Nitrogen fixation is a widespread metabolic trait in certain types of microorganisms called diazotrophs. Bioavailable nitrogen is limited in various habitats on land and in the sea and, accordingly, a range of plant, animal, and single-celled eukaryotes have evolved symbioses with diverse diazotrophic bacteria, with enormous economic and ecological benefits. Nitrogen fixation is a widespread metabolic trait in certain types of microorganisms called diazotrophs. Bioavailable nitrogen is limited in various habitats on land and in the sea and, accordingly, a range of plant, animal, and single-celled eukaryotes have evolved symbioses with diverse diazotrophic bacteria, with enormous economic and ecological benefits. Until recently, all known nitrogen-fixing symbionts were heterotrophs, such as nodulating rhizobia, or photoautotrophs, such as cyanobacteria. In 2016, the first chemoautotrophic nitrogen-fixing symbionts were discovered in a common family of marine clams, the Lucinidae. Chemosynthetic nitrogen-fixing symbionts use the chemical energy stored in reduced sulfur compounds to power carbon and nitrogen fixation, making them metabolic “all-rounders” with multiple functions in the symbiosis. This distinguishes them from heterotrophic symbionts that require a source of carbon from their host, and their chemosynthetic metabolism distinguishes them from photoautotrophic symbionts that produce oxygen, a potent inhibitor of nitrogenase. In this review, we consider evolutionary aspects of this discovery, by comparing strategies that have evolved for hosting intracellular nitrogen-fixing symbionts in plants and animals. The symbiosis between lucinid clams and chemosynthetic nitrogen-fixing bacteria also has important ecological impacts, since they form a nested symbiosis with endangered marine seagrasses. Notably, nitrogen fixation by lucinid symbionts may help support seagrass health by providing a source of nitrogen in seagrass habitats. These discoveries were enabled by new techniques for understanding the activity of microbial populations in natural environments. However, an animal (or plant) host represents a diverse landscape of microbial niches due to its structural, chemical, immune, and behavioral properties. In the future, methods that resolve microbial activity at the single cell level will provide radical new insights into the regulation of nitrogen fixation in chemosynthetic symbionts, shedding new light on the evolution of nitrogen-fixing symbioses in contrasting hosts and environments. Chemosynthetic symbioses are classically defined as intimate associations between eukaryotic hosts and either of two types of bacterial symbionts: (i) chemolithoautotrophic bacteria, which fix inorganic carbon using the energy released by oxidation of reduced chemical compounds such as hydrogen sulfide, or (ii) methane-oxidizing bacteria, which synthesize their biomass from the single-carbon compound methane –3. Both –Riftia pachyptila, which lacks a digestive system, relying entirely on its intracellular symbionts for nutrition. In contrast to the host’s absolute dependency on the symbionts as adults, the symbionts can make a living by themselves in the surrounding environments and are acquired from the environment by juvenile hosts during development  known to power chemosynthetic symbioses for the first 30 years of research on these associations, but this limited picture has now also been overturned with first the discovery of hydrogen-powered symbioses in the deep sea, as well as subsequent discoveries revealing an expanding range of energy sources that fuel chemosynthetic primary production, which include carbon monoxide and potentially also reduced iron  only chemosynthetic symbiont was brought into pure culture only recently . Most efc growth –24. This growth –. Similarced iron –30.Sedimenticola and, surprisingly, also the endosymbionts of deep-sea Riftia tubeworms  shallow marine habitats that can be reached with SCUBA or snorkel rather than the oceangoing vessels and large robotic instruments needed for deep-sea exploration , 12. Coa68–Seagrass beds are a common habitat for burrowing clams of the family Lucinidae. Lucinids can promote seagrass health through a “nested” association between seagrasses and the lucinid bivalves and their intracellular sulfide-oxidizing symbionts (see below). Experimental evidence showed that both lucinids and seagrasses grow better together than apart . This beLoripes orbiculatus from the Mediterranean and Codakia orbicularis from the Caribbean  either the common free-living ancestor of lucinid (and Riftia) symbionts was capable of nitrogen fixation, and this was lost independently on multiple occasions, or (ii) these genes have been gained multiple times independently by horizontal gene transfer. Considering that the closest free-living relative, Sedimenticola thiotaurini has nif genes , the functions and benefits of chemosynthetic symbionts may shift with changing seasons , 81.nif gene expression between the symbiont populations of different clam individuals. At the transcriptome level, nif genes were some of the most highly expressed, as high as those involved in sulfide oxidation, but only in two of five individuals sequenced. In the other three, nif genes were barely or undetectable in the metatranscriptomes  oxygen flux to intracellular symbionts. Leguminous plants use intracellular oxygen-binding proteins, called leghemoglobins, to control oxygen flux to the symbionts. Animals that host chemosynthetic symbionts also make hemoglobin proteins to bind oxygen. Lucinid clams in particular encode multiple distinct hemoglobins to bind either sulfide or oxygen –108. The–113–Sedimenticola thiotaurini, also a diazotrophic sulfide-oxidizing chemolithoautotroph, is more tolerant to oxygen when supplemented with organic carbon (Rhodobacter capsulatus (One major difference between diazotrophic symbioses in plants and in lucinid clams is the direction of carbon flow . Plant sc carbon . From thpsulatus .The all-rounder in sport is a flexible player capable of carrying out multiple functions or of playing multiple roles or positions equally well, but the all-rounder is almost by definition not the best performer in any of these roles. Flexibility comes at the cost of peak performance. Does this apply to chemosynthetic symbiotic all-rounders? If they are routing metabolic energy gained from sulfur oxidation to nitrogen fixation, then this must reduce their maximum capacity to fix carbon. Future efforts to quantify symbiont activity at the single-cell level will tell us whether the symbiont population of a single host animal is a team of genetically identical individuals divided up into metabolic specialists, and if so, how these fit together to form a functioning symbiotic partnership.


Traumatic brain injury (TBI) is a leading cause of injury-induced disability in young children worldwide, and social behavior impairments in this population are a significant challenge for affected patients and their families. The protracted trajectory of secondary injury processes triggered by a TBI during early life—alongside ongoing developmental maturation—offers an extended time window when therapeutic interventions may yield functional benefits. This mini-review explores the scarce but promising pre-clinical literature to date demonstrating that social behavior impairments after early life brain injuries can be modified by drug therapies. Compounds that provide broad neuroprotection, such as those targeting neuroinflammation, oxidative stress, axonal injury and/or myelination, may prevent social behavior impairments by reducing secondary neuropathology. Alternatively, targeted treatments that promote affiliative behaviors, exemplified by the neuropeptide oxytocin, may reduce the impact of social dysfunction after pediatric TBI. Complementary literature from other early life neurodevelopmental conditions such as hypoxic ischemic encephalopathy also provides avenues for future research in neurotrauma. Knowledge gaps in this emerging field are highlighted throughout, toward the goal of accelerating translational research to support optimal social functioning after a TBI during early childhood. Persistent social deficits are common after traumatic brain injury (TBI) during childhood, and their impact on quality of life is increasingly recognized . Social While a TBI sustained at any age has the potential to impair psychosocial function, the pediatric injured brain appears to be particularly susceptible to social behavior deficits. This vulnerability may be attributed to an immature state at the time of injury, such that injury disrupts not only the developing neural networks that underpin social cognition, but also the acquisition of new social skills , 8. Socivia the administration of compounds that provide broad neuroprotection—for example, by targeting a range of secondary injury mechanisms that underpin progressive neuropathology and the development of social deficits. Alternatively, targeted treatment with drugs known to promote affiliative behaviors may be effective at reducing the impact of social dysfunction after TBI.Rehabilitation for survivors of TBI is both multifaceted and interdisciplinary, and broadly aims to facilitate neurocognitive and functional recovery . SupportThis mini-review describes the current state-of-the-field in the development of such therapies, with a focus on pre-clinical modeling in pediatric TBI. Drugs and targets with demonstrated potential in other early life neurodevelopmental disorders such as hypoxic-ischemic (HI) injury are also described where relevant. Knowledge gaps are highlighted throughout, and our goal is to drive toward accelerated translational research to support the optimal social functioning after pediatric TBI.Historically, the pre-clinical neurotrauma field has focused on assessments of sensorimotor and cognitive outcomes . Over thSemple and Noble-Haeusslein in 2012 first employed such methods to investigate social behavior changes in a model of severe TBI in mice at postnatal day (p) 21. Male mice were found to exhibit normal social behaviors at 2 weeks post-injury, approximately adolescence—but showed aberrant social interactions and social recognition memory by early adulthood (around 8 weeks post-injury) . SimilarSeveral pre-clinical neurotrauma studies have subsequently incorporated measures of social behavior into their study designs, considering how social functioning may be altered after injuries sustained across a lifespan –31. The With this expanding body of literature characterizing social behavior changes after pediatric TBI, the field is poised to now trial novel therapies for their potential to rescue or prevent such deficits. Although the field remains in its infancy, this mini-review will highlight the few studies conducted to date in this context. Where appropriate, we have extended the scope to other early life insults such as HI injuries, modeling encephalopathy of pre-maturity or perinatal stroke depending on the nature of the insult and timing , 44. OthOxytocin and vasopressin are evolutionarily conserved neuropeptides with important roles in the control and regulation of social behaviors . In miceModulation of both the vasopressin and oxytocin signaling pathways has generated promising findings to date as a means to improve social deficits in human conditions in which aberrant social behaviors are a feature, such as ASD , 51. ForTargeting oxytocin or its receptor has also demonstrated broad neuroprotection in the context of acquired prenatal and perinatal brain insults, including models of pre-maturity, fetal asphyxia, and fetal growth restriction –57. HoweIn pediatric TBI, Runyan et al. have recently investigated the potential of oxytocin treatment to ameliorate social behavior deficits following moderate TBI in p11 rats . IntranaA wide range of social behaviors including parental care, social interactions, play, aggression, and sexual behaviors, are influenced by gonadal hormones, including testosterone, estradiol and progesterone . NeuroenFundamental differences between sexes remain to be fully elucidated, with only one study in the p21 mouse reporting sex-specific phenotypes in social and sociosexual behaviors after severe TBI, as well as neuronal morphology in the prefrontal cortex and hippocampus, two brain regions with known roles in social functioning . As our The potential for hormonal manipulation to modulate social dysfunction after brain injury can be gleaned from models of HI injury in the rodent. The p10 rat exhibits a reduction in same-sex social play behaviors in both male and female injured rats at 4–5 weeks post-injury . HoweverFinally, the steroid hormone progesterone has been extensively studied in models of adult TBI for its multiple mechanisms of purported neuroprotection , 71. ProThe neuroinflammatory response induced by a TBI has long been considered integral to functional and neuropathological outcomes , 77. WhiIn the pediatric injured brain, inflammation is similarly implicated in outcomes; however, several reports have demonstrated age-specific differences in the innate immune response in the immature injured brain –81. For Another promising therapeutic with widely touted neuroprotective and anti-inflammatory properties is erythropoietin (EPO). With a primary role in erythroid development and maturation during hematopoiesis, EPO is now well-known for additional effects on the central nervous system, ranging from stimulation of neurogenesis through to prevention of oxidative stress, inflammation and cell death , 85. RecOther potential therapeutic targets to alleviate social deficits have been revealed in models of perinatal or early postnatal brain insults. Adapting the well-established Rice-Vannucci model of HI injury in mice at either p5 or p10 , Dupré et al. found that p5 injuries resulted in pronounced hyperactivity by adulthood, whereas injuries at p10 resulted in reduced social investigation . In contIt is noteworthy that several of these studies to date have detected social behavior deficits and hyperactivity concurrently in the same animals after injuries to the pre-term, term, pediatric or adult brain , 91, 92.via a range of mechanisms, including neuroinflammation (Broad neuroprotective agents with differing biological mechanisms may also influence social outcomes ammation . In addiammation ; the Trkammation ; minocycammation ; and antammation . In thisIn summary, pediatric TBI results in pronounced impairments in social interactions in rodent models, recapitulating a subset of aberrant social outcomes that are commonly observed after TBI in young children. Therapeutic targeting to improve social outcomes in experimental models are very limited to date. Findings regarding endogenous oxytocin treatment in the immature injured rat brain are promising , and sevThe combination of pharmacological targeting and rehabilitation strategies also deserves consideration. Although scarce, a few pre-clinical studies have evaluated the potential benefit of rehabilitation-based strategies in the aftermath of an early-life TBI; although social outcomes have not been evaluated , 101. FuAltogether, the prospect of treating social behavior impairments with novel therapeutics after pediatric TBI is an exciting one, and we forecast significant advances in the field in the coming decade. Even incremental or subtle improvements in social functioning after pediatric TBI have the potential to significantly improve quality of life for survivors, through increased participation in society, peer friendships, family life, school and work activities.BS and RR conceptualized and designed the manuscript. BS wrote the first draft, then BS and RR edited and revised the manuscript. Both authors approved the final submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Kidney failure requiring dialysis is associated with high symptom burden and low health-related quality of life (HRQL). Patient-reported outcome measures (PROMs) are standardized instruments that capture patients’ symptom burden, level of functioning, and HRQL. The routine use of PROMs can be used to monitor aspects of patients’ health that may otherwise be overlooked, inform care planning, and facilitate the introduction of treatments. Incorporating PROMs into clinical practice is an appropriate strategy to engage patients and enhance their role in decisions regarding their care and outcomes. However, the implementation of PROMs measurement and associated interventions can be challenging given the nature of clinical practice in busy hemodialysis units, the variations in organization and clinical workflow across units, as well as regional programs. Implementing PROMs and linking these with actionable treatment aids to alleviate bothersome symptoms and improve patients’ wellbeing is key to improving patients’ health. Other considerations in implementing PROMs within a hemodialysis setting include integration into electronic medical records, purchase and configuration of electronic tools , storage and disinfection of such tools, and ongoing IT resources. It is important to train clinicians on the practical elements of using PROMs, however there is also a need to engage clinicians to use PROMs on an ongoing basis. This article describes how PROMs have been implemented at in-centre hemodialysis units in Alberta, Canada, addressing each of these elements. Patients with kidney failure requiring dialysis experience a high symptom burden and severity and low health-related quality of life (HRQL) . HoweverHistorically, Alberta Health Services (AHS) has delivered kidney care through two programs covering the northern and southern regions of the province. In 2010, Alberta Kidney Care – North (AKC-N) introduced PROMs to in-centre hemodialysis units with the initiation of a supportive care program. This program was intended to identify patients with high palliative care needs who may benefit from additional supportive or palliative care services . This inAlberta Kidney Care – South (AKC-S) had not implemented PROMs until recently, when AKC-N partnered with AKC-S and the Ontario Renal Network to design, pilot, and rigorously evaluate PROMs interventions for in-centre hemodialysis care. These interventions were designed based on successes from PROMs studies in oncologic settings, 11 intePROMs are administered to adult patients undergoing chronic hemodialysis who are willing and able to complete the questionnaires. Patients with cognitive impairment, undergoing acute dialysis, or transiently dialyzing are not given PROMs. In some locations, PROMs were translated into the top three languages represented in the Alberta renal programs: Simplified Chinese, Punjabi, and Vietnamese. Patients with other language barriers either have a family member help, or the health system interpretive services are used. For patients with eyesight issues, their family members or nurses provide assistance. Most often, PROMs can be completed within 5 min but can vary depending on additional support required.Edmonton Symptom Assessment System Revised: Renal  (for AKC-N)Integrated Palliative care Outcome Scale – Renal  (for AKC-S)EQ-5D-5LPROMs can be generic or disease-specific, and each has several advantages and disadvantages . GenericThe Edmonton Symptom Assessment System (ESAS) is a clinically validated and reliable symptom measurement tool, extensively used in chronic diseases including cancer, heart disease, and kidney disease . It was The Palliative care Outcome Scale (POS) was developed in 1999 for use Both renal programs in AHS implemented the EQ-5D-5L. The EQ-5D-5L is a generic preference-based measure of HRQL and has been selected as the PROM of choice in many clinical areas and settings. The EQ-5D-5L includes a health status classification system with five dimensions , each with five levels of problems , describing 3125 distinct health states . An indeThe presence and severity of symptoms can change considerably over a short period of time in hemodialysis patients, emphasizing the need for regular surveillance. Within the EMPATHY Trial protocol, patients complete PROMs every two months. However, post-trial, the frequency of measurements may vary between renal programs. In AKC-S, PROMs are administered electronically through a tablet and automatically entered into the local EMR. Electronic collection includes many considerations such as tablet purchase, configuration, storage, disinfection, and staff and patient comfort. Tablet displays had to be intuitive for clinicians and patients, and investments in ongoing information technology (IT) support and training were required. In AKC-N, patients complete PROMs on paper and nurses manually enter patients’ responses into the EMR. The collection process varied across units. Some units applied a schedule for PROMs administration, some coordinated PROMs to accompany blood draws, while some units employed a practice that patients’ primary nurse would administer and follow-up on PROMs. Allowing for variability within the local unit workflow was necessary for successful implementation.The implementation and integration of PROMs assessments into hemodialysis clinics requires a feasible system to capture and report PROMs. This was facilitated by the development of PROM report cards to present easily interpretable results to clinicians and patients, similar to clinical laboratory measures. Both renal programs’ EMRs generate a PROM report card with a symbol scheme for follow up , and EMR data entry. A toolkit for using PROMs in the hemodialysis unit was developed and shared with all clinicians in the hemodialysis units. A train-the-trainer model was adopted, where clinical nurse educators received training and subsequently provided on-site training to other nurses. Initial training was administered in a series of webinars and workshops over the course of two months. Figure The implementation of PROMs measurement and associated interventions can be challenging given the nature of clinical practice in busy hemodialysis units, the variations in organization and clinical workflow across units, as well as regional programs. Incorporating PROMs into clinical practice is an appropriate strategy to engage patients and enhance their role in decisions regarding their care and outcomes. However, it is not enough to simply collect PROM measurements from patients. Implementing PROMs and linking these with actionable treatment aids to alleviate bothersome symptoms and improve patients’ wellbeing is key to improving patients’ health. Other considerations in implementing PROMs within a hemodialysis setting include integration into electronic medical records, purchase and configuration of electronic tools , storage and disinfection of such tools, and ongoing IT resources. It is important to train clinicians on the practical elements of using PROMs, however there is also a need to engage clinicians to use PROMs on an ongoing basis.The EMPATHY trial is intended to comprehensively evaluate the effectiveness of disease-specific and generic PROMs-based interventions, the adoption and implementation within real-world hemodialysis settings, as well as the cost-effectiveness of PROMs in routine hemodialysis care. Along with planned qualitative assessments, the information gained from this implementation trial will provide valuable information for the regional dialysis programs in enhancing and sustaining PROM interventions.


Researchers are looking into techniques to intervene sooner and earlier in the disease process thanks to advances in disease genetics, etiologies, and prenatal diagnosis. We conducted a literature search in PubMed-indexed journals to provide an overview of the evolution of gene therapy, rationale for prenatal gene therapy, uses and risks of gene therapy, and ethical issues following the usage of gene therapy. Recent animal research has revealed that transmitting genetic material to a growing fetus through viral and non-viral vectors is conceivable besides proving how gene-editing technology is achieved by various mechanisms that utilize zinc finger nucleases, TAL effector nucleases, and clustered short palindromic repeats-Cas9 complex. This review offers an overview of the current knowledge in the field of prenatal gene therapy, as well as potential future research avenues. In addition, it weighs the risks of prenatal gene therapy, such as oncogenesis, genetic mutation transfer from mother to child, and fetal disruption, against the expected benefits, such as preventing the development of severe early-onset illness symptoms, targeting previously inaccessible organs, and establishing tolerance to the therapeutic transgenic protein, all of which lead to permanent somatic gene correction.This review discusses the scientific, ethical, legal, and sociological implications of these groundbreaking genetic disease prevention techniques, as well as the parameters that must be satisfied for a future clinical application to be considered. Gene therapy involves delivering genetic material to a cell to elicit a therapeutic effect, such as correcting an error or giving cells a new function. This is accomplished by using a vector to deliver the genes to the correct cell, where the expression of the protein produces a therapeutic effect ,2.Gene therapy became popular in the 1980s, and the first human gene therapy trials began more than a decade ago. However, despite ongoing technological advancements, most clinical outcomes have been poor. There are several reasons for this, including difficulty in identifying the proper organ, a strong immunological reaction to therapy in adults, and low levels of expression of therapeutic gene products. Many of these issues can be avoided if therapy is applied to the fetus .Prenatal gene therapy has been proposed for life-threatening diseases for which prenatal gene delivery offers an advantage over cell transplantation or postnatal gene therapy, and for which satisfactory treatments are currently not available . A growiEvolution of gene therapyRole of Vectors in Fetal Gene TherapyThe transfer of genes into fetal cells is accomplished with the help of vectors ,7. The cTypes of VectorsVarious types of vectors are used in gene transfer in clinical trials. They can be broadly classified into viral and non-viral vectors ,8-10. ThRetroviruses: These are ssRNA viruses that integrate with the host genome and have low immunogenicity. Retroviruses were considered successful vectors in animal experiments until the early 2000s. However, research with first-generation gene therapy in children with severe combined immunodeficiency (SCID) revealed that retroviruses are carcinogenic, putting an end to their use as vectors .beta-globin gene into humanized mouse models with beta-thalassemia, Shangaris et al. demonstrated that normal hemoglobin levels could be achieved postnatally [Lentiviruses: These are a subclass of retroviruses. They are capable of transducing in dividing and quiescent cells . After itnatally . Howevertnatally .Adenoviruses: These are non-enveloped dsDNA viruses with an icosahedral capsule . They reAdeno-associated viruses (AAVs): These are single-stranded, non-enveloped DNA viruses with an icosahedral capsule. AAV is episomal and can be used in both dividing and non-dividing cells . They arDelivery of Naked DNA MaterialNaked DNA particles can be injected directly into the tissue or organ of interest. However, this method of gene delivery is vulnerable to endonuclease degradation. To improve gene delivery, physical methods such as electroporation, gene guns, ultrasound, and hydrodynamics are used ,12.Electroporation: This is a method of applying an electrical voltage to transfer genes by improving cell permeability through the destabilization of the cell membrane. Skin, muscle, and neurons are specific targets employed in this method ,12. HoweGene gun: Ballistic transfection can be used for gene therapy. In this approach, gold nanoparticles (AuNPs) serve as plasmid DNA (pDNA) carriers. Using this kind of gene delivery, it is possible to transport genes into tissues such as muscle, skin, and liver . AuNPs aUltrasound-mediated transfection: Ultrasound pulses are used to transfer genes into cells by increasing cell permeabilization ,14. It aHydrodynamic delivery: This method of gene delivery consists of applying a hydrodynamic force to transfer DNA into target cells from circulating blood. This technique is influenced by factors such as the strength of the hydrodynamic force, capillary structure, and the structure of the target tissue. Using this method, gene transfer to muscles and the liver can be carried out by injecting pDNA into the femoral artery with adequate force .The transfer of genes cannot be accomplished by injecting naked DNA without the application of hydrodynamic force because it is rapidly degraded by nucleases and cleared by monocytes and immune cells ,14. ThisPolymers: Various polymer-based nanoparticles, such as polyethyleneimine (PEI), poly-L-lysine (PLL), and chitosan, are used in gene transfer. These polymers encapsulate nucleic acids and ensure the targeted delivery of genes to specific sites. The use of PEI and PLL in prenatal gene therapy is limited due to their cytotoxicity . Poly-b-Liposomes: Lipid nanoparticles (LNPs) are less immunogenic and are one of the safer modes of gene delivery. Fetal plasma is known to have a higher proportion of high-density lipoprotein and, subsequently, apolipoprotein E (ApoE). LNPs can be of great use, particularly in metabolic and hematopoietic disorders, due to their tendency to accumulate in the liver . LNPs caGene Editing in Fetal Gene TherapyGene editing is used in the treatment of monogenic disorders in fetal gene therapy ,15. GeneGene editing can be done using different delivery platforms, such as AAV, with greater packaging ability and higher rates of gene transfection, or physical methods, such as LNPs, electroporation, and AuNPs ,10. To dIn utero gene editing can be done at various stages of fetal development: mid-to-late gestation somatic cell gene editing  and preiEx Vivo in Utero Gene EditingIn this method, autologous cells are harvested from the fetus and transplanted back to the fetus after gene editing is done in vitro. While it offers the advantage of not exposing the mother to therapy, it confers certain challenges, such as harvesting and maintaining the differentiation of an adequate number of fetal cells. These challenges can be overcome by hematopoietic stem cell transplantation . It alsoViral Versus Non-Viral Gene DeliveryViral vectors have a large packaging ability but carry the risks of immunogenicity and tumorigenesis . Non-virModes and Timing of Prenatal Gene TherapyThe timing and routes of transduction administration play a key role in the distribution and efficiency of transduction ,9.Routes of AdministrationIntravenous: Intravenous access to the umbilical vein can be obtained through ultrasound guidance. The umbilical vein also acts as a pathway for the systemic route. Umbilical vein injections can be used to target the liver, which is one of the sites of hematopoiesis in the fetus .Intra-amniotic: The distribution of transduction by intra-amniotic injection is influenced by changes in epithelial differentiation . After tIntracardiac: This route is highly specific but associated with an increased risk of the procedure. Other routes of administration include intramuscular, intraperitoneal under ultrasound guidance, and intraparenchymal injections to target the lungs ,10.Factors To Be Kept in Mind for Efficient Gene DeliveryThe vector must be safe for both the mother and the fetus. It should reach the target organ after administration and should not be rapidly removed from the bloodstream. After the vector reaches its target organ, it undergoes receptor/ligand-mediated uptake, resists endosomal lysis, and reaches the nucleus to express the protein. Moreover, it should not alter the expression of nearby genes or inactivate transgenes .Timing of Prenatal Gene DeliveryDifferent routes of administration achieve a highly variable degree of distribution, even when injected at the same gestational age. Stem cells can be easily accessed at an earlier gestational age rather than at later stages, thereby increasing the efficiency of transduction at earlier stages of development ,9. For eFuture of Prenatal Gene TherapyPrenatal gene therapy can treat monogenic disorders even before the pathology of the disease begins, thereby significantly decreasing morbidity and mortality ,10. SeveThe challenges encountered in the way IUGT becomes a clinical reality include safety, insertional mutagenesis, and oncogenesis. It is also crucial to minimize the risks of fetal intervention, such as fetal demise, injury, and premature birth . PlacentUses and risks of fetal intrauterine gene therapyRationale for Prenatal Gene TherapySeveral aspects of fetal development make treatment delivery more efficient, safe, and long-lasting.Small fetal size : First, Accessible proliferating progenitor cells : Second,Tolerogenic fetal immune system : FinallyUsesThe hazards of prenatal gene therapy are not fully understood, and its efficacy has yet to be demonstrated, as with any other treatment approach. In this regard, according to the NIH’s Recombinant DNA Advisory Committee, prenatal gene therapy should be limited to the following disorders that are linked with substantial morbidity and mortality concerns for the fetus, either in gestation or after birth: (1) Have a bad outcome with existing postnatal therapies or do not have effective postnatal therapy . (2) TheRecent gene therapy studies using various vector systems have yielded notable results for hemophilia B, retinal blindness, and SCID, ushering in a new era for a technology that has so far failed to live up to its promise. Candidates for IUGT are likely to have well-characterized monogenic abnormalities that cause severe prenatal or early childhood morbidity. Other disorders include those for which an accurate molecular diagnosis can be achieved before birth and those for which there is no effective postnatal therapy .IUGT might be used to treat a wide range of illnesses. Hemoglobinopathies, for example, have a worldwide carrier frequency of 7%, with alpha-thalassemia major (Bart’s hydrops) being the most lethal without treatment. Other examples include perinatally lethal clotting disorders such as factor VII and X deficiency, lysosomal storage disorders  that cause significant perinatal morbidity and mortality, and hereditary blindness such as Leber congenital amaurosis . PreclinHemophiliaAdult gene therapy techniques have focused on muscle or liver application, with sustained expression of factor IX in adult hemophiliac dogs or mice after intramuscular or intravascular injection of AAV vectors. The AAV serotype is crucial, according to Chao et al. When AAV serotype 1 was injected into mice, it resulted in 10 times larger amounts of canine factor IX than serotype 2. Only short-term and low-level factor IX expression has been reported in clinical studies using AAV2hFIX vectors in patients with hemophilia B, which was likely caused by a cell-mediated immune response to transduced hepatocytes. In adults with hemophilia B, a clinical trial using a self-complementary AAV8 vector serotype is currently underway, which may generate a lower immune response .At 16 days of gestation (term = 22 days), Waddington et al. showed permanent phenotypic correction of immune-competent hemophiliac mice by intravascular injection of a lentivirus vector expressing human factor IX (hFIX) protein . In the Hemophilia A and B normally do not show up until after delivery. However, a lack of certain clotting factors can result in life-threatening infant central nervous system hemorrhage. For example, in approximately 20% of patients with a homozygous or compound heterozygous genotype, congenital factor VII deficiency, the most prevalent autosomal bleeding condition, severe, or deadly bleeding occurs. They show up to 1% of normal levels and frequently have central nervous system hemorrhage . A treatThalassemiaHMGA2 gene expression dysregulation in myeloid stem/progenitor cells, or it could be a random and serendipitous event. More studies are needed to see if this clone is homeostatic or if its growth is a precursor to multistep leukemogenesis, an issue that has been observed in ex vivo autologous stem cell gene therapy for SCID [An adult patient with severe b-thalassemia who had been reliant on monthly transfusions because infancy recently showed the possibility of an ex vivo gene transfer technique for the treatment of thalassemia. He was transfusion-free for at least 21 months after autologous transplantation of gene-modified hematopoietic stem cells using a lentivirus expressing b-globin. The blood hemoglobin level was maintained between 9 and 10 g/dL, with one-third of the blood containing vector-encoded b-globin. The therapeutic impact is attributable to the dominance of a myeloid-based cell clone. The vector’s integration into this clone’s host chromosomal location AT-hook 2 (HMGA2) resulted in transcriptional stimulation of high mobility group protein (HMGA2) production in erythroid cells and truncation of an inhibitory microRNA binding site in this gene. The observed cell growth could be the consequence of for SCID .Cystic FibrosisNon-viral gene transfer agents, such as PEI, cationic lipid 67 (GL67), and DNA nanoparticles, are being used in the current clinical study for cystic fibrosis (CF), which can result in a lower immune response. Proof-of-principle investigations in the nasal epithelium reveal a 25% repair of molecular deficiency, and sheep transfected with a human CFTR plasmid complexed with GL67 exhibiting expression of hCFTR . One of Fetal Growth RestrictionPlacenta-directed gene therapy induces the production of a therapeutic transgenic protein for a brief period, which might be effective in the treatment of fetal growth restriction (FGR). In preclinical FGR tests, adenoviral vascular endothelial growth factor gene therapy delivered to the placenta via maternal uterine arteries increased fetal growth velocity, and placental insulin-like growth factor gene therapy may enhance glucose and amino acid transport while also increasing fetal development . Gene thNeuronal Disorderssurvival of motor neuron 2 (SMN2) gene, a nearly identical copy gene of 4.4.2. spinal muscular atrophy (SMA) SMN1, which generates just 10% of the full SMN RNA/protein, can partially protect affected persons. This shows that SMN2 may have disease-modifying properties and might be a target for disease-modifying gene therapy [The existence of an elevated copy number of the  therapy .SMN gene. Multiple intramuscular injections of a RabG-EIAV lentivirus vector harboring the human SMN gene boosted SMN protein levels and decreased motor neuron death in SMA type 1 fibroblasts and SMA mice [Autosomal recessive childhood variants can be divided into three categories based on their severity. Type 0 of the illness affects fetuses in utero, causing decreased fetal movements and arthrogryposis. Type I SMA causes neuronal degeneration and death throughout pregnancy, making prenatal gene therapy an attractive approach. In vitro and in vivo, vectors derived from adenovirus, herpes simplex virus, AAV, and lentivirus can transduce neurons. Neuroprotective factors such as corticotrophin 1 and antiapoptotic proteins such as B-cell lymphoma-extra large (Bcl-xL) can be employed; for example, adenovirus Bcl-xL has been found to prevent neuronal cell death in rat cell cultures. It is also possible to replace the SMA mice .SCIDEarly in pregnancy, before the immune system matures, prenatal gene therapy could theoretically eliminate the requirement for marrow conditioning or the need for a human leukocyte antigen-matched donor . PrenataRisks of IUGTThe most important risks of IUGT include the ability to disrupt normal fetal development ; carry aThe ability to disrupt normal fetal development: Due to the change in the accessibility of the vector to tissues and stem cell compartments, as well as the fluctuation in cellular receptor expression throughout early fetal development, Endo et al. group revealed the temporal dependency of transgenic expression after prenatal gene administration in fetal mice. The same group discovered adenomatous abnormalities in the lungs of rat fetuses after prenatal overexpression of fibroblast growth factor 10 . GonzagaCarry an elevated risk of germline transfer of the genetic modification: The risk of gene therapy constructing germline transfer is frequently mentioned, with the misunderstanding that it is the goal of fetal gene therapy. It is critical to emphasize that prenatal somatic gene therapy, similar to postnatal gene therapy, is intended solely for the treatment of individual patients, not their children. Any gene therapy study will try to avoid accidentally transferring the therapeutic gene to the treated patient’s germ cells as much as feasible. However, there is a legitimate fear that in utero gene transfer may raise the possibility of unintentional germline gene transfer. Human prenatal gene therapy is unlikely to be performed before seven weeks of pregnancy because germ cells are compartmentalized on the genital ridge during this period .Induce genotoxicity and/or oncogenesis: The incidence of lymphoproliferative illness in five of the 21 children treated with gene therapy using retroviral vectors for the fatal immune deficiency X-SCID shows that the integration of gene therapy vectors can lead to tumor development. This potential for oncogenesis is due to the vector’s random insertion into active genes in the host genome, which can result in oncogene activation or tumor-suppressor gene inactivation. Most of these genes play a role in the regulation of growth and differentiation processes in the body and are particularly active during fetal development. Consequently, they would be ideal integration sites for prenatally administered gene transfer vectors, and in utero, gene therapy might be particularly vulnerable to oncogenesis as a result of their integration with vectors .Ethical issuesIn utero gene therapy offers the potential to cure or reduce the severity of a wide range of genetic illnesses for which there are currently no effective therapies. However, there are ethical and practical concerns with prenatal gene therapy that do not arise with postnatal gene therapy. As with any fetal procedure, two patients, the mother and the child, participated in the intervention. The timing of the intervention during the development of the fetus involves critical questions. At the Foreign Forum, a recent panel discussion with international specialists was held at the Society for Fetal Transplantation and Immunology yearly conference, and it summed up many of these .It is important to note that complications such as infection, preterm labor, and fetal loss are theoretically possible with in utero gene therapy; however, strong clinical data are supporting a fetus’s ability to tolerate multiple minimally invasive accesses via intravascular, intraperitoneal, or intra-amniotic routes with very low risks related to the procedure. As previously stated, maternal safety is of paramount importance. Continued animal research is needed to examine the likelihood of maternal exposure to viral vectors and transgenic products as well as any potential maternal immunological response to the injectate. Finally, before the therapeutic use of prenatal in vivo gene augmentation and gene editing, the incidence and effect of viral vector integration and/or off-target consequences must be thoroughly characterized. Fortunately, no substantial germline integration/editing, or off-target effects have been observed in animal investigations thus far. Because these off-target evaluations for in utero gene editing were conducted in a biased manner, future robust and unbiased investigations are needed .Intrauterine fetal gene therapy provides minimally invasive approaches to preventing genetic disorders. Although various viral and non-viral methods are employed to deliver gene therapies, it is also essential to determine the optimal gestational age and delivery route. Non-viral vectors are advantageous over viral vectors regarding safety and usability. Unfortunately, some vectors, such as naked DNA injection, are restricted in clinical settings due to low transfection efficiency. Nevertheless, research has broadened our knowledge of possible hazards and benefits, like every novel technology we come across. Once prenatal gene therapy has been proven to be safe for treating target disorders, and the strategy of counseling families and implementing in utero gene therapy has been perfected, it can be considered for broader use. We believe that fetal intrauterine gene therapy is the future of fetal and neonatal medicine to improve quality of life and potentially cure monogenetic disorders before irreversible pathology occurs. Soon, humankind will start accepting progress and be a part of it.


