The placenta plays a vital role in fetal development during pregnancy. Dysfunction of the placenta can be caused by oxidative stress and can lead to abnormal fetal development. Preventing oxidative stress of the placenta is thus an important measure to ensure positive birth outcomes. Research shows that tryptophan and its metabolites can efficiently clean free radicals (including the reactive oxygen species and activated chlorine). Consequently, tryptophan and its metabolites are suggested to act as potent antioxidants in the placenta. However, the mechanism of these antioxidant properties in the placenta is still unknown. In this review, we summarize research on the antioxidant properties of tryptophan, tryptophan metabolites, and metabolic enzymes. Two predicted mechanisms of tryptophan's antioxidant properties are discussed. (1) Tryptophan could activate the phosphorylation of p62 after the activation of mTORC1; phosphorylated p62 then uncouples the interaction between Nrf2 and Keap1, and activated Nrf2 enters the nucleus to induce expressions of antioxidant proteins, thus improving cellular antioxidation. (2) 3-Hydroxyanthranilic acid, a tryptophan kynurenine pathway metabolite, changes conformation of Keap1, inducing the dissociation of Nrf2 and Keap1, activating Nrf2 to enter the nucleus and induce expressions of antioxidant proteins (such as HO-1), thereby enhancing cellular antioxidant capacity. These mechanisms may enrich the theory of how to apply tryptophan as an antioxidant during pregnancy, providing technical support for its use in regulating the pregnancy's redox status and enriching our understanding of amino acids' nutritional value. During pregnancy, the placenta's nutrient transport and barrier functions have been shown to affect the development and health of the fetus . BecauseIn the placenta, syncytiotrophoblast mainly serves invasion and endocrine functions but may also be an important part of the placental barrier, ensuring optimal fetal development and pregnancy. During gestation, various factors can induce oxidative stress in syncytiotrophoblast, trophoblast, and other portions of the placenta, thus disturbing the placenta's overall functioning; even worse, these can induce pathological pregnancy . For insIn addition to a myriad of external factors, there are numerous internal factors that can affect placental stress. The addition of antioxidants into a diet during pregnancy is important to improve antioxidant capacity of the placenta and fetus and is beneficial for the mother's health. Some traditional antioxidant nutrients (such as vitamin C and vitamin E) are used in the diet and can relieve oxidative stress during pregnancy . HoweverTryptophan (Trp) is an essential amino acid in animals. It is also a precursor to many active molecules, such as serotonin, melatonin, kynurenic acid, NAD, and NAPD . StudiesThese studies reveal that Trp can act as an effective antioxidant. However, the mechanism of how it works is still poorly understood. Accordingly, this review discusses the potential antioxidation mechanisms of Trp in the placenta and other extrahepatic tissues based on related research, with the intent of providing theoretical support and practical reference to improve the antioxidant capacity and reproductive performance of pregnant mammals by controlling Trp metabolism.in vitro culture tests of human glioma cells [In previous research, Trp has been found to be an important antioxidant in certain foods, such as eggs , yacon , and potma cells . Under hma cells . These sin vivo is not directly attributable to the radical scavenging activity of Trp molecules per se, but rather the radical scavenging activity of some Trp metabolites [To investigate the mechanism of Trp's antioxidation, Perez-Gonzalez et al. showed that Trp cannot be oxidized by hydrogen peroxide across a variety of chemical environments based on quantum mechanical and chemical detection methods. The antioxidant property of Trp abolites , 27 or tabolites .In the body, tryptophan (Trp) is catabolized through two primary pathways and generates numerous bioactive molecules in the following ways: (1) by synthesizing indole derivatives, containing serotonin and melatonin, through the biosynthesis pathway of melatonin, