in vivo ,25. Thesin vivo by disruption of the input from the medial septum [In addition, despite the presence of these intrinsic hippocampal generators, external input plays a major role and hippocampal theta is severely attenuated l septum –30 and fl septum . The conl septum , but thel septum ). Non-rhl septum –36 and il septum , but hasin vivo. Contributions from two theta generators were dependent on cholinergic state. Low cholinergic states engaged the recurrent connections amongst pyramidal cells for theta generation, while high cholinergic states utilized the OLM-pyramidal cells subnetwork, indicating that the low and the high cholinergic states had distinct mechanisms for theta generation, with specific cholinergic effects fostering the engagement of certain theta generators.We used our previously developed biophysical computational model of the hippocampus  that incWe investigated theta generating mechanisms in the hippocampus using our published biophysical model that included networks for entorhinal cortex (EC), dentate gyrus (DG), and CA3 regions, and their interconnections . The sinResults reported below represent data averaged over 10 instantiations of the network with different random seeds for initial cell membrane potentials, synaptic connections, synaptic weights, and random external inputs. To examine the non-rhythmic component of medial septal and EC input, we used an input layer with 30 spiking neurons with no spike-frequency adaptation (referred to as ‘EC’). External input arrived as Poisson spikes to either this layer or directly to pyramidal cells depending on the experiment setup, at rates constrained to produce reported firing rates of hippocampal place cells during active locomotion [The full model  generatePrevious studies have suggested multiple potential theta generators in the hippocampus ,49, so wThe simplest case examined had isolated pyramidal CA3 cells . The celTo further explore the relationship between spike-frequency adaptation and spiking oscillations, we lowered the time constant of adaptation from 100 ms to 25 ms , we continued to observe theta oscillations . To explNext, we examined the effects of inactivating individual theta generators, across different cholinergic states. We started with the full model with external random inputs arriving at EC (DG area and the h-current remained inactive). We then inactivated each theta generator individually and observed changes in the relative theta power (ratio of power in the 4–12 Hz range to the entire spectrum from 0 to 250 Hz). To inactivate the spiking oscillations of pyramidal cells we removed spike-frequency adaptation by lowering the adaptation time constant to 10 ms  state affected the cells and synapses of the network and took values from 0 (lowest) to 2  received external input as trains of Poisson-distributed spikes, triggering an influx of AMPA and NMDA currents into the cell. We studied two model cases: one with external input arriving at EC, and the other with input arriving directly at CA3 pyramidal cells. The two types of inputs differed in the weight of the associated input synapses, and the base rate of the Poisson spike trains arriving at these synapses. Input to EC arrived at synapses with a 100% spike transmission rate to ensure that EC firing pattern was dictated by the Poisson input, whereas input to CA3 pyramidal cells had a lower weight value with parameters matching the EC to CA3 synapses .To determine the base rates of the Poisson processes generating these input trains, we considered place cells in CA3. Place cells respond to certain areas in the environment and their firing rates approximate a lognormal distribution  with an For spectral analysis, we summed the spikes of all cells of each type in a region  in 0.1 ms bins and computed the fast-Fourier transform of the resulting vector, using the Matlab function psautospk.m , with a The coherence measure used was implemented as described by Wang and Buzsaki . BrieflyThe model was developed using the NEURON software package  and run


Oxidative and nitrosative stress are common problems when handling gametes in vitro. In vitro development in mammalian embryos is highly affected by culture conditions, especially by reactive oxygen species (ROS) and reactive nitrogen species (RNS), because their absence or overproduction causes embryo arrest and changes in gene expression. Melatonin in gamete co-incubation during in vitro fertilization (IVF) has deleterious or positive effects, depending on the concentration used in the culture medium, demonstrating the delicate balance between antioxidant and pro-oxidant activity. Further research is needed to better understand the possible impact of melatonin on the different IVP steps in humans and other mammals, especially in seasonal breeds where this neuro-hormone system highly regulates its reproduction physiology. The protocols for in vitro maturation, fertilization, and embryo culture in assisted reproductive techniques (ART) have been greatly improved during the last decade. However, only a few embryos produced by ARTs are capable of carrying out development to full term. This is mainly due to the lack of optimal in vitro conditions that cannot mimic the in vivo conditions, leading to several differences between both conditions such as increased levels of ROS or RNS . Both fr+2 in oocytes [There is a duality in the role of ROS and RNS. Physiological levels are needed in several contexts: ROS are necessary in human follicles to establish pregnancy , as pote oocytes , and for oocytes . In sper oocytes , sperm c oocytes ,12,13,14 oocytes , and the oocytes .RNS, on the other side, are necessary for the development of large antral follicles ,18, to sWhen an imbalance between pro-oxidant molecules occurs due to the increase of ROS/RNS levels or the reduction of the antioxidant defense mechanisms, the phenomenon called oxidative or nitrosative stress is triggered ,30.2) is an essential element for aerobic organisms because oxidative metabolism represents the main energy source. The partial reduction of O2 results in ROS formation; these are molecules that contain one oxygen atom in their structure and possess at least one highly reactive unpaired electron in an outer orbital [•O2−) and hydroxyl radical (•OH), and molecules such as hydrogen peroxide (H2O2) [•O2− is the initial step for the formation of ROS, which is generated by the acceptance of an electron by O2, catalyzed by NADPH oxidase or xanthine oxidase. This radical can be converted into H2O2 by the action of the superoxide dismutase (SOD) enzyme, and then degraded to H2O and O2 by catalase or glutathione peroxidase [•OH is generated during the Haber-Weiss reaction which produces more toxic free radicals through the interaction between •O2− and H2O2 [+3 and Fe+2), the Fenton reaction, can also generate •OH [Oxygen  . The proroxidase . •OH is and H2O2 . Alternarate •OH .2O2 during in vitro embryo production [2O2 levels could induce stress tolerance in these embryos, determined by the embryo development, quality, and gene expression pattern [In pathological events, ROS have been involved in patients with endometriosis . The totoduction . We obse pattern .d-or l-arginine [•O2− yielding peroxynitrite (ONOO−), which is a potent oxidant inducing protein, lipid, and DNA damage [Like ROS, RNS such as nitric oxide (NO) act as signaling molecules modulating various aspects of the reproductive physiology ; they inarginine . High anarginine , leadingarginine . The cheA damage .Previously, we investigated nitrosative stress tolerance in oocytes by the in vitro incubation of oocytes adding NO donors during in vitro embryo production in bovine. However, no differences in the embryo quality or resistance to nitrosative stress were observed for incubation with either added 3-morpholinosidnonimine (SIN-1)  or addedCurrently, the administration of antioxidants is recommended for counteracting oxidative and nitrosative stress in cells. This review synthesises the experimental data that has been published and the advances in the knowledge on the effects of melatonin on gametes and the different steps of in vitro reproduction. Here, we argue that the application of melatonin should be considered for improving the efficiency and outcomes of reproductive biotechnologies in humans and domestic animals due to its capacity as a powerful antioxidant counteracting ROS/NOS induced damage.N-acetyl-5-methoxytryptamine) is a multifunctional molecule secreted by the pineal gland in response to changes in light levels and other tissues [Melatonin  and vitamin E (600 mg/day) treatments . The fer2O2 exposure [MnSOD expression [Spermatozoa are sensitive to oxidative stress, leading to an apoptosis-like process. Melatonin can decrease mitochondrial ROS production when sperm is exposed to oxidative stress . Thus, iexposure , increaspression , glutathpression , and glupression ; preventpression , and thepression . Additiopression , which ipression . This anpression . Melatonpression .Our experience during the supplementation of IVF medium with melatonin shows that this antioxidant has a dual effect over sperm function and embryo development in bovine : lower cCryopreservation is a highly stressful process that significantly reduces the potential for embryo development. Melatonin added to the culture medium increases cleavage, blastocyst, and hatching rates ,134. It Melatonin has been described to reduce nitrosative/oxidative stress in many different tissues and organelles . These dMelatonin has been shown to be protective for gamete handling in vitro. It has been proposed that melatonin inhibits the activity of the pro-oxidative enzyme nitric oxide synthase (NOS) in the Graafian follicle . MelatonA beneficial effect on male fertility has been described for humans and domestic animals: melatonin induces a significant decrease in the intracellular NO concentration in human sperm, increasing sperm motility and viability . The NO ARTs can induce vascular dysfunction and arterial hypertension related to epigenetic alterations of the regulation of the eNOS gene. However, this can be prevented by the addition of melatonin during the in vitro culture of embryos, which has doubled the success rate of IVF . Melatonin has been found to protect the fetus and placenta from oxidative stress due to ROS and RNS . ClinicaDespite the fact that more specific studies regarding the melatonin effect against nitrosative stress in reproductive biotechnologies are scarce, melatonin has demonstrated direct and indirect beneficial effects against ROS. Therefore, considering that ROS can generate RNS, we can deduct that melatonin could have a protective action over nitrosative stress during gamete and embryo handling in the laboratory, as has been demonstrated in other tissues.There is a long list of studies that support the use of melatonin against oxidative stress; however, much remains to be investigated regarding the role that melatonin might have on nitrosative stress during the in vitro manipulation and cryopreservation of gametes and embryos. Therefore, the evidence is clear that melatonin is involved in the protection against oxidative/nitrosative stress by scavenging free radicals, inducing the activity of antioxidant enzymes and preventing the induction of the mitochondrial pathway of apoptosis, improving gamete and embryo quality, both in human and domestic animals during ARTs.


Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are reversevoltage-dependent, and their activation depends on the hyperpolarization of themembrane and may be directly or indirectly regulated by the cyclic adenosinemonophosphate (cAMP) or other signal-transduction cascades. The distribution,quantity and activation states of HCN channels differ in tissues throughout thebody. Evidence exhibits that HCN channels play critical roles in the generationand conduction of the electrical impulse and the physiopathological process ofsome cardiac diseases. They may constitute promising drug targets in thetreatment of these cardiac diseases. Pharmacological treatment targeting HCNchannels is of benefit to these cardiac conditions. Thehyperpolarization-activated funny current (If) is a major determinant of cardiacdiastolic depolarization and plays a key role in controlling heart rate. The expression of HCN channel isage-related, the expression has difference in distinct growth stages, HCN genes areinactivated in ventricular myocytes cells following maturation whereas theyexpressed in these cells in the fetal and/or neonatal heart-8. Three isoforms  are expressed in cardiactissues, with HCN2 and HCN4 the dominant subtypes. In the healthy adult heart, HCNchannels are predominantly expressed in the conduction system, especially in thesinoatrial node, HCN4 has been determined as the principal HCN isoform in sinoatrialnode cells. However, some studies demonstrate a significant expression of HCN1 andalso a small amount of HCN2 beneath HCN4 in murine sinoatrial cells at the proteinlevel, indicating that the three subunits together contribute to If in thesecells,9. HCN2 has long been known to be expressed incardiac ventricular myocytes,and recently, the expression of HCN4 and low amounts of HCN1 and in addition afunctional role of HCN3 in ventricular myocytes has been demonstrated,12, but the expression of HCN channels in healthy adultventricular myocardium is generally weaker than in the conduction system, so Ifcurrents are rarely detectable in normal ventricular myocytes,14. The kinetics of HCN1 is the fastest, followed by HCN2,and the slowest is HCN4. cAMPcan make the potential of HCN channel open probability curve more positive. HCN2 andHCN4 channels increased 10-20 mV, and HCN1 only 5 mV. HCN channel is also affectedby the endocrine system. Studies have demonstrated that HCN2 and HCN4 genetranscription is controlled by thyroid hormone, so it is speculated that HCN2 andHCN4 gene down-regulation level is an important mechanism for bradycardia induced byhypothyroidism. Positive inotropic effect of thyroid hormone may be associated withupregulation of the channel molecules,17.The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels comprise afamily of cation channels activated by hyperpolarized membrane potentials andstimulated by intracellular cyclic nucleotides. The members of the family are fourhuman isoforms (HCN1-4). Upon hyperpolarization, all four isoforms generate a inwardcurrent (If) in the heart and in the nervous system. Undernormal conditions, HCN channels are poorly expressed outside the cardiac pacemakingand conduction system,18-20, but it changes during cardiac disease. Duringhypertension and heart failure, If activity is observed in the ventricularmyocardium due to re-expression of HCN genes,21-23, several studies proved that If current densityand occurrence is significantly greater in hypertrophic cardiomyocytes and end-stagefailing hearts and this is directly related to the arrhythmias,21,24-27. Basing on the presence of If current inventricular myocytes isolated from severely hypertrophied rat hearts, the currentarrhythmogenic role in cardiac hypertrophy and failure has been inferred, and its density is larger inhuman ventricular myocytes isolated from the hearts of patients with ischemic thanin those with dilated cardiomyopathy. Stillitamo et al. demonstrated for the first time comparing the mRNA andprotein expression of HCN subunits in the human atrial and ventricle under normaland heart failure conditions in human heart failure, an upregulation of ventricularHCN2 and HCN4 underlies the increase in functional If current, Michael etal.studied the modelwith ventricular HCN2/4 knockout and identified the enhanced If as an importantcontributor to the typical electrophysiological alterations observed in thehypertrophic heart, including prolonged ventricular action potentials and lengthenedQT intervals. So the overexpression of If may contribute to increased ventricular propensity to arrhythmias which leading to sudden death caused byventricular tachycardia or fibrillation in heart failure and hypertrophic heart.Cardiac dysfunction is a complex and important problem for people with epilepsy.Recently, a study has showed that cardiac electrophysiology was significantlyaltered in adult rat models of genetic or acquired epilepsy, with slower heart rate,shorter QRS duration, longer QTc interval, greater standard deviation of RRintervals, and bradycardia, molecular analysis demonstrated significant reductionsin cardiac HCN2 mRNA and protein expression in models, providing a molecularcorrelate of these electrophysiologic abnormalities.A large number of studies have found that in heart failure, atrial fibrillation,myocardial hypertrophy and myocardial infarction, atrial and ventricular HCN2 andHCN4 channels gene express unusually causing atrial or ventricular myocyte Ifcurrent rise or fall, and all of these may be associated with heart disease fatalarrhythmia. Atrialfibrillation is the most common sustained tachyarrhythmia and is one of the majorcauses of cerebrovascular accident,32. Clinicalelectrophysiology studies in patients have demonstrated that rapid focal activityoriginating from PV can trigger and maintain atrial fibrillation-35. A study found that the pacing-induced spontaneous cationpotential and larger If current were observed in the PVs cardiac myocytes fromcanine using RAP , and a slow diastolic depolarization in thePVs cells. Meanwhile If current densities were significantly higher. As mentionedabove, delayed ventricular repolarization and prolonged QT intervals exist inhypertrophic heart, and they can promote early afterdepolarizations and electricalinstability, so ventricular HCN channels may play a significant role in the diseasedheart by increasing the risk for severe ventricular arrhythmias,37. For the patients with advanced atrioventricular block orsick sinus syndrome, pacemakers are implanted. Biological pacemakers have severaladvantages , so the efforts to use gene therapy to create a biologicalpacemaker have been ongoing for many years. And most of the efforts focused on HCNgene family of channels. Plotnikov et al. has proved that HCN212 chimeric channel is noteworthy,therefore it resulted in tachycardia when overexpressed in the canine left bundlebranch to achieve the desired physiological range.HCN channels are essential for cardiac pacemaker and electric conduction. The cardiachyperpolarization activated cation current (If) is known to be present in regionwith primary or second pacemaker activity, which is found in nonpacemaking regionsof the heart. In pacemaker regions, If is believed to contributed to spontaneousdiastolic depolarization. HCN1-knockout animals display a dysfunction of thesinoatrial node resulting in sinus dysrhythmia, sinus pauses and a reduced cardiacoutput, demonstrating that HCN1 contributes to a stable cardiac rhythmgenerationanalyzed the effect of ivabradine on survival and rhythmicity in a transgenic mousemodel (dnNRSF) of dilated cardiomyophathy, and the founding is although an increasedcardiac expression of If accompanied by ventricular tachyarrhythmias and suddenarrhythmic death, the survival was improved significantly when ivabradine wasadministrated at low doses without affecting heart rate. Ivabradine can markedlyreduce If currents in the RAP  cells in aconcentration-dependence manner. It has been proved that Ivabradine can reduce heart ratewithout exerting negative inotropic or vasodilatory effects. A recent clinical trial, theSystolic Heart failure treatment with the If inhibitor ivabradine Trial (SHIFT),showed the outcomes of patients with resting heart rates ≥70 bpm weresignificantly improved with the heart rate decreased. A subgroup analysis of BEAUTIFUL trial suggested a specificbenefit for patients with heart rates over 70 bpm, and echocardiography revealed that the reductionin the left ventricular systolic volume index observed in the ivabradine treatmentwas related to the degree of heart rate reduction, although the large study showedthe composite endpoint was not significantly different between the ivabradine andthe placebo groups. All of the results suggest that ivabradine improves cardiacdiseases outcome depends on its reduction of heart rates. In the treatment ofchronic heart failure, β-receptor blockers are used widely, but somepatients cannot tolerant β-receptor blockers , thus,ivabradine maybe the alternative. Another study examined the effects of ivabradineon survival and arrhythmicity in transgenic mice (dnNRSF-Tg). In this study,ivabradine was used 7 mg/kg per day orally, and the results showed it significantlyreduced ventricular tachyarrhythmias and improved survival among dnNRSF-Tg micewhile having no significant effect on heart rate or cardiac structure or function.Ivabradine inhibited the abnormal automaticity induced by β-adrenergicstimulation and prevented the increase in automaticity in dnNRSF-Tg mice. It is well known that theincidence of sinoatrial node dysfunction increases with age and is accompanied byatrial fibrillation (AF) and other tachyarrhythmia. AF is one of the most common arrhythmias. It isestimated that 84% of the patients with AF are over 65 years old, and the incidencerate of AF doubles every 10 years, the incidence rate of AF is 10% in people over 85years old. It can lead to congestive heart failure, thromboembolism, andcerebrovascular accident. So the mortality rate of AF is very high,46. Recent animal study showed that dogs with agerelated AFinduced by rapid atrial pacing administered ivabradine for intervention, andivabradine could increase the effective refractory periods in the left atrium andleft superior pulmonary vein, reduce the duration of AF after induction, andsignificantly decrease the inducing rate of AF (the incidence rate of AF decreasedfrom 50% to 25%). Therefore, ivabradine has potential effects in preventingage-related AF. Amiodarone isone of the most frequently used anti-arrhythmic drugs. Amiodarone was shown toinhibit HCN channel currents in oocytes and HEK-cells. To determine the effects ofamiodarone under pathological conditions, a study monitored If under the amiodaronetreatment in ventricular myocytes from spontaneously hypertensive rats with leftventricular hypertrophy using the whole-cell patch-clamp technique. The findings ofthe study showed that the incubation of myocytes with amiodarone significantlysuppressed If density and downregulated HCN2 and HCN4 expression at both the mRNAand protein level. This is the first study indicating that amiodarone inhibits Ifunder hypertrophied condition.It is well known that slow heart beat is good for the heart. Therefore, HCN channelsbecome the main target of the drug study controlling rapid arrhythmia. Recently,there are many studies about ivabradine, the first selective blocker of If current.A work done by Kuwabara et al.in vitro andin vivo gene transduction experiments proved the feasibility ofconstructing cardiac biological pacemaker, but its clinical application and safetyissues is yet to be confirmed further. In order to avoid side effects of HCN channelblockers, the subtype-selective HCN channel blocker is promising.With the development of the study on HCN channels, the importance of HCN channels onheart diseases attracted more attention. The biological pacemaker based on HCNchannels has attached much attention, and a lot of


Mitochondrial health is critically dependent on the ability of mitochondria to undergo changes in mitochondrial morphology, a process which is regulated by mitochondrial shaping proteins. Mitochondria undergo fission to generate fragmented discrete organelles, a process which is mediated by the mitochondrial fission proteins , and is required for cell division, and to remove damaged mitochondria by mitophagy. Mitochondria undergo fusion to form elongated interconnected networks, a process which is orchestrated by the mitochondrial fusion proteins , and which enables the replenishment of damaged mitochondrial DNA. In the adult heart, mitochondria are relatively static, are constrained in their movement, and are characteristically arranged into 3 distinct subpopulations based on their locality and function . Although the mitochondria are arranged differently, emerging data supports a role for the mitochondrial shaping proteins in cardiac health and disease. Interestingly, in the adult heart, it appears that the pleiotropic effects of the mitochondrial fusion proteins, Mfn2  and OPA1  may play more important roles than their pro-fusion effects. In this review article, we provide an overview of the mitochondrial fusion and fission proteins in the adult heart, and highlight their roles as novel therapeutic targets for treating cardiac disease. Mitochondrial health is critically dependent on the ability of mitochondria to move and change their morphology. By undergoing fission they generate fragmented discrete mitochondria, a process which is regulated by the mitochondrial fission proteins, dynamic-related peptide-1 (Drp1), human fission protein-1 (hFis1), mitochondrial fission factor (Mff) and mitochondrial dynamics proteins 49 and 51 (MiD49 and 51). Mitochondrial fission is essential for cell division and is required to remove damaged mitochondria by mitophagy. In contrast, the fusion of mitochondria generates elongated interconnected networks, a process which is orchestrated by the mitochondrial fusion proteins, Mitofusins 1 and 2 (Mfn1 and Mfn2), and optic atrophy protein-1 (OPA1), thereby enabling the replenishment of damaged mitochondrial DNA .Fi.Fimdivi-Emerging studies suggest that changes in mitochondrial morphology occur in different cardiac disease and pharmacological targeting of the mitochondrial fusion and fission proteins may provide a novel therapeutic strategy for treating cardiac diseases such as acute myocardial infarction, LVH, PAH, and heart failure. However, it is important to take into consideration the pleiotropic roles of the mitochondrial shaping proteins especially the fusion proteins, Mfn2 and OPA1, when targeting the mitochondrial shaping proteins as a treatment for cardiac disease. Furthermore, although acute inhibition or activation of the shaping proteins may be beneficial in certain cardiac diseases, the chronic inhibition or activation of either mitochondrial fusion or fission proteins may have detrimental off-target effects.


As such, this article aims to review study results which evaluated effects of cannabinoids from plant, synthetic and endogenous origins on cancer development in preclinical animal models and to examine the current standing of cannabinoids that are being tested in human cancer patients.Cancer is the second leading cause of death in the United States with 1.7 million new cases estimated to be diagnosed in 2016. This disease remains a formidable clinical challenge and represents a substantial financial burden to the US health care system. Therefore, research and development of novel therapeutics for the treatment of cancer is of high priority. Cannabinoids and their derivatives have been utilized for their medicinal and therapeutic properties throughout history. Cannabinoid activity is regulated by the endocannabinoid system (ECS), which is comprised of cannabinoid receptors, transporters, and enzymes involved in cannabinoid synthesis and breakdown. More recently, cannabinoids have gained special attention for their role in cancer cell proliferation and death. However, many studies investigated these effects using  Cannabinoids are a class of hydrophobic molecules that typically bind to G-protein-coupled cannabinoid receptors, cannabinoid receptor type 1 (CB1R) or cannabinoid receptor type 2 (CB2R). Endogenously synthesized cannabinoids (endocannabinoids), cannabinoid receptors, and enzymes involved in cannabinoid formation and breakdown are collectively known as the endocannabinoid system (ECS). This system has been shown to impact different neurological and immunological processes Zhu, . AdditioAnimal tumor models have become a mainstay in preclinical cancer research due to their ability to produce fairly predictable tumor growth. The first tumor model used to study tumorigenesis was the chemical carcinogenesis model. This model employs the use of two known carcinogens, an initiator and promotor. Initiators are typically employed in a single dose to cause irreversible DNA damage. A promotor is given in repeated doses to increase cell proliferation and induce subsequent DNA damage  cancers in 2016  is a potent inducer of glioma death in vivo. Utilizing a C6 glioma cell xenograft model, it was demonstrated that tumor growth was significantly reduced by Δ9-THC  in which tumor development was reduced in p8+/+ animals treated with Δ9-THC compared to vehicle treated animals. In contrast, no significant difference in tumor growth was observed in Δ9-THC or vehicle treated p8−/− mice suggesting that p8 was needed for Δ9-THC-mediated tumor death. In a subsequent study by this group, endoplasmic reticulum stress-mediated autophagy and cell death was observed in Δ9-THC treated glioma. Specifically, Δ9-THC caused an increase in ER stress protein, TRB3, autophagy protein, LC3, and apoptosis as well as a decrease in mTORC protein, p-S6 in U87MG xenograft tumors   or Δ9-THC + CBD + TMZ significantly reduced tumorigenesis compared to the individual drugs  can promote or prevent tumor invasion and metastasis by degrading extracellular matrix and other cell-associated proteins  exhibited limited toxicity and did not affect animal survival, health, behavior or hematopoietic parameters  and activation of the CB2 receptor was involved in the anti-tumor effect. However, unlike Δ9-THC, CBD toxicity did not appear to be regulated by CB1 or ceramide. Furthermore, in an orthotopic and subcutaneous tumor model, CBD significantly decreased U251 cell tumor growth  antioxidant signaling as inhibition of this target decreased tumor cell survival and augmented the antitumor activity of CBD. A different study revealed that CBD potentiated the killing effect of ionizing radiation (IR), a common clinical therapy for glioblastoma. Using an orthotopic xenograft model, CBD + Δ9-THC + IR significantly decreased the size of GL261 gliomas compared to CBD + Δ9-THC or IR alone  is a non-psychotropic phytocannabinoid whose effect on tumor growth is being extensively explored. CBD significantly reduced U87MG xenograft growth when compared to vehicle control animals . However, additional experimentation in animals is needed to define the pharmacokinetic properties of cannabinoids before large scale human studies are conducted. It will be particularly important to examine drug accumulation in tumors and drug disposition in body compartments subsequent to drug administration via different routes as most of the glioma studies conducted thus far employ intra/peri-tumor drug delivery. Such investigations may identify more clinically convenient routes of administration, establish the extent of drug stability and metabolism while providing clues about potential adverse effects of these agents.The bulk of our knowledge about the effect of cannabinoids on cancer growth in Digestive system cancers include colon, stomach, liver, and pancreatic cancer. In the United States, these combined malignancies account for the highest incidence of cancer besides skin cancer and are the second leading cause of death for both sexes. In 2016, an estimated 304,930 new cases of digestive system cancer will be diagnosed in the US with 153,030 estimated deaths  or CB2 (Cannabis extract)  is the most lethal type of digestive cancer followed by pancreatic and then liver cancer. Recent studies advocate that the ECS plays a critical role in CRC development and is therefore considered an appropriate target for CRC inhibition. The levels of the endocannabinoid, anandamide (AEA), the enzymes participating in AEA biosynthesis and degradation, as well as its molecular target CB2, are elevated in tumor specimens of CRC patients  were mediated by the activation of CB2 receptors followed by increased ceramide, ER-stress, PPAR-γ activity, and eventually the induction of autophagy , activation of Wnt/JNK signaling (in vitro), and recruitment of the Fas death receptor into lipid rafts (in vitro) , which originates in hepatocytes and accounts for approximately 75% of liver cancers and cholangiocarcinoma, which develops within the bile ducts of the liver  increase tumor progression, high concentrations (>10 uM) reduce cancer cell proliferation  and/or progesterone receptors (PR); HER2-positive breast cancer, which expresses human epidermal growth factor receptor ; and triple negative breast cancer (TNBC), a type of breast cancer which is difficult to treat due to the lack of ER, PR, and HER2 expression. Cannabinoid receptor expression differs between these breast cancer sub-types. CB1 receptors were reported to be present in 28% of all breast carcinoma patients, with 14% being HER2 positive. On the other hand CB2 receptors were found in 72% of breast carcinomas and 91% of these tumors were HER2 positive, suggesting a possible link between HER2 and CB2 expression  and one TNBC xenograft model  and HER2 positive xenografted mice  or Akt similar to phytocannabinoids  mouse model , an anti-metastatic protein which has been suggested to suppress tumor growth via an immuno-surveillance mechanism  and melanoma, respectively. Skin cancer is the most common cancer in the United States with approximately 5.4 million new lesions (3.3 million cases) of NMSC and more than 76,000 new cases of melanoma diagnosed each year , Glodde's group demonstrated that the anti-tumor effect of Δ9-THC on HCmel12 xenografts was dependent on cannabinoid receptors  and wild type (CB1/2+/+) mice, it was determined that CB1 and CB2 receptors were required for papilloma formation initiated by UVB-light. In contrast, similar studies with CB1/2+/+ and CB1/2−/− mice in the DMBA/TPA model demonstrated that cannabinoid receptors were not required for skin tumorigenesis. Data from these two different studies indicate that the involvement of ECS in skin cancer development is dependent on the type of the stimulus . Following a washout phase , the safety and tolerability of Δ9-THC was then compared to placebo . The primary outcome measured was the incidence of adverse effects in patients receiving the combination of Δ9-THC and Temodar®. Secondary measures were the progression free survival at 6 months and overall survival. Additional study outcomes have not yet been reported. Δ9-THC was found to reduce tumor cell growth in vitro from 2 study participants and reduce tumor cell immunostaining in these same patients. Importantly, intracranial administration of Δ9-THC was found to be a safe and tolerable approach with no apparent psychoactive effects (Gúzman et al., in vivo animal data demonstrating anti-tumor effects of cannabinoids, offer promise for a clinical role for cannabinoids in the eradication of tumors.Guzman and colleagues were the first group to report a Phase I human study of the anti-metastasic effects of Δin vivo animal studies discussed here indicate that cannabinoids from plant, synthetic and endogenous origin are capable of effectively decreasing tumor growth and invasion (Table To gain an understanding regarding the translational and therapeutic potential of cannabinoids, this review focused on examining the overall efficacy of these molecules in animal and human studies. The majority of on Table . Furtherin vivo animal studies with in vitro experiments in order to examine proposed cannabinoid mechanisms of action and to identify specific cellular targets. The studies reviewed herein indicate that cannabinoids elicit activity through cannabinoid receptor dependent and independent pathways. Moreover, processes such as ceramide production, ER-stress, autophagy, angiogenesis and matrix remodeling also appear to regulate the anti-tumor activity of cannabinoids. Hence, these investigations shed light on the role of cannabinoids on tumor growth in vivo and may ultimately pave the way for the development of novel cannabinoid therapeutics for cancer treatment.This review also sought to couple DL, ES, LG, and RV contributed to the conception and design of the study, the acquisition of data, and the analysis and interpretation of the data. All authors participated in drafting or revising the manuscript, and all authors approved the final version of the manuscript for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Anal cancer is uncommon neoplasm with an incidence of 2 new cases per 100,000 per year in the USA, accounting approximately 0.4 % of all tumors and 2.5 % of gastrointestinal malignancies. An early detection of the anal cancer is crucial for the patient management, whereas the diagnosis at an early stage allows conservative management with sphincter sparing, on the contrary a delays in diagnosis might lead to an advance cancer stage at presentation with worst survival. According to National Comprehensive Cancer Network (NCCN) Anal Carcinoma guidelines the patients should be subjected to a careful clinical examination, including a digital rectal examination (DRE), an anoscopic examination, and palpation of inguinal nodes. The guidelines recommended for the assessment of T stage, only a clinical examination, while the role of imaging techniques, as Magnetic Resonance imaging (MRI) is limited to the identification of regional nodes. Instead, the endoanal ultrasound (EAUS) is not recommended. This paper presents an overview and some updates about 3D EAUS and MRI in detection, staging and assessment post therapy of anal cancer patients. Anal caner is uncommon neoplasm with an incidence of 2 new cases per 100,000 per year in the USA . CompareMost primary cancers of the anus are squamous cell carcinomas . LymphatBoth the 3-dimensional (3D) EAUS and MRI allows to perform a detailed evaluation