Autophagy is an evolutionary conserved catabolic pathway essential for the maintenance of cellular homeostasis. Defective proteins and organelles are engulfed by autophagosomal membranes which fuse with lysosomes for cargo degradation. In neurons, the orchestrated progression of autophagosome formation and maturation occurs in distinct subcellular compartments. For synapses, the distance from the soma and the oxidative stress generated during intense neuronal activity pose a challenge to maintain protein homeostasis. Autophagy constitutes a crucial mechanism for proper functioning of this unique and vulnerable cellular compartment. We are now beginning to understand how autophagy is regulated at pre-synaptic terminals and how this pathway, when imbalanced, impacts on synaptic function and -ultimately- neuronal survival. We review here the current state of the art of “synaptic autophagy”, with an emphasis on the biogenesis of autophagosomes at the pre-synaptic compartment. We provide an overview of the existing knowledge on the signals inducing autophagy at synapses, highlight the interplay between autophagy and neurotransmission, and provide perspectives for future research. Macroautophagy, hereafter ‘autophagy’, is a conserved, regulated pathway that cells use to turn-over organelles and proteins, and create new bio-molecules. Damaged material is engulfed by a double-membrane and directed to lysosomes for hydrolytic degradation.In the early 1990’s, Yoshinori Ohsumi was the first to characterize the genes essential for autophagy . By now,Like other cell types, neurons employ autophagy to maintain homeostasis and ensure survival. This is particularly important because most neurons are not regenerated during adult life and they need to maintain functional over many decades. Furthermore, autophagy also serves a more direct role in neuronal physiology, like neurotransmission , synaptiThe unique physiology of synapses makes them susceptible to protein and organelle damage. Synaptic transmission is an energy demanding process. The production of energy by mitochondria results in the accumulation of reactive oxygen species (ROS), which might affect protein functionality. Additionally, synapses are often located far from the soma. Although local translation in distal axons of mature neurons has been reported , other sDrosophila neuromuscular junction (NMJ) synapses showed with higher spatial resolution that autophagosome biogenesis takes place at the pre-synaptic compartment  stores . Specifivia altered ER calcium buffering, several studies have reported the opposite effect upon autophagy induction   neurons .C. elegans shows that ATG9 undergoes exo-endocytosis cycles and that disruption of ATG9 trafficking impairs activity-dependent synaptic autophagy P-binding protein ATG18a/WIPI2 on the membrane of nascent autophagosomes, and the consecutive failure in ATG8/LC3 recruitment  to decipher how the cycling of vesicles at synapses signals autophagy induction, 2) to unravel how these two vital pathways are cross-regulated in pathological conditions, 3) to uncover novel synapse-specific modulators of this pathway, 4) to define the cargo, and thus the purpose of synapse-specific induction of autophagy.


Mycosporine-like amino acids (MAAs), are secondary metabolites, first reported in 1960 and found to be associated with the light-stimulated sporulation in terrestrial fungi. MAAs are nitrogenous, low molecular weight, water soluble compounds, which are highly stable with cyclohexenone or cycloheximine rings to store the free radicals. Microalgae are considered as a good source of different kinds of MAAs, which in turn, has its own applications in various industries due to its UV absorbing, anti-oxidant and therapeutic properties. Microalgae can be easily cultivated and requires a very short generation time, which makes them environment friendly source of biomolecules such as mycosporine-like amino acids. Modifying the cultural conditions along withmanipulation of genes associated with mycosporine-like amino acids biosynthesis can help to enhance MAAs synthesis and, in turn, can make microalgae suitable bio-refinery for large scale MAAs production. This review focuses on properties and therapeutic applications of mycosporine like amino acids derived from microalgae. Further attention is drawn on various culture and genetic engineering approaches to enhance the MAAs production in microalgae. Gloeocapsa sp. strain under high UV radiation [Secondary metabolites are specialized natural products which are not directly involved in general metabolism of an organism, but they have specialized functions such as antibiotic, pharmacological, photoprotective, anti-inflammatory activities, etc. . Severaladiation . The moladiation .Palythoa tuberculosa is of mycosporine-glycine, which was established by Ito and Hirata [MAAs play a vital role in maintaining the osmotic equilibrium of many organisms especially in photosymbiotic partnerships. The structure of the first mycosporine-like amino acid (MAA) isolated from zoanthid d Hirata . The strd Hirata .The two biosynthesis pathways of MAAs include the shikimate pathway and pentose phosphate pathway, but most of the produced MAAs come through the shikimate pathway. MAAs are well known for their UV (Ultra violet) rays absorbing properties, where these compounds can accommodate their free radicals in their core ring structure . Hence, Many groups of organisms like macroalgae, microalgae, marine heterotrophic bacteria, fungi and various other marine organisms are known to produce mycosporine-like amino acids which are involved in some of their survival processes and stress defense mechanisms. They also act as an intracellular nitrogen reservoir in some species. In aquatic organisms like algae and various marine organisms, these biomolecules are involved in the protection against harmful solar radiation. Specifically in certain microalgae, these MAAs protect them from the inhibition of motility by UVB radiation exposure . MycospoAlexandrium excavutum, Phaeocystis pouchetii, and the diatom Guinardia striata, it was found that there is a strong increase in MAAs content when exposed to sunlight. In certain other species, it was also observed that the accumulation of MAAs in their cells has a strong correlation with the spectral composition of the light [Different conditions and parameters were shown to have a significant impact on MAAs expression in certain species of microalgae. In some species like he light ,11.It was found that about 152 species of marine microalgae produce MAAs, in which the highest ratio of these compounds was observed in the bloom forming algal groups like dinoflagellates, prymnesiophytes, raphidophytes, and cryptomonads. However, in some cases, irrespective of their taxonomic affiliation, the expression of MAAs molecules in these marine organisms solely depended on their exposure to UV radiation and high intensity light . Table 1In addition to the traditional MAAs extraction techniques, identification of genes involved in MAAs biosynthesis and the application of genetic engineering can be utilizedto increase the expression of MAAs in microalgae. Also, various environmental parameters and culture conditions can be modified to increase the accumulation of MAAs in algal cells .Alexandrium tamarense, Alexandrium catenella and Alexandrium minutum showed induced MAAs synthesis on exposure to high light conditions (65 ± 5 µmol quanta m−2 s−1 PAR) [Microcystis aeruginosa [Scytonema cf. crispum (UCFS10 and UCFS15) under alternative 12-h of day and night cycle [Chlorogloeopsis fritschii PCC 6912 [Desmodesmus sp. was grown under high saline conditions, it was able to accumulate more MAAs in its cells, which helped it to survive under salt stress [Aphanothece halophytica, a halotolerant cyanobacterium [Chlorogloepsis when exposed to increased temperature or cold shock, downregulation of MAAs was observed, but when they were exposed to UV rays and salt stress, MAAs synthesis was induced [It was found that the spectral composition of light in which the algae are exposed have a significant effect on the MAAs content in them. Blue light and UVA radiation was observed to have more effect in increasing the MAAs production compared to red and green light . Study ps−1 PAR) . Similarruginosa  under coht cycle . Light cPCC 6912 . They wePCC 6912 . When frt stress . Similaracterium . Furtheracterium . In cont induced . Moreove induced ,31,32. TAccording to a review on MAAs published by Bhatia et al. (2011) , isolatiStereum hirsutum. The structure 1,2-methoxy-3-bis (hydroxymethyl) methylamino-5-hydroxy-5-hydroxymethyl-2-cyclohexen-1-one has been credited to mycosporine P310. Mycosporines were earlier depicted as differentiation or reproduction markers in fungal spores [Mycosporine-like amino acids are multi-beneficial compounds with molecular weight less than 1.2 kDa . These ml spores . Nowadayl spores  activitil spores . Figure MAAs are denoted as microbial sunscreens which are natural and well known for its UV absorbing property ,44,45,46MAAs are mostly seen as solutes in cytosolic space, but in some terrestrial cyanobacterium, it was also reported as derivatives of mycosporines linked to amino acids and oligosaccharides secreted by their cells . MAAs leMAAs are highly stable because of their zwitterionic form, which is stabilized by resonance between equivalent structures and also due to its hydrogen bonds. Most of the MAAs have hydrogen bonded 3D structure with more than 12 bonds . ExtraceMycosporines, have a common cyclohexenone ring system, which shares equal spectral characteristics and absorption maxima irrespective of their different molecular structures . They arFurther, the photoprotective role of MAAs is based on photostability, lack of fluorescence and radical production . Anoxic The mycosporine-glycine is not stable under aerobic conditions and they have to be converted to methyl esters. In acidic media many MAAs are converted to methyl esters with HCl methanol treatment . TemperaTrichothecium roseum [Chlorogloeopsis confirmed shikimic acid pathway for MAAs production. The presence of exogenous tyrosine can act as the repressor and glyphosate can act as the inhibitor of shikimic acid pathway [MAAs synthesis starts from an intermediate point of two different pathways, shikimic acid pathway and pentose phosphate pathway . It is bm roseum . Radiola pathway ,59.This  pathway . The pentose phosphate pathway is not the major pathway involved in MAAs production . This waMonosubstituted cyclohexenones are formed by conjugation of glycine molecules and this forms the common intermediate in production of disubstituted imine type MAAs. It is said that amino acids are modified to form MAAs by condensation, dehydration, hydroxylation and decarboxylation. MAA sulphates and glycolates are also formed by adding required side chains to it .Biosynthesis of essential amino acids in most of the organisms is via shikimate pathway. This pathway is present in algae, fungi, bacteria etc., but not in animals. They acquire MAAs through food chain ,62. EachMicroalgal species are good sources of MAAs. MAAs are utilized in various cosmetic and pharmaceutical industries and application of strategies such as genetic engineering can greatly enhance the productivity of MAAs in microalgae. Further, the transcriptomic studies to explore different genes involved in the biosynthesis of MAAs and its alternate pathways can help in these experiments.Changing/applying various external parameters can have great influence on the expression of bioactive molecules like MAAs in microalgae. Along with the modification of cultural conditions, improving the metabolic pathways can increase the yield of desired metabolites. Thus, the identification of the genes responsible for the MAAs biosynthesis is crucial for future genetic engineering applications. However, possible consequences and environmental risks that can happen as a result of such genetically engineered organisms’ usage should be considered beforehand and their impact should be estimated ,66.Nannochloropsis, Chlamydomonas, Haematococcus, Dunaliella, and Phaeodactylum are proven to be capable of nuclear transformation which make them suitable species for the application of genetic engineering. The use of proper codons, strong endogenous promoters, and insertion of 5′, 3′ and intron sequences, which are species specific can impact their expression stability. Thus, access to the microalgal genetic sequences have great importance in genetic engineering experiments. Biolistic or electroporation techniques are mainly utilized for the transformation of genes into microalgal cells and the transformants are selected using appropriate selection markers. Generating knockdown lines using RNA silencing technique is another approach for target gene deletion in microalgae along with homologous recombination [Some microalgae like bination ,67. Recebination ,68. HoweIn cyanobacteria, MAAs biosynthesis involves a four-enzyme pathway ,69. HoweScytonema cf. crispum (UCFS10 and UCFS15) were used and putative mys gene clusters (mys ABC), important for mycosporine-glycine biosynthesis were identified [Escherichia coli led to the exclusive production of shinorine, which confirmed that Anabaena type mys cluster is responsible for the shinorine production in these species . They were able to overexpress the mysABCE gene in E. coli, which resulted in more shinorine production [In one of the studies, two cyanobacterial strains of entified . The hetoduction .As MAAs are known to be multifunctional compounds, we tried to list some of the most prominent applications and uses of different types of MAAs.Stylophora pistillata, induced high catalytic activity, and high amount of superoxide dismutase was produced while disrupting photosynthesis machinery. Intracellular mycosporine-glycine amounts were decreased because of oxidative stress although MAAs other than mycosporine-glycine were not affected. Precursors of MAAs; 4-deoxygadusol also have a very strong antioxidant property. Imino-mycosporine compounds like shinorine, porphyra-334, asterine-330 cannot be oxidized [Natural or artificial compounds that can prevent damage caused by free radicals are referred as antioxidants or free radical scavengers. The first antioxidant activity of MAAs were reported in mycosporine-glycine in 2004 . High teoxidized ,42,43. Soxidized .Energy metabolism and production requires oxidation and it is linked to ROS production which serves as cell signaling molecule, triggering cellular processes such as cell division, inflammation and stress responses . OxidatiHarmful human activities can cause ozone depletion and influx of UV radiation to earth. UVA and UVB reaches the ground level but UVC radiations are absorbed by the atmosphere. Strict photosynthetic active radiation requirement may increase the UV exposure, which can be detrimental to some organisms. Most of the highly energetic UV reaching the surface of earth can be absorbed by MAAs. They absorb UV radiations and release energy in the form of heat without producing ROS.Melanin is a natural skin protection pigment placed above the nucleus and takes part in UV absorption and also acts as an antioxidant. It plays a key role in protecting keratinocyte cells against UV radiation. However, when exposure to UV radiation is excessive, an extra protection using topical applications such as sunscreen is required. These sunscreen compounds can be inorganic or organic but most of them have bad effects on the skin health. MAAs are the best choice of sunscreen which are compatible with biological systems Shinorine, porphyra-334 and mycosporine-glycine doesn’t show any toxicity in murine fibroblasts, which was confirmed by second longer term direct incubation assay in the same cell line . These MThe Keap1 (Kelch-like ECH-associated protein 1) - Nrf2 (erythroid 2–related factor 2 protein) pathway is an important regulator of oxidative stress in cells. Reactive oxygen species (ROS) are recognized by the Keap1, which leads to the release of Nrf2, and this Nrf2 is involved in the activation of oxidative stress related cytoprotective genes . MAAs liGenomic mutations by the production of DNA photoproducts are one of the most serious damaging effects of UV rays’ exposure . ProlongOverexpression of biomarkers of skin leads to conditions like psoriasis. Topical application of mycosporine-glycine, shinorine and porphyra-334 are studied against these conditions and it was found that these can be controlled with high concentration of mycosporine-glycine and low concentrations of shinorine .Long term UV rays’ exposure leads to wrinkles and sagging in skin where matrix metalloproteinases are activated . These mP. yezoensis cells [Antiproliferative activities on neoplastic cells put MAAs into the group of anticancer agents. Murine skin melanoma cells when administered with red algal extracts rich in various MAAs like shinorine, porphyra-334, etc. showed dose dependent inhibition of cell proliferation . Presencis cells . Wound healing proceeds in an organized way, which includes homeostasis, where wounds clot, constrict to resist blood flow and platelets stick to seal the cut part. Inflammation is the other phase in which localized swelling occurs. Proliferative phase and maturation phase are where rebuilding and remodeling occur respectively. These processes require many factors including epidermal growth factors. MAAs were also reported to promote wound healing in HaCaT human keratinocytes cells. MAAs stimulates the activation of tooting pathway of focal adhesion kinases (FAK) and mitogen activated protein kinases (MAPK). When MAAs are administered FAK phosphorylation occurs, which, in turn, facilitates the activation of MAPK’s extracellular signal regulated kinase .In this review, we summarized all the important properties, structure and applications of MAAs. Due to their high photoprotective and antioxidant activities, they are being utilized in pharmaceutical and cosmetic industries nowadays. Unfortunately, utilization of their biological sources for the mass production of these economically important molecules has not been explored very well. Thus, this review focused mainly on microalgae as a potential biological source of MAAs and, the prospects and challenges involved in their genetic manipulation for the increased MAAs expression.Microalgae are microscopic algal species, which are known to be a good source of various bioproducts. As they are easily culturable and require less generation time, microalgae are considered to be a good option for the production of mycosporine-like amino acids. As microalgae are unicellular, photosynthetic organisms, they can reduce the environmental pollution to some extent, increasing the quality of air we breathe. This reassures the fact that microalgae are one of the best sources for MAAs production.In microalgae, extraction of MAAs using solvent, which only consists of water and a volatile additive like methanol, followed by their detection and quantification using HPLC or LC/MS is the common procedure used. This resulted in the detection of great diversity in the occurrence of different MAAs in various microalgal species. Combination of HPLC and LC/MS or NMR and LC/MS are some common protocols followed today to increase the detection and quantification efficiency of MAAs.In order to make microalgal species capable of producing large amounts of MAAs, certain improvements in the culture conditions or external parameters can be utilized. Modifying the temperature, salinity, nitrogen concentration and UV/fluorescence exposure during the culturing of microalgae in such a way that they are exposed to stressful environment can induce the overexpression of MAAs in them. However, as these are stressful conditions for the microalgae, they can lower their cell division, decreasing thus the overall productivity of the biomolecules.Application of biotechnology in the production of MAAs from microalgae at an industrial level is the most promising future prospect in the case of these biologically important molecules. Identification of the genes and regulatory mechanisms involved in MAAs biosynthesis in various microalgal species, using certain bioinformatics tools and multiple omics approaches is important for this purpose, as only limited information regarding the genomic sequences of microalgal species are currently available. Various genetic engineering techniques can be utilized for the manipulation of these genes, particularly mys cluster of genes, involved in MAAs biosynthesis in certain microalgal species. Heterologous cloning techniques are utilized for the overexpression of these genes for high production of certain MAAs like shinorine. Molecular cloning techniques and, genome editing tools like zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) can be great tools in the genetic manipulation (knock-down or knock-in) of these genes in microalgae. However, low efficiency, cytotoxicity and restricted knowledge in the genomic information of microalgal species are the main limitations in these applications. Techniques like CRISPR-Cas9 have great scope in future for the mass production of MAAs from microalgae because of its simplicity and accuracy, if the limitations associated with their application in them are solved. Genome editing in high biomass producing microalgae for the overexpression of MAAs biosynthesis genes (enhancing their promoters and pathway regulation strategies) or genetic manipulation in high MAAs producing microalgal species to increase their biomass production (increasing photosynthetic efficiency) are two alternative routes in which genetic engineering can be carried out for the increased MAAs production in microalgae. Genetic engineering of the organisms like microalgae for the mass production of MAAs can bring great future prospects and applications for this high valuable compound. Thus, the challenges involved in the culturing and genetic engineering of the microalgae for the industrial production of MAAs need to be addressed very well in the future.