of the multilayer wall of the anus, to detect the lesion, to stage the lesion, to identify the relations with adjacent structures and the presence of lymphadenopathy .This paper reports an overview and some updates about 3D EAUS and MRI in detection, staging and assessment post therapy in anal cancer patients.Data for this review were identified by searches of the PubMed database using a multimodal strategy. The following search terms were employed: endo sonography in anal cancer, three Dimensional-Endoanal-sonography in anal cancer, Magnetic Resonance in anal cancer, functional Magnetic Resonance in anal cancer, assessment post treatment of anal cancer, Magnetic Resonance after chemotherapy in anal cancer, endo sonography after neoadjuvant therapy in anal cancer. The inclusion criteria were: clinical study evaluating anal cancer, clinical study evaluating new functional imaging criteria in the MR study of patients with anal cancer, and clinical study evaluating follow-up after chemoradiotherapy of patients with anal cancer. Articles published in the English language from January 1989 to June 2016 were included. The references of these articles were also analyzed to identify original studies that were not identified by the search of the data. Exclusion criteria unavailability of full text and absence of original research data .A Pubmed search yielded 9 articles for key endo sonography in anal cancer, 2 articles for key 3 Dimensional-Endoanal-sonography in anal cancer, 442 articles for key Magnetic Resonance in anal cancer, 25 articles for key functional Magnetic Resonance in anal cancer, 31 articles for key assessment post treatment of anal cancer, 60 articles for key Magnetic Resonance after chemotherapy in anal cancer, 1 articles for key endo sonography after neoadjuvant therapy in anal cancer. According to inclusion and exclusion criteria, 26 articles were included at the end.3D-EAUS is a valuable tool to evaluate the normal anatomy and diseases of the anal canal Fig. . It is eThe advent of new therapies, more and more personalized to the patients and the recent improvements of neoadjuvant therapies, radiotherapy and chemotherapy can cause a down staging of the lesion, such as to make operable lesions that were not resectable before treatment –14. In a3D-EAUS has some limitations since it is highly operator dependent and it does not allow a reliable distinction between tumor and fibrosis.MRI is the gold standard in oncological pelvic examination, providing morphological and functional data. MRI leads to an excellent imaging of the rectal and anal wall, so to obtain an accurate evaluation of cancer stage including tumour infiltration degree, involvement of the internal and external sphincters, of the intersphincteric plane, and an effective assessment of lymph nodes status thanks to improvement of phased array coils and endorectal coils. A standard MRI protocol for anal cancer staging consists of turbo spin-echo MR sequences T2-weighted in the 3 spatial planes  with high spatial resolution Fig.  25, 26], 2625, 2MRI is a valuable diagnostic tool in anal cancer staging, although the major limitation is an incorrect detection of T1 patients , neitherAlthough the most widely used imaging tool for guidance of interstitial radiotherapy in anal cancer brachytherapy (BT) is EAUS , since iMR imaging plays an important role in therapeutic assessment, properly stratify patients into responders or non responders to neoadjuvant treatment, surveillance after surgery, and evaluation of suspected disease fall-out , 26. TheIn clinical practice, the recognized criteria for the assessment after therapy are response evaluation criteria in solid tumor (RECIST), which are based on size criteria. Although the morphological evaluation on MR images can identify a patient as responder to therapy, based on decreased of maximum diameter of the lesion,, it does not establish if the remaining tissue is cancer or fibrosis, and it does not seem to be linked to patient outcome. Goh et al.  evaluateSeveral studies evaluated the DCE-MRI as promising tool to monitor assessment after neoadjuvant therapy thanks to the link between tumor growth and angiogenesis , 41. It Many clinical trials showed that angiogenesis inhibition could increase the treatment effectiveness. Imaging techniques is able to assess tumour vascularization and the capability to improve the treatment management in oncologic patients , 41.DCE-MRI consists of a multiple T1-weigthed images acquired before and after contrast medium administration in specific time linked to study temporal resolution. It measures the rate of contrast movement between the intravascular and extra-cellular extravascular space. This rate reflects tissue microvasculature permeability and perfusion. In order to assess tissue perfusion by means of DCE-MRI, several approaches to analyse Time Intensity Curve (TIC) were proposed in literature. The most commonly used in the radiological practise is the visual inspection of TIC . AccordiAlthough the anal caner is rare neoplasm, the incidence of the tumor shows an incremental trend that reflects an increase in infection rates of HPV and HIV and immunosuppression state. The real rule of imaging techniques in detection, staging and follow-up of tumor is unclear. 3D EAUS and MRI are the best diagnostic tools in detection of the lesion, although 3D EAUS is more accurate than MRI for T1 stage Table . However


A number of intravital techniques originally developed for studying diverse biological events have recently been adapted to monitor axonal transport in real-time in a range of live organisms and are providing novel insight into this dynamic process. Here, we highlight these methodological advances in intravital imaging of axonal transport, outlining key strengths and limitations while discussing findings, possible improvements, and outstanding questions.Axonal transport is the active process whereby neurons transport cargoes such as organelles and proteins anterogradely from the cell body to the axon terminal and retrogradely in the opposite direction. Bi-directional transport in axons is absolutely essential for the functioning and survival of neurons and appears to be negatively impacted by both aging and diseases of the nervous system, such as Alzheimer’s disease and amyotrophic lateral sclerosis. The movement of individual cargoes along axons has been studied This bi-directional axonal transport is governed by the kinesin and cytoplasmic dynein motor proteins, respectively, and is essential for neuronal survival and function2. Given the large distances over which these processes must occur, it is perhaps unsurprising that disturbances in axonal transport have been linked to both ageing and many severe nervous system diseases, including Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS)5. Emphasising the importance of efficient axonal transport to nervous system health, mutations in a number of motor proteins have been identified as causative in neuronal disorders7.Neurons are highly polarised, excitable cells with long, thin axons whose integrity requires specialised mechanisms to transport cargoes such as organelles (e.g. mitochondria) and molecules (e.g. proteins and RNA) in anterograde  and retrograde  directionsex vivo tissue axons10; however, there is evidence to suggest that these artificial environments do not consistently reflect thein vivo situation14. Differences in transport dynamics, such as average speeds and pause frequencies11, could be attributed to limitations inherent to thesein vitro andex vivo platforms. Cultured primary neurons lack the array of cells with which neurons normally physically and chemically interactin situ; for example, cultured motor neurons are not myelinated, do not contact target muscle cells, and lack a network of regulated excitatory and inhibitory input onto their cell bodies. Myelination17, target-derived signals19, and activity23 are all known to impact axonal transport, whereas non-cell autonomous and age-dependent disease mechanisms are difficult to accurately modelin vitro. Mouse primary cultures are often derived from embryonic animals25 not currently analysedin vivo, which may also cause discrepancies, as could the current intrinsic variability of human induced pluripotent stem cell (hiPSC)-derived neuronal cultures26. Furthermore, the artificially controlledin vitro environment could affect subcellular energy demands and transport kinetics27, as might stress caused by axotomy and the continual growth of primary cultures. Intravital analysis of axonal transport of individual cargoes  remain unchanged45Consistent with sciatic nerve explant dataDrosophila marginal nerve45Reduced levels of the dynein co-factor Lissencephaly-1 caused an increase in the percentage of motile mitochondria in theDrosophila neurons; conversely, upregulating mitochondrial transport correlates with a delayed appearance of protein aggregates45Defective mitochondrial transport results in an increase of protein aggregation in58In zebrafish retinal ganglion cells (RGCs), disruption of Kif5A, a neuron-specific anterograde motor, resulted in increased frequency of retrograde mitochondrial transport but not synaptophysin-containing vesicles59 and Parkinson’s disease60 showed mitochondrial motility defects in disease-relevant nerve cellsZebrafish models of Charcot-Marie-Tooth disease type 2A60During larval development (2–5 days post-fertilisation), the percentage motility of mitochondria and the ratio of anterograde to retrograde movements progressively decreased in zebrafish central nervous system (CNS) dopaminergic neurons, whereas run length increased, but speeds remained stable70, display pre-symptomatic defects in the transport speeds of signalling endosomes in sciatic nerve axons67; mitochondrial transport is also perturbed in both SOD and TDP-43 models of ALS68Amyotrophic lateral sclerosis (ALS) mice, but not spinal and bulbar muscular atrophy mice71, which varies from the age-related transport deficiencies reported in different experimental settings at similar time points90Retrograde axonal transport kinetics  of signalling endosomes in the sciatic nerve remain unchanged from 1 to over 13 months77Bi-directional defects in the transport of both mitochondria and peroxisomes are detected in spinal cord axons of acute and chronic multiple sclerosis mouse models before major symptom onset12In mouse RGCs, the number of moving mitochondria, but not run length, was decreased prior to cell death in a glaucoma model, whereas the duration and distance of mitochondrial transport were both diminished with age (23–25 months)14The vast majority of mitochondria in neonatal and adult cortical pyramidal neurons remain stationary over periods of up to 20 minutesBox 2. What is the direct biological significance of altered cargo pausing and transport speeds?Are the defects in axonal transport observed in myriad neurological disease a cause of neuronal dysfunction or the consequences of a dysfunctional neuron?Will therapeutics targeting axonal transport ameliorate symptoms of these diseases?Does ageing impact all neuronal and cargo subtypes equally?What mechanisms underlie cargo-specific disturbances in transport versus global transport deficiencies?91 and motor proteins1 often manifest in a nervous system-specific pathology?Why do mutations in mitochondrialWhy are neurons particularly vulnerable to trafficking defects?What causes the axonal transport of distinct cargoes to be differentially affected by ageing and