Liquid biopsy includes non-invasive analysis of circulating tumor-derived substances. It is a novel, innovative cancer screening tool that overcomes the limitations of current invasive tissue examinations in precision oncology. Circular RNA (circRNA) is a recent, novel, and attractive liquid biomarker showing stability, abundance, and high specificity in various diseases, especially in human cancers. This review focused on the emerging potential of human circRNA in body fluids as the liquid biopsy biomarkers for cancers and the methods used to detect the circRNA expression and summarized the construction of circRNA biomarkers in body fluids for treating human cancers and their limitations before they become part of routine clinical medicine. Furthermore, the future opportunities and challenges of translating circRNAs in liquid biopsy into clinical practices were explored. Cancer is one of the leading causes of death worldwide, with more than 10 million people deaths estimated in 2020, and the morbidity and mortality rates are increasing. The development of “omics” technology has facilitated the progress of precision oncology, which includes tailoring treatment plans based on the molecular characteristics of each patient’s tumor , pleuralCircRNA stems from spliceosome-mediated non-sequential backsplicing of precursor mRNA (pre-mRNA); that is, the downstream 5′ splice donor site is connected to the upstream 3′ splice acceptor site . They weCompared to the traditional tumor tissue biopsy biomarkers, circRNAs are novel, convenient, and non-invasive liquid biopsy biomarkers. Their high abundance and stability , abundanThe potential functions of circRNAs have been extensively studied. It can regulate fundamental biological processes and display an essential function in various human illnesses, including cancers –34. One via sponging miR-326 /Ras homolog family member A (RhoA) signaling pathway, suggesting that exosomal circ-133 can be a surveillance biomarker for progression of CRC . They showed that the expression level of circ-RanGAP1 in GC and plasma was closely connected with the advanced tumor-node-metastasis (TNM) stage, lymph node metastases, and low survival rate. Circ-RanGAP1 has been shown to accelerate tumor-cell growth by mediating the miR-877-3a/vascular endothelial growth factor A (VEGFA axis) . Likewisn of CRC . This phn of CRC . Chen etn of CRC . The resA good clinically significant biomarker must fit the analytical and clinical validity and clinical utility standards . As a neCircRNAs have broad application prospects in monitoring the treatment efficacy and assessing cancer prognosis. Li et al. identified more than 1,000 circRNAs in human serum exosomes for the first time and proposed that these circRNAs could distinguish colon cancer patients from the healthy controls . The decIn addition to plasma, serum, and exosome, circRNAs were also found in some other body fluids such as saliva , 68, 69,CircRNAs have also been used to evaluate the effectiveness of tumor-treatment methods and supervise tumor stress. For instance, Xie et al.  demonstrThe methods suitable for detecting circRNAs must distinguish circRNA from linear RNA species and be sensitive enough to allow reliable quantification of the closed-loop structure of circRNAs. The currently available technologies for circRNA assay are far from perfect Figure 2Compared with the traditional circRNA detection methods, reverse transcription rolling circle amplification (RT-RCA) assay has the advantages of simple operation, low cost, and high sensitivity . It can Cationic liposome nanoparticle (tCLN) biochips are advanced over the previous technologies. The positively charged chips trap the negatively charged exosomes in solution by attaching tCLN coated with a specific molecular probe to the chip. The molecular probes in nanoparticles can recognize specific substrates in exosomes and generate corresponding fluorescence signals. This method is simple and cost-effective, has good repeatability, and can effectively detect the differences between different exosomes compared to the currently available methods. Although it is mainly used to detect the microRNA (miRNA) or messenger RNA (mRNA) content in vesicles to predict cancer , 106, thPLK1) gene for liver cancer treatment have completed phase I clinical trials  or antisense oligonucleotide (ASO). Earlier research data have confirmed that the siRNA can treat various solid tumors, including liver cancer, pancreatic cancer, lung cancer, prostate cancer, breast cancer, and ovarian cancer. Among them, two lipid carrier-based siRNA drugs ALN-VSP02 and TKM-080301 targeting the vascular endothelial growth and Polo-like kinase 1 , ASO, and CRISPR/cas9  effectively  and good therapeutic effects of RNA-targeted therapy have attracted widespread interest from academic research institutions and pharmaceutical companies (Orna Therapeutics); increased revenues from approved RNA therapies have led to increased investments in this field  and the National Natural Science Foundation of China (81772270).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Hepatocellular carcinoma (HCC) is the sixth most common cancer and fourth leading cause of cancer-related death worldwide. The number of HCC cases continues to rise despite advances in screening and therapeutic inventions. More importantly, HCC poses two major health disparity issues. First, HCC occurs more commonly in men than women. Second, with the global increase in non-alcoholic fatty liver diseases (NAFLD), it has also become evident that HCC is more prevalent in some races and/or ethnic groups compared to others, depending on its predisposing etiology. Most studies on HCC in the past have been focused on genetic factors as the driving force for HCC development, and the results revealed that genetic mutations associated with HCC are often heterogeneous and involve multiple pathogenic pathways. An emerging new research field is epigenetics, in which gene expression is modified without altering DNA sequences. In this article, we focus on reviewing current knowledge on HCC-related DNA methylation changes that show disparities among different sexes or different racial/ethnic groups, in an effort to establish a point of departure for resolving the broader issue of health disparities in gastrointestinal malignancies using cutting-edge epigenetic approaches. Gastrointestinal (GI) cancers include some of the most common cancers worldwide, such as colorectal cancer , gastric cancer (5th), liver cancer (6th), esophageal cancer (8th), and pancreatic cancer 13th), based on GLOBOCAN 2018 data . An. An6-metR: 0.06) , which sGlutathione S-transferase (GST) is a family of enzymes responsible for detoxifying electrophilic compounds using glutathione, a key cellular antioxidant . The expThe study by Herath et al. also used a combined bisulfide restriction analysis to compare the methylation status of five CpG islands  in HCC tumor tissues between an Australian cohort (74% Caucasian and 26% Asian) and a South African cohort (100% black) . There ahttps://www.cdc.gov/obesity/data/prevalence-maps.html, accessed on 26 February 2021). Therefore, to effectively analyze the possible primary contributions of DNA modification to racial/ethnicity HCC disparities, study designs need to take into account differences in HCC etiologies and risk factors present in the populations studied. At present, initial studies support a potential role of differential promoter methylation of APC, GSTP1, and SOCS in Asian HCC patients in possibly accounting for some of the racial group differences in HCC incidence. It remains to be determined whether these differences are truly oncogenic or mere passengers of broader genomic and epigenetic instability. Furthermore, whether these promoter methylation changes are the result of known HCC etiopathological conditions such as chronic HCV infection remains to be established. A summary of the above-mentioned genes/pathways and studies is listed in Similar to sex-related disparities in HCC, different racial/ethnic groups also vary in their prevalence rates of different HCC risk factors and/or etiologies. The prevalence of HCV infection in Asia and Africa is ~3.5%, which is much higher than that in Europe (<1%) and the United States (~1%). In line with the HCV infection prevalence, HCV infection-associated HCC cases are higher in Asia and Africa compared to other regions. In the United States, HCV had the largest population attributable fraction (PAF) among NHBs (36.1%) and Asians (29.7%), whereas metabolic disorders had the largest PAF among Hispanics (39.3%) and NHWs (34.8%) [The development of HCC is linked to well-identified etiological risk factors. The contribution of specific risk factors to HCC development varies greatly by geographic location. Currently, chronic HBV infection is the predominant risk factor for HCC in Southeast Asia and Africa, whereas chronic HCV infection is the main HCC risk factor in Japan, Latin America, the United States, and Western countries. In the United States, NAFLD/NASH make up the largest component of HCC patients, followed by HCV infection, heavy alcohol consumption, and cigarette smoking . ExposurFrom a scientific standpoint, the concept of race itself is problematic, due to the lack of validated biological markers for racial assignment. While race plays a role in the self-identity of many individuals as a sociological concept, there can be a wide range of genetic/epigenetic variation within a given “race” as currently defined. Furthermore, the self-identification of individuals with a specific race may be at variance with their ancestral genetic heritage, which may, for instance, include contributions from multiple races. Currently, self-reported race/ethnicity is widely used in epidemiological studies as a record of genetic background origin. However, self-reported race/ethnicity may not accurately represent their genetic composition, especially in mixed populations, such as Hispanic, which are admixed with European, African, and Native American ancestry. Genetic composition varies greatly from individual to individual in mixed populations. From a scientific standpoint, race is defined better by germline  ancestry than by self-identified ancestry, as genetic and epigenetic components may be mixed in a continuous spectrum within individuals self-identified as one race. Given the complexity in defining germline  ancestry using objective measurements  and that epi/genetic signatures may or may not exist to clearly define race, major challenges remain in the research of race-related health disparities.6-MGMT, may be limited by heterogeneity within the tumor samples obscuring genuine methylation differences between groups. In select cases, further probing of preliminary negative findings may be warranted. Confirming that methylation changes reflect shifts in gene expression, and assessing the oncogenic contribution (if any) of specific differentially methylated genes remain substantial tasks to be addressed in the near term.Several questions remain unanswered regarding the role of methylation and other epigenetic changes in total HCC incidence, as well as in accounting for sex- and race-based HCC disparities. As mentioned above, apart from several meta-analyses on APC, GSTP1, SOCS, and P14, some of the identified methylation changes between groups require validation or have conflicting reports. In addition, it should be noted that studies finding no significant differences between different racial or sex groups, such as the papers examining OOne area that has been virtually untouched is examining gene methylation differences between sexes or racial groups in healthy individuals, and assessing whether these differences may result in divergent levels of risk from the same exposure. An initial step towards addressing these questions was done by McCormick and co-workers using animal models, who catalogued sex-based methylation differences between isogenic mice across multiple tissues . DiffereFurthermore, it is likely that substantial numbers of differentially methylated genes remain to be discovered. Whole genome bisulfite sequencing (WGBS) at the single nucleotide resolution level enhances the possibility of discovering novel predictive and prognostic markers, not limited by our prior knowledge. To understand the totality and dynamics of the disease, it may be necessary to do WGBS on a spectrum of patients representing both sexes and different racial/ethnic groups across a wide range of disease states. The potential findings will likely provide new perspectives on our understanding of HCC as well as new avenues for clinical intervention. In the past, most DNA methylation analyses were performed using tissue samples collected from precancerous and/or cancerous livers. The invasive nature of tissue biopsy poses a serious ethical issue that limits the source of available samples as well as the feasibility of applying the discovered DNA methylation markers as a diagnostic tool in the clinic. In recent years, liquid biopsy based on circulating cell-free DNA (cfDNA) has emerged as a new research, diagnostic, and/or screening platform to determine genetic and epigenetic changes in patients previously inaccessible due to ethical considerations ,89. SeveWe foresee several clinically relevant outcomes from studies of HCC methylation differences between sexes and different racial groups. Knowledge of underlying epigenetic differences tied to differential sensitivity to given HCC risk factors can be used to inform public education and lifestyle intervention efforts in a preventive medicine context. These can also be used, in principle, in screening efforts to generate tailor-made individual risk assessments . Furthermore, epigenetic changes linked to specific genes that are correlated with differential survival of HCC patients of different sexes or racial groups can hold diagnostic or prognostic value, and perhaps become incorporated in treatment as well as health policy decisions. Finally, development of therapeutics targeting key epigenetic changes hold great hope for overcoming several drawbacks of traditional cancer therapies. Since epigenetic changes are reversible (in contrast to genetic mutations), there is a higher chance that oncoepigenetic changes acquired over the course of tumor development may be fully reversed by epigenetic-targeting therapies. Furthermore, it may be possible to design epigenetic-targeting approaches that reduce tumor genomic instability, thereby reducing the tumor’s capability of adapting to a therapeutic cocktail by developing resistance. Finally, it is possible that epigenetic-targeting therapies may be less toxic to non-tumor tissues than traditional cancer therapeutics, reducing the severity or extent of treatment side effects.