disease-associated mutations?Caenorhabditis elegans andDrosophila melanogaster allow the specific targeting of fluorescent proteins to vesicles and organelles such as mitochondria. When coupled with the ever-expanding repertoire of neurological disease-relevant worm and fly models34, these reporter lines provide a powerful system for analysing the axonal transport of assorted cargoes in both ageing and disease40. Transport studies inDrosophila have largely been performed in filleted larval preparations rather than adult flies, limiting the time period over which analyses can be performed and the developmental stage of the neurons under investigation. Third instar larvae are typically dissected for imaging of fluorescent cargoes predominantly in motor axons42. Alternatively, microfluidic devices that physically immobilise intactDrosophila larvae afford a non-invasive approach to image axonal transport44.The sophisticated genetic toolboxes ofDrosophila wing has been developed 45. Given the translucency of the wing and the accessibility of the marginal nerve, rapid and non-invasive light microscopy can be performed on different wing regions of flies expressing fluorescently tagged proteins specifically in neurons. Motivated by previous work in which the same nerve was used to evaluatein vivo responses to neuronal injury50, the GAL4-UAS system was implemented to visualise mitochondria and dense core vesicle (DCV) transport dynamics in flies up to 30 days old 45. Flies showed an early peak in the number of bi-directional moving mitochondria during early adulthood and subsequent decline with age linked to misfolded axonal protein accumulations; however, the dynamic properties of the moving mitochondria did not change with time. Intriguingly, reduced levels of the dynein co-factor Lissencephaly-1 (Lis1) caused a doubling in the number of motile mitochondria across time-points (without increasing their number) and reduced the age-associated build-up of protein in the axon45. Contrasting with mitochondria, the percentage of motile DCVs remained steady across ages in wild-type flies and was unaffected by reduced Lis1 levels, which is indicative of an organelle-specific perturbation rather than a global transport defect. Although theDrosophila marginal nerve cannot be used for whole-mount staining or large-scale biochemistry and has no direct counterpart in humans, its simple preparation permits quick and non-invasive analysis of anterograde and retrograde axonal transport in sensory nerves of adult flies. Without constraints on fly age, extended experimental time-points can be incorporated, facilitating the study of ageing and neurodegeneration inDrosophila.A novel technique to analyse transport dynamics in sensory axons of theveloped , which pDanio rerio) possesses many of the advantages of the invertebrate species, such as short generation time and lower maintenance costs, with the added benefit of being a vertebrate with myelinated axon tracts51. Also, like worms and flies, zebrafish are highly genetically tractable, with an array of reporter lines expressing fluorescently tagged proteins in specified organelles in subsets of cells. Zebrafish larvae are translucent, which obviates the need for invasive imaging techniques and allows repeated, longitudinalin vivo measurements throughout development. Several studies have probed cargo movement in the axons of anaesthetised zebrafish, the first of which assessed mitochondrial dynamics in sensory nerves called Rohon-Beard (RB) neurons found in the tail tip52. These transgenic “MitoFish” were created using the GAL4-UAS system to specifically express fluorescent proteins in the mitochondria of single RB neurons. A number of other groups have created similar fluorescent fish to assess the dynamics of RB mitochondria53 and endosomes55, as well as lysosomes in mechanosensory axons57.As a genetic model, the zebrafish  were created58. Disruption of the neuron-specific anterograde motor Kif5A increased the number of small motile synaptophysin-containing vesicles at early developmental stages without altering the ratio of anterograde to retrograde transport. In contrast, the percentage of mobile mitochondria was unaffected in–/–Kif5 animals, but mitochondria were transported more frequently in the retrograde direction, which likely causes the observed depletion of axonal mitochondria. Similar to the aboveDrosophila study45, these experiments once again highlight that transport of distinct organelles can be differentially impacted depending on the type and stoichiometry of the motor proteins driving their movement. The results also emphasise the importance of measuring multiple transport parameters (59 and CNS dopaminergic neurons60 (59 and Parkinson’s disease (PD)60 display perturbations in mitochondrial motility in disease-relevant neuronal subtypes; CMT2A motor nerves had a reduced percentage of motile mitochondria with unchanged speeds, while PD dopaminergic neurons displayed multiple transport defects dependent on the dose of toxin (MPP+) used to model the disease. The percentage of motile mitochondria and the ratio of anterograde to retrograde movements decreased during larval development (2–5 days post-fertilisation) in wild-type dopaminergic neurons, while speeds remained stable and run lengths increased60. Given the assortment of neurons now available for imaging, zebrafish provide an exciting platform for thein vivo analysis of axonal transport during development, with the caveat that once zebrafish reach adulthood, they become opaque, abrogating their utility for post-larval analyses.Until recently, the bulk ofameters , as diffrons60 . These fBox 3. Speed71Frequency of frame-to-frame speeds 11,67,71Individual cargo average velocities 7192 and maximum cargo speeds  can be determined92Immobile cargoes can be either included or omitted, and analysed separatelyMotility45 and number92 of motile cargoes in a given time Percentage60Percentage of time motile cargoes are moving56 and longest45 run distances (run length)Average39Run durationPausing71Percentage of cargoes that pause 67,7171 and length92 of time that motile cargoes remain stationary  could miss brief pauses, resulting in the recording of slower “moving” speeds for individual cargoes and fewer pauses. Rapid frame rates may provide more accurate information but must be offset against how rapidly a sample bleaches, potential phototoxic changes to specimen physiology, and the signal-to-noise ratio. Frame rate also directly impacts the labour required for analysis if manual tracking of cargoes is carried out.Imaging time: the longer an axon is imaged, the greater the chance that stationary organelles, particularly mitochondria, will become motile. The impact of phototoxicity should also be considered. Imaging over several orders of magnitude can circumvent this problem: for example, imaging at 2 Hz for 1 minute followed by 0.2 Hz for 10 minutes95.in vivo, including the response to spinal cord injury61, retinal degeneration62, and cortical function and development64. Similar to the animal models mentioned above, experimentalists working with mice can use an extended library of transgenic fluorescent reporter strains facilitating live imaging65. Consequently, there has been a recent flurry of publications adapting thesein vivo techniques for the analysis of axonal transport in live mice.Intravital imaging techniques have been developed in mice to study a range of biological processes66. Mitochondria are labelled throughout most of the “MitoMouse” nervous system, permittingin vivo analysis across a broad spectrum of neuronal subtypes provided they can be accessed in live animals. In this publication,in vivo axonal transport of individual organelles was imaged for the first time in a live mammal66. Mitochondrial kinetics were analysed in single motor and sensory axons of surgically exposed sciatic nerves using time-lapse confocal microscopy , the dynamics of a second type of organelle, the signalling endosome, were assessed in the sciatic nerve of SOD1G93A mice  could also help to overcome this issue. Alternatively, the mainly motor femoral nerve72 or primarily sensory sural72 or saphenous nerves20 could be imaged either in fluorescent reporter strains or by altering the HCT injection site to ensure fluorescent signalling endosome transport in the appropriate nerve. However, these alternative peripheral nerves are more technically challenging to image on an inverted microscope because of their anatomical distribution and size. Indeed, the sciatic nerve is a large, superficial collection of peripheral nerve axons, in which the dynamics of various cargoes can be imaged, with the useful possibility for longitudinal analysis in the same animal72.Adaptations of this technique have since been developed and expounded upon by a number of different laboratories. The Schiavo group crossed the “MitoMouse” with the SOD13A mice 67. Fluoin vivo75, permitting longitudinal and location-specific comparisons. Mice expressing fluorescent proteins in only a small percentage of sensory neurons have been used to assess axonal degeneration caused by spinal cord lesion in individual large, myelinated sensory axons found in the dorsal aspect of the spinal cord61. Numerous synthetic vital dyes that label structures such as myelin and microglia can also be used to aidin vivo analysis of the spinal cord76. This technique was recently implemented to assess axonal transport in acute and chronic mouse models of multiple sclerosis (MS)77. Crossing these strains with “MitoMice” and specifically generated “Thy1-PeroxiYFP” mice, both mitochondria and peroxisomes, respectively, were imaged in spinal cord and dorsal root axons. Pervasive defects in anterograde and retrograde transport of both cargo types were observed in MS mouse spinal cord axons before the onset of additional deficiencies, suggesting that axonal transport defects represent an early and important pathological sign in this disease77. These defects were not seen in dorsal root axons (sensory nerves), suggesting that subcellular location (i.e. proximity to the soma) has a bearing on axonal transport defects. However, the sensory identity of the axons imaged in the spinal cord was only presumed  and not experimentally confirmed, so the potential site-specific transport issue could instead be a product of neuron subtype (e.g. motor versus sensory).In contrast, the mouse CNS is inherently more difficult than the relatively accessible peripheral nervous system to image directly; nevertheless, axonal transport has now been successfully tracked in a number of CNS neurons. Axons within the spinal cord can be surgically exposed by dorsal laminectomy and imaged across time and several spinal segments12. RGC axons extend into the nerve fibre layer of the eye, parallel to the ocular surface of the sclera, permitting the visualisation of organelles after subtle opening of the surrounding skin and conjunctiva. The number of moving mitochondria, but not their run length, was diminished before RGC death in a model of glaucoma, whereas in aged mice (23–25 months), the duration and distance of mitochondrial movement were both diminished12. Interestingly, counter to results from retinal explants in the same study, this method showed that mitochondrial transport is highly dynamic in the mouse CNSin vivo12. Unfortunately, as fluorescence microscopy could impact the activity of light-sensitive retinal neurons, this observation may be confounded by artefactual alterations in