New approaches to deal with the growing concern associated with antibiotic-resistant bacteria are in high demand. For many years, antibiotics have been the gold standard for treating infections. However, their excessive consumption and misuse have contributed to the rise of microorganisms resistant to antibiotic action, leading to a global health crisis. Engineering new drug delivery platforms and uncovering alternative biomolecules with antimicrobial and regenerative potentials is becoming extremely urgent. This Special Issue aims at expanding our understanding of the antimicrobial action of specialized biomolecules, recently engineered or chemically modified from their ancient origins, and to introduce a broad audience to new systems of drug delivery. Microsporum canis [Acinetobacter baumannii, unveiling a new vehicle of delivery for conventional antibiotics capable of improving their effectiveness [In this collection of research, many important findings can be highlighted, namely the emergence of flavonoid-coated gold nanoparticles  and the um canis . Anotherum canis , while Ltiveness . Discovering new pharmaceutical strategies to fight infection is a very challenging and time-consuming process. Considering DNA gyrase and topoisomerase IV are known targets for novel antibacterial drug design, Saleh et al. demonstrated the effectiveness of diphenylphosphonates as DNA gyrase inhibitors, with great potentially for new pharmaceutical formulations . FurtherRosa roxburghii against powdery mildew, being highlighted as a potential green, cost-effective and environmentally friendly strategy to raise production yields [Even though most of the focus of this Special Issue is on developing and unveiling new antimicrobial cues capable of mitigating or irradicating infection in humans, plants and animal products are also affected by similar issues, with pesticides and other chemical agents becoming highly ineffective against microbial plagues. Assessments of pathogenic resistance have been made by Lianou et al. on bulk-tank milk of goat herds, to identify potentially dangerous factors affecting the product quality . These an yields . Finally, recent advances in antibiotic- and heavy-metal-loaded titanium surfaces were examined as potential mechanisms for preventing local infection in bone-related applications , while f


Primary nasopharyngeal mycobacteriosis is a rare disease. We present a case in which skull base bone erosion appeared and was alleviated during the course of the treatment. Bone complications occur in osteoarticular mycobacteriosis, but their occurrence in primary nasopharyngeal mycobacteriosis has not been reported. A 77-year-old immunocompromised Asian woman presented with a right occipitotemporal headache. An ulcerative mass covered with a thick yellowish discharge was found in the roof and posterior walls of the right nasopharynx. Because histopathological examination indicated the presence of mycobacterial infection, we began using antituberculosis medication for the treatment because of the possibility of primary nasopharyngeal tuberculosis. However, this was followed by glossopharyngeal and vagus nerve paralysis. Computed tomography (CT) showed a diffuse enhancing mucosal irregularity in the nasopharynx with bony erosion of the external skull base. Deep tissue biopsy was repeated to differentiate it from malignant lesions, and drainage of pus from the right nasopharynx was confirmed. Subsequently, the headache, neurological findings, and the yellowish discharge disappeared, and the bony erosion of the external skull base was alleviated. Surgical intervention should also be considered for drug-resistant mycobacteriosis. We concluded that mycobacteriosis should also be considered apart from carcinoma even if CT shows a diffuse enhancing mucosal irregularity with bone destruction in the nasopharynx. Mycobacterium tuberculosis (TB) nor nontuberculous mycobacteria (NTM).Nasopharyngeal mycobacteriosis is a rare disease, and the irregular and swollen mucosal lesion requires differentiation from nasopharyngeal carcinoma. In previous reports of nasopharyngeal mycobacteriosis, lesions were usually found to be confined to the nasopharyngeal area without invasion into the surrounding structures such as the skull base , 2, wherA 77-year-old Asian woman presented with a right occipitotemporal headache for 1 week (day 1) but reported no other aural or nasal symptoms. Her medical history included diabetes mellitus, hypertension, rectal cancer, colon cancer, liver cirrhosis, and hepatocellular carcinoma.Bacillus culture test and polymerase chain reaction test of the sputum and nasopharyngeal smear were negative, but the T-SPOT.TB test was positive (both panels A and B > 50 spots). Cervical computed tomography (CT) revealed neither a mass shadow in the nasopharynx nor cervical lymphadenopathy , granulomatous inflammation , and autoimmune disease  . In the The most characteristic feature of the present case was bony erosion of the skull base, which appeared and alleviated during the treatment course . SrivaniIn the present case, antituberculosis medication alone was insufficient treatment; thus, the infection spreaded to the parapharyngeal space, formed an abscess, and resulted in glossopharyngeal and vagus nerve paralysis . BacteriAlthough a drug susceptibility test could not be performed, we presumed the causative bacteria as a drug-resistant TB or an NTM based on the clinical course and the patient's medical history. In such cases, the potential benefits of the surgical procedure as an adjunctive treatment for drug-resistant mycobacteriosis have been suggested , 14. In This report describes a case of primary nasopharyngeal mycobacteriosis with bony erosion of the external skull base. Differential diagnosis considering both carcinoma and mycobacteriosis should be undertaken, even when CT shows a diffuse enhancing mucosal irregularity with bone destruction in the nasopharynx. Surgical intervention should also be considered as an adjunctive treatment for drug-resistant mycobacteriosis.


Alternative lengthening of telomeres (ALT) is a telomerase-independent mechanism used by some types of malignancies, including pancreatic neuroendocrine tumors, to overcome the issue of telomere shortening, thus supporting tumor growth and cell proliferation. This review is focused on the most important achievements and opportunities deriving from ALT assessment in PanNET onco-pathology, highlighting the most promising fields in which such biomarker could be implemented in clinical practice.DAXX and ATRX. Recent advances in tumor biology permitted to uncover important roles of ALT in the landscape of PanNET, potentially relevant for introducing this biomarker into clinical practice. Indeed, ALT emerged as a reliable indicator of worse prognosis for PanNET, helping in clinical stratification and identification of “high-risk” patients. Furthermore, it is a very specific marker supporting the pancreatic origin of neuroendocrine neoplasms and can be used for improving the diagnostic workflow of patients presenting with neuroendocrine metastasis from unknown primary. The activation of this process can be determined by specific FISH analysis.In pancreatic neuroendocrine tumors (PanNET), ALT is strongly correlated with the mutational status of two chromatin remodeling genes, ALT should be introduced in clinical practice for identifying “high-risk” PanNET patients and improving their clinical management, and as a marker of pancreatic origin among neuroendocrine tumors. Since somatic cells are unable to polymerize all DNA during physiological replication, each round of cell division determines telomere shortening ,2. This Physiologically, telomere shortening is counterbalanced by the specific activity of the enzyme telomerase, which is a DNA polymerase that uses reverse transcription of a RNA template to produce de novo synthesis of telomeric DNA . InteresALT has been detected in different tumor types, including soft-tissue sarcomas and brain tumors ,10. In sIDH1-mutated astrocytomas [In brain tumors, ALT appears as the major telomere maintenance mechanism in ocytomas . Furtherocytomas . These fInterestingly, ALT has also been detected in pancreatic neuroendocrine tumors (PanNET), with important diagnostic and prognostic implications. Here we will analyze and discuss more in depth the most important findings related to ALT in PanNET, shedding light on its potential applications in clinical practice.DAXX) and α-thalassemia/mental retardation X-linked (ATRX) genes [DAXX/ATRX mutations [DAXX mutations were more commonly reported than ATRX mutations in PanNET [ATRX were markedly prominent in sarcomas [The first description of the presence of the ALT process in a significant proportion of PanNET was provided by Marinoni et al. in 2014 [X) genes . Subsequutations ••, 19. In PanNET ••, 19, wsarcomas •.DAXX/ATRX mutations can be studied not only with a direct molecular analysis but also with immunohistochemistry, which represents a reliable surrogate of their mutational status. Indeed, in the case of mutation, there is a total loss of expression of the corresponding protein at the immunohistochemical level  vs. active surveillance (ALT negative).A recent study assessed the feasibility of ALT determination on materials from fine needle aspiration (FNA), as well as of immunohistochemistry in determining DAXX and ATRX expression . InvestiIn the case of neuroendocrine metastasis from unknown primary, which represents a non-negligible proportion of cases, Dogeas et al. indicated that the presence of the activated ALT mechanism could represent an important tool in supporting the pancreatic origin of a neuroendocrine tumor . Indeed,A last insight regards the possibility to predict the ALT status on the basis of radiological findings. McGovern and colleagues showed that some features identified at computed tomography were significantly associated with the ALT phenotype, such as tumor necrosis, vascular invasion, pancreatic duct dilation, and liver metastasization. These findings should be read in the context of multidisciplinary and precision medicine, where both pathology and radiology can integrate their information guiding the best clinical management of PanNET patients.The assessment of the ALT status can be very useful to improve the clinical practice for PanNET patients: (i) to stratify prognosis of resected patients, with immediate implications for patients management; (ii) in the pre-surgical setting, serving as an additional tool to select patients for surgical resection; and (iii) as a marker of pancreatic origin, improving the diagnostic workflow of patients presenting with neuroendocrine metastasis from unknown primary. Globally considered, these represent three major reasons that call for launching ALT determination in the PanNET clinical practice.