mitochondrial dynamics (i.e. caused by increased activity20). Furthermore, this technique is restricted to albino strains because uveal pigmentation prevents imaging, and is more technically challenging with gaseous anaesthetics because of face access requirement. Nonetheless,in vivo comparisons of organelle transport in proximal and distal regions of RGC axons allow for repeated, longitudinal analyses.Multi-photon microscopy has also been used to assess mitochondrial transport dynamics in the axons of RGCs of anaesthetised “MitoMice” 12. RGC 14. Rather than using transgenic fluorescent strains, plasmids were unilaterally electroporated into embryosin utero to target the expression of fluorescent proteins to both the mitochondria and the cytoplasm13 or membranes14 of cortical progenitor cells (the latter to aid the identification of successfully transfected neurons). The process of electroporation restricts the fluorescence to a small proportion of axons, facilitating the assessment of individual collaterals. Similar to previousin vivo reports from zebrafish RB sensory52 and CNS dopaminergic60 neurons and mouse sciatic nerve axons66, the percentage of immobile mitochondria in cortical axons was very high over short time periods: >99% in 2 minutes in both pups (P10–13) and adults (P70–120)14 and ≈90% at P10–12 over 10–20 minutes13. The discrepancy between these two studies could reflect the different but overlapping cortical layers that were imaged; however, it is more likely caused by the 5- to 10-fold difference in imaging periods. Indeed, over 95% of mitochondria were reported as stationary in mature cortical neurons in culture when imaged for 30 minutes, which drops to ≈75% using a 12-hour imaging window13. This highlights a key problem in the study of axonal transport: to track individual cargo movements, rapid frame rates are required, but biologically relevant transport events may occur separated by much longer periods (minutes to hours), which is particularly challenging forin vivo imaging , and neuronal morphology . With recent developments discussed here, we are beginning to acquire a diverse and very powerful arsenal ofin vivo experimental systems across model organisms that will greatly enrich our understanding of transport. It is now vital to implement these intravital methods to tackle the questions of when (age), where (neuron category and subcellular location), what (cargo type), and how axonal transport deficiencies manifest in neuronal dysfunction.The active transport of organelles and molecules along axons is critical to neuronal health, function, and survival. Deficiencies in this process appear to be intricately linked to ageing and neurodegeneration, but whether they play a causal role or are simply a consequence of a pathologically affected tissue remains to be fully elucidated in each setting . There ain vivo has challenges in common with allin vivo imaging experiments  as well as formidable problems unique to the phenomena being studied: namely, relevant scales of measurement that span several orders of magnitude, both in time and distance. The difficulty in labelling cargoes specifically and with a labelling density allowing a sufficient signal-to-noise ratio for transport analysis is highlighted by the small number of axonal cargoes currently being studied, which are almost exclusively membranous organelles. Using bright, photoactivatable fluorophores is a particularly helpful strategy for studying transport, as it allows the tracking of subpopulations of cargo otherwise too dense to study individually or sparse cargoes over long time periods13. However, there is an urgent need to adopt new labelling strategies and fluorescent reporters to understand the behaviour of non-membrane-bound organelles, particularly in the field of RNA transport. Much of what has been learnt about organelle transport is garnered from experiments on mitochondria30; however, their movement within axons, which is characteristically interspersed by long pauses and thus perhaps more characteristic of slow axonal transport, is not reflective of all cargo types. This is perhaps because they have distinct axonal roles, rely on specific subsets of adaptor and motor proteins2, and are unique, highly dynamic, network-forming organelles. To have a thorough and informed understanding of axonal transport in health and disease, it is therefore of paramount importance that multiple cargo types are analysed.Progress in imaging axonal transport86 could be incorporated into these analyses in order to provide a more global assessment ofin vivo axonal transport in health and disease. Moreover, as imaging techniques become more sophisticated, allowing high-speed, multi-channel acquisition at greater tissue depths88, we will be able to simultaneously monitor the dynamics of different organelles in their native environment and reliably assess the transport of organelles, such as RNA granules, for which similar robust protocols are currently lacking.Most of the methods discussed here are adaptations of intravital techniques initially developed to study other biological processes. It is therefore likely that imaging of additional neuronal subtypes or subcellular locations


Although growing evidence points to the potential therapeutic effects of transcranial Direct Current Stimulation (tDCS), there is still no consensus on the most appropriate protocol to be used in specific neurological and neuropsychological symptoms. This case report evaluated the neuromodulatory therapeutic effects of two 15-day courses of tDCS on an elderly female patient, aged 78 years with mild neurocognitive disorder, chronic pain and depression-related symptoms. Results indicated an overall significant improvement of cognitive and executive functions, as well as reduction in both depression and chronic pain symptoms. These results highlight the potential of tDCS as a safe and useful neuromodulatory clinical tool in the rehabilitation of elderly patients. However, there is still no consensus about the most appropriate tDCS protocol to be used or on its comparative efficacy in special situations such as in elderly patients.A 78-year-old female patient, with 12 years of education, graduated in Economics, returned to the NA Neuroscience Clinic, Brasilia-DF, Brazil, 7 months after an initial consultation in which she had been diagnosed as having mild neurocognitive disorder, depression, and chronic pain. The patient was accompanied by her adult daughter, who reported that her mother's symptoms had worsened over the past 7 months.overall cognition was assessed by the Mini-Mental State Exam (MMSE); [2] comprehension and reasoning were evaluated by the Comprehension, Similarities and Vocabulary Tests ; [3] executive and attention functions were assessed by the Digit Span (forward and backward), Arithmetic (WAIS-III), and Victoria Stroop Color-Word Test; [4] episodic memory and short-term memory were evaluated by the Rey Auditory Verbal Learning Test (RAVLT); [5] verbal and visuoconstructive abilities were assessed by the Rey-Osterrieth Complex Figure immediate recall Test (ROCFT); [6] perceptual organization was evaluated by the Block Design and Matrix Reasoning (WAIS-III); [7] processing speed was evaluated by the Digit Symbol (WAIS-III); [8] verbal, phonemic and semantic fluency were assessed by the Animal Categories, Fruit Categories and Letters FAS Tests; [9] functionality was scored by Pfeiffer's Functional Assessment Questionnaire (FAQ); and [10] depression was scored by the Beck Depression Inventory (BDI). Additionally, [11] chronic neuropathic pain was scored using a Visual Analog Scale (VAS), and a self-report scale pain assessment.The patient was diagnosed with mild neurocognitive disorder, depression and neuropathic pain . Selective serotonin reuptake inhibitors were prescribed with no clear benefit. Diagnosis was based in anamnesis and scores derived from neuropsychological evaluations by a board-certified neuropsychologist using the following instruments and scales: [1] ® 20 mg), and anti-inflammatory and/or analgesic agents (Feldene® 20 mg) in this case, together with the reported benefits of tDCS on executive function by several authors, compassionate use of tDCS was prescribed. A commercially available transcranial direct current stimulator  was used to deliver 2.0 mA for 30-min per session through saline-soaked sponge electrodes (surface 35 cm2). Electrode placement on the scalp was determined using the International 10-20 EEG system. The anode was positioned over the left prefrontal dorsolateral cortex (F3), and the cathode was positioned over the contralateral scalp (Fp2). Location and polarity of the electrodes, number of sessions, and current intensity were chosen on the basis of previously reported results demonstrating that anodal tDCS over the left prefrontal dorsolateral cortex (F3) for 30 min is capable of improving executive function.Given the lack of effect of other treatment modalities including selective serotonin reuptake inhibitors  and the second (March 2015) courses. During the first course, the patient was additionally submitted to cognitive training (CT), once a day, for 60 minutes, which was carried out on the same days, but not simultaneously with CT.tDCS sessions were very well tolerated by this elderly patient and may have played a role in the significant improvement on neuropsychological tests. After the first ten sessions of the first tDCS course, she also subjectively reported feelings of well-being and a decrease in chronic pain.Besides the therapeutic effects of tDCS, ameliorating one or more symptoms related to mild neurocognitive disorder, depression and chronic pain, our protocol also improved several functions, including executive and attention functions (Digit Span), verbal fluency (Letters – FAS), comprehension and reasoning (Similarities – WAIS), episodic recall (RAVLT), nonverbal recall (ROCFT) which were already relatively well preserved in this elderly patient before the first tDCS clinical trial.In line with our results, tDCS over the DLPFC has been proposed as a promising tool for restoring cognitive function in the context of memory decline related to both mild and severe neurocognitive disorder.,,,The potential for tDCS to increase learning and cognition may well lead to the development of enhanced therapeutic interventions. For example, previously reported studies have also shown therapeutic effects of tDCS in executive functions with anodal tDCS over the left DLPFC,,The antidepressant-like effects observed were probably due to tDCS treatment since cognitive training (CT) is not intended to treat depression, although it may have some effect on some aspects, for example on ruminant thoughts. A number of studies have linked depression with reduced activity in the left DLPFC, which may account for the negative emotional bias, that is, the tendency of depressed individuals to show enhanced attention to and preferential memory for negative information. Although there are suggestions of an important synergy of these two therapeutic modalities,,tDCS has also been used to alleviate chronic pain.In conclusion, this report illustrates potential benefits of anodal tDCS sessions over the left DLPFC in a 78-year-old patient with mild neurocognitive disorder and complaints of depression and chronic pain. Further controlled studies involving a larger number of patients are required to ascertain the potential benefits of this non-invasive technique in mild neurocognitive disorder cases.


During the hours that human cells spend in the DNA synthesis (S) phase of the cell cycle, they may encounter adversities such as DNA damage or shortage of nucleotides. Under these stresses, replication forks in DNA may experience slowing, stalling, and breakage. Fork remodeling mechanisms, which stabilize slow or stalled replication forks and ensure their ability to continue or resume replication, protect cells from genomic instability and carcinogenesis. Fork remodeling includes DNA strand exchanges that result in annealing of newly synthesized strands , controlled DNA resection, and cleavage of DNA strands. Defects in major tumor suppressor genes BRCA1 and BRCA2, and a subset of the Fanconi Anemia genes have been shown to result in deregulation in fork remodeling, and most prominently, loss of kilobases of nascent DNA from stalled replication forks. This phenomenon has recently gained spotlight as a potential marker and mediator of chemo-sensitivity in cancer cells and, conversely, its suppression - as a hallmark of acquired chemo-resistance. Moreover, nascent strand degradation at forks is now known to also trigger innate immune response to self-DNA. An increasingly sophisticated molecular description of these events now points at a combination of unbalanced fork reversal and end-resection as a root cause, yet also reveals the multi-layered complexity and heterogeneity of the underlying processes in normal and cancer cells. Among classic causes of replication stress are DNA damage during S phase and unbalanced or reduced levels of intracellular nucleotide pools, both of which can be elicited by exogenous agents such as chemotherapy drugs. Other recognized causes include conflicts with transcription, epigenetic abnormalities, and defective cell cycle regulation. Prolonged stalling of forks during stress can cause fork collapse, i.e. DNA damage and loss of protein machinery of the fork, which can jeopardize complete duplication of the genome. Replication stress is considered one of the major drivers of genomic instability as well as premature senescence and carcinogenesis. For in-depth review of these topics, see 12345678The three billion base pairs of the human genome are replicated in about six to eight hours of the S phase of the cell cycle. At the height of S phase, there are hundreds of replication forks running through chromosomes, each carrying on its three-way DNA junction anywhere from dozens to perhaps low hundreds of proteins that service every aspect of chromatin duplication. When replication forks experience disruptions to their normal mode of operation, which manifests as forks’ stalling or slowing, the condition is referred to as Eukaryotic cells have evolved elaborate mechanisms to buffer replication fork activity against stress. These mechanisms can be divided into two categories: those that enable bypass of damage, and those that allow stable pausing and on-demand resumption of replication once the obstacle is cleared or conditions are normalized. It is the latter group that we will focus on in this review. For additional discussion of the subject, we recommend these excellent recent reviews 10111213remodeling to refer to these processes. Evidence suggests that reversal and resection are actively regulated, and not only are compatible with but in fact enable stabilization of forks and contribute to their ability to resist disruptions of DNA synthesis (see next section). Fork collapse may be viewed as an exhaustion or breakdown of fork remodeling over time. Likely however, it is an actively triggered pathway of last resort initiated by cleavage of one arm of a fork by the MUS81 endonuclease 16171823It is helpful to distinguish three processes that can occur at a stalled replication fork and involve DNA strand separation/annealing, degradation, and breakage. These are, respectively, fork reversal, resection, and collapse  and repriming, we recommend a recent review Figure 1B, 2), in which two nascent strands pair with one another to form a forth branch have first been observed by electron microscopy (EM) in the preparations of genomic DNA decades ago in vivo after treatment with several different fork-interfering agents 30Table 1). In vitro, the RAD54 DNA translocase 32in vitro and in vivo193637383940414243Reversed forks  364647in vitro and triggers fork collapse and DNA damage in vivo, further supporting the notion that fork reversal is a protective mechanism 47Table 1 and 50Figure 3.FBH1 is a 3’-5’ DNA helicase capable of targeting its binding partners for ubiquitination in vivoin vitro and in vivo in vivo is orchestrated by counteracting activities and is regulated by a master-switch PARP1 that prevents premature restoration of a reversed fork.While failure to reverse forks has deleterious consequences for fork activity and genome stability overall, studies find that excessive fork reversal, e.g. due to overexpression of SMARCAL1 or RAD51, can also result in DNA damage and threaten genome stability 52535456prior to fork arrest . This phenotype was present in cells defective for breast cancer susceptibility genes BRCA1 and BRCA2, or Fanconi Anemia genes FANCD2, or FANCA, but not in the wild type cells. The authors implicated the MRE11 nuclease known for its role in DNA end resection during double strand break (DSB) repair, in the loss of nascent DNA at forks. Moreover, they provided evidence that nucleolytic attack by MRE11 was upregulated without BRCA2 because no ssDNA/RAD51 filament was there to protect the fork. In particular, BRCA2 C-terminal domain involved in stabilizing RAD51 filament on ssDNA was critical for preventing degradation. Overexpression of BRC4 domain of BRCA2, which binds RAD51, or of the K133R mutant of RAD51, which forms hyper-stable filaments, respectively, enhanced and suppressed nascent strand degradation.Schlacher and colleagues in the Jasin lab were the first to put degradation of nascent DNA at stalled forks in the spotlight 60et al. thereby referred to this distinct pathway as fork protection to highlight the protective functions of RAD51 and BRCA/FANC genes. Notably, BRCA2 absence did not inhibit fork restart after HU removal, though it increased DNA damage and genomic instability associated with HU treatment. Though BRCA2 C-terminal mutants were later found defective in fork restart 4219The C-terminus mutant of BRCA2 that failed to protect forks from degradation was able to maintain homology-dependent DNA repair, suggesting, crucially, that this function of BRCA2 is at least somewhat separate and different from its DSB repair function. Schlacher Overall, stalled fork condition emerged as a counterbalance of protective activity of RAD51 and nucleolytic activity of MRE11, a kind of a battle between a hero and an anti-hero. However, several observations complicated the picture. For example, RAD51 depletion does not always result in upregulated nascent strand degradation at stalled forks . The extent of resection or reversal as well as prevalence of one over the other may vary depending on relative abundances and activities of the involved proteins. In theory at least, reversal may precede and/or follow resection.To begin, resection and reversal can be viewed as interlocked, mutually constraining fork remodeling processes  can be initially exposed due to failure of DNA synthesis and excessive unwinding ahead of the fork by the replicative helicase, and then expand due to resection. Appearance of nascent ssDNA  strongly suggests reversal with subsequent resection. RAD51, MRE11, and other proteins may act either on the single-stranded portion of an extruded nascent strand duplex, i.e. the "forth" arm of a reversed fork , or on the parental DNA over a single-stranded gap (structure e); the latter mode may be followed by reannealing of the parental duplex (structure f). Techniques such as Proximity Ligation Assay (see BOX1 for detail) can now begin to distinguish between these scenarios and will help delineate specific sequences of events in individual model systems. The order or prevalence of reversal versus resection may be the reason the phenotype of RAD51 deficiency can be either protective or sensitizing for resection. That is, RAD51 depletion will suppress resection if RAD51 acts only at reversal stage , and resection is strictly dependent on prior reversal (i.e. a →e path is not active) 1962The timing of appearance and prevalence of single-stranded (ss)DNA at forks is a critical readout of fork reversal and resection. Figure 3 lists the proteins that contribute to remodeling of stalled forks. Some of these proteins were already mentioned in the previous sections. These and other proteins will be the subject of the discussion below.Historically, many proteins that are active on stalled forks were first recognized for their roles in the repair of two-ended DSBs in DNA. MRE11 is no exception. Much of what is known about MRE11 at the molecular level comes from the studies of DSB end processing in the context of competition between homologous recombination (HR)-mediated repair and non-homologous end joining (NHEJ). MRE11’s ssDNA endonuclease activity initiates resection by introducing a nick internal to a DSB end. The nicked strand is then degraded in the 5’-3’ direction by the EXO1 exonuclease and in the 3’-5’ direction by MRE11’s exonuclease activity 7881The extent of contribution of MRE11 exonuclease to resection appears to vary between experimental systems 83At replication forks, the forth arm that forms upon fork reversal bears resemblance to a one-ended DSB, and is expected to be a substrate for resection in a manner analogous to the above. Some evidence is also consistent with MRE11 targeting nascent DNA ends that flank ssDNA gaps within newly replicated parental/daughter DNA duplexes 163186878862At DSBs, BRCA1 both promotes and limits strand resection  suppress mitotic MUS81-coupled BIR after fork stalling It should be noted that recent work also points at mechanistically important differences between BRCA1 and BRCA2 in the ways these proteins contribute to the fate of forks RAD51 is recruited to a stalled fork and is one of the factors that reverses it or stabilizes an already reversed fork 591943et al. found that the T131P mutation of RAD51 that prevents it from forming a stable nucleofilament et al. observed that B02 inhibitor of RAD51, which blocks its binding to DNA The dual role of RAD51 before/during and after fork reversal was supported by two observations. Mijic Figure 3). That said, the possibility that nascent strands can also be degraded in non-reversed forks by MRE11 or other nucleases is not ruled out, particularly for the case of BRCA1/2-proficient cells. If so, such nucleolytic activity will expose parental ssDNA that can serve as a substrate for RAD51, as it does during DSB repair. The final resolution of the question will require data on when parental and/or nascent ssDNA accumulate during fork remodeling, and which of these ssDNA are bound by RAD51, MRE11, and other proteins.In sum, there is now a very strong case for a reversed fork as an entryway for nascent strand degradation by MRE11 and EXO1 , and MRE11 and DNA2 were epistatic with respect to nascent strand degradation, suggesting a crosstalk between these nucleases As we have seen, MRE11 and EXO1 are demonstrably active in fork resection in the BRCA1/2-deficient backgrounds. In contrast, Lemacon BRCA-proficient cells On the other hand, DNA2/WRN and not MRE11 or EXO1 carried out resection in cases where nascent strand degradation was upregulated in et al. adds a helpful hint to this puzzle nascent ssDNA in SV40-transformed human fibroblasts . To our knowledge, this is the demonstration of restrained resection limited to the reversed arm. Moreover, both nascent ssDNA and nascent strand degradation phenotypes were suppressed by depletion of MRE11 (or EXO1) or by inhibition of MRE11 exonuclease activity. One possibility suggested by these data is that there is a regulatory link connecting DNA2/WRN to MRE11, EXO1 at stalled forks. Here it is worth mentioning that WRN has been associated with promoting RAD51 binding to stalled forks in two studies 72A study by Iannascoli Figure 4). Their relative prevalence may depend on the BRCA status and/or yet unknown genetic determinants that define the balance between fork reversal/restoration. Once DNA2/WRN are engaged, this inhibits MRE11 and EXO1. This model thus depicts a scenario very different from the HR-mediated DSB repair and will require the support of additional data for its validation.Based on the data reviewed in this and previous sections, we therefore hypothesize that there are two, potentially interconnected, pathways that are capable of resecting nascent DNA at reversed forks  Remarkably, Mijic et al. showed that loss of RAD52 in BRCA2-deficient cells has a phenotype similar to PTIP loss: nascent strand resection at stalled forks is suppressed while fork reversal is unaffected. Moreover, without RAD52 less MRE11 is bound at stalled forks RAD52 is known for its involvement in the HR-mediated DSB repair, and becomes critical for cell survival in the absence of BRCA1/2 . Some of these proteins directly act on RAD51, while the role of others is more indirect - for example, they may compete with RAD51 for the same substrates.The list of proteins whose role involves upregulating or downregulating the level of RAD51 at stalled forks in vitro110111112RECQ helicases RECQ5 and BLM were perhaps the first to be demonstrated to disassemble RAD51/ssDNA filament in vitroin vivo activities of FBH1 are consistent with this role of a RAD51 restrainer: disruption of FBH1 has antirecombinogenic phenotype (ibid.), and also suppresses the phenotypes of two facilitators of fork protection, WRNIP and BOD1L (see below). However, FBH1 also reverses forks FBH1, an F-box-containing 3’-5’ DNA helicase, was also shown to displace RAD51 from ssDNA in vitro and has anti-recombinogenic phenotype in vivoPARI is another protein that can disrupt RAD51/DNA association Constraints on RAD51 activity are counterbalanced by several proteins that either load RAD51 independently of BRCA2, or protect loaded RAD51. In Chinese hamster cells, deficiency in RAD51 paralogs RAD51C, XRCC2, or XRCC3 upregulates nascent strand degradation and exposes excess of parental DNA in single stranded form at stalled forks In vitro, FANCD2/FANCI can stabilize RAD51/ssDNA filament and protect the ds/ss DNA junction behind the 3’ ssDNA overhang from nucleolysis Fanconi Anemia proteins FANCD2 and FANCI likely enable RAD51 by protecting it on DNA. FANCD2 deficiency results in excessive nascent strand degradation at HU-arrested forks The following proteins contribute positively to fork protection, however, it is not yet clear, whether they facilitate RAD51 and/or restrain the nucleases.In the absence of BOD1L, nascent strand degradation at forks stalled by HU is upregulated, as are the levels of parental ssDNA, albeit assayed using a different replication-stressing agent, mitomycin C 121Figures 3, 4). Addressing this will require further research.Depletion of the ATPase WRNIP1 known for its association with WRN, generates a phenotype very similar to that of BOD1L with respect to its interactions with BRCA2 and FBH1 The list of proteins that influence RAD51 binding and/or modulate resection will likely continue to grow. Among new additions are REV1 The overview of the proteins engaged in stalled fork remodeling suggests that many of them either contribute to both sides of the "standoff" between DNA protectors and DNA degraders, or are subject to counteracting activities of other proteins. In a system with so many moving parts, the exact balance between fork reversal, protection, and resection may depend on the sub-nuclear distribution, mutual ratios, and relative activity of the proteins involved, which may vary with the genetic and epigenetic background. The nature of the experimental perturbation may also play into this. For example, the level of RAD51 may influence how stringently fork reversal depends on it, while the degree of RAD51 depletion may affect its ultimate phenotype at forks. That is, only a complete loss of RAD51 may abolish fork reversal while partial depletion will only disrupt its function in fork protection.It should be appreciated that most of the cell lines used in fork remodeling studies are transformed or cancer-derived, and fork remodeling in cancer cells may be already rewired or relaxed under the pressure of chronic replication stress. For example, lines HCT116 and U2OS exhibit baseline nascent strand degradation 131in vivo and in vitro selection for cisplatin or olaparib resistance in BRCA2 or BRCA1-deficient backgrounds readily yields tumors or cell line clones in which the parental nascent strand degradation at forks is now suppressed (see refs. below). These important findings brought replication fork remodeling to the frontline of cancer chemotherapy. Collectively, the data point at the equilibria between fork reversal and restoration and between resection and protection as "breeding grounds" of chemo-resistance. However, intensifying research by many labs also poses additional questions about the nature of the lethality of PARP1 inhibition in BRCA-deficient cells, and about the role of PARP1 in nascent strand degradation.Lethality of BRCA1 or BRCA2 deficiency when combined with PARP1 inhibition or cisplatin is perhaps the most famous of synthetic lethal interactions discovered 134Table 2). Also in agreement with this hypothesis is the observation that PARPi resistance in BRCA2-deficient cells can emerge due to secondary mutations that also suppress nascent strand degradation Table 2): PARP1 deficiency or inhibition suppress nascent strand degradation and reduce MRE11 binding to forks in BRCA2-deficient cells 43138et a.l observed that PARP1-/-,BRCA1-/- double knockout mouse B cells exhibited a suppressed nascent strand degradation compared to BRCA1-/- cells Excessive nascent strand degradation correlates with increased DNA breakage 60et al. observed a mild increase in chromatid breaks and gaps after forks stalling in BRCA2-depleted cells treated with olaparib et al. found that chromosomal aberrations and DNA damage were reduced upon depletion of SMARCAL1 or ZRANB3 in BRCA1 or BRCA2-depleted cells These findings are more in line with the consensus that PARP1 facilitates recruitment of MRE11 to stalled forks 762843restoration of reversed forks rather than blocking reversal in the first place, as the SNF2 family members or RAD51 do. Second, at least for some cases, trapping of PARP1 on DNA by olaparib may explain how a differential phenotype may emerge from PARP1 inhibition versus depletion in BRCA2-deficient cells 1034299First, the unique nature of PARP1 loss may be in that it causes inappropriate It should also be noted that the variability of PARP1 phenotypes may reflect its multiple, often counteracting inputs at the fork. For example, PARylation facilitates recruitment of BRCA1 to DSBs 140144145Perhaps the best testimony in favor of the association between fork protection and chemo-resistance is the accumulation of data that demonstrate this association in diverse, independently derived genetic backgrounds. For example, overexpression of FANCD2 was shown to suppress nascent strand degradation and confer PARPi resistance in a subset of BRCA1 or BRCA2-deficient cases suppressing recruitment of MUS81. Overall, our understanding of the epigenetic control of fork protection is far from complete, but it is safe to assume that it will reveal further heterogeneity and plasticity of fork protection mechanisms of cancer cells.Discovery of the involvement of MLL3/4 implicates epigenetic regulation by histone modification in fork protection, adding another layer of complexity to the already elaborate system. Another prominent epigenetic regulator found to affect nascent strand degradation is CHD4, a member of the NuRD complex involved in gene regulation via nucleosome remodeling and histone deacetylation In this last section we would like to bring attention to a developing, exciting area of research that may well improve our understanding of the effects the deregulation of fork remodeling has on cancer and normal cells. This area is innate immune response.Briefly, innate immune response is a universal cell-intrinsic response to bacteria and viruses. One of its jobs is to recognize and react to foreign DNA found in the cytoplasm, which involves multiple sensor molecules. Over the past decade, studies have begun to make connections between DNA damage response and the immune response to self-DNA 155158159160162163et al. detected a spike in short cytoplasmic ssDNA in cells treated with ionizing radiation (IR), mitomycin C or cisplatin, which activated innate immune response. Depletion of resection nucleases DNA2, EXO1, and BLM, but, interestingly, not MRE11 and CtIP, suppressed these responses, and TREX1 deficiency exacerbated them Several studies have now made explicit associations between DNA resection during DNA repair or fork stalling and innate immune response to self-DNA. Erdal Pasero and colleagues identified SAMHD1 as a cofactor of MRE11 at stalled forks. SAMHD1 is a dNTPase with roles in cell-intrinsic antiviral response, and one of the genes mutated in AGS et al. In another study, RAD51 depletion was correlated with elevation of cytoplasmic ss and dsDNA, upregulation of innate immune response including STING, and enhanced nascent strand degradation at forks after IR We hope to have shown that fork remodeling mechanisms emerge as balancing acts of many counteracting processes - a game of substrates that revolves around degradation versus protection of nascent DNA strands, and whose original main characters, MRE11 and RAD51, are surrounded by a whole cast of others. Moreover, fork remodeling moves into the spotlight as a major contributor to carcinogenesis, chemotherapy resistance, and inflammation.We have seen that many HR and NHEJ proteins are involved in fork protection and curiously, often in a somewhat "off-label" way that is not exactly identical to their roles in DNA repair. We have also seen that close inspection of fork remodeling reveals cell line-dependent heterogeneity and malleability under selective pressures of carcinogenesis. While complete understanding of these complexities is still farther down the road, a few general considerations may guide us on the way.First, it is worth remembering that fork remodeling phenotypes, as typically measured, are likely an average of heterogeneous responses to fork stalling in different genomic and epigenomic regions, next to actively transcribed genes or not, with different configurations of parental and daughter strands , for different periods of time since the onset of fork-arresting treatment; and in early versus late S phase of the cell cycle. Aggregated together, these responses may create an impression of one too many proteins contributing semi-redundantly, while in reality cells may use "local" solutions to fork remodeling with only a subset of proteins involved in each case. To bring just one example, only a particular subset of stalled forks - those with leading strand gaps - may actually be reversed by SMARCAL1 Second, it should be considered that the phenotypes often measured in perturbation studies of fork protection may not be a simple consequence of a protein’s absence, but a re-balancing act that the whole network undergoes because of it, i.e. an absence of function of one may actually cause a gain of function of several others.And lastly - even if variability is widespread, the functional variables that define how fork remodeling is configured in cells are still few, and common. These are: 1) robustness and efficiency of loading of RAD51 at forks and the extent of its dependency on BRCA1/2; 2) robustness and dominance of fork reversal over fork restoration; 3) robustness and efficiency of nucleolytic activity at forks. The additional variables that are less studied at present but may come to the fore in the future are: efficiency of loading of NHEJ proteins as protective factors, the degree of nucleosome mobility and histone turnover at forks, and the efficiency of innate immune response. As the data show, while chemotherapeutic resistance in the fork protection-defective cancer cells arises frequently, it seems to work by adjusting the above-mentioned common variables. As such it is predictable - and thus can be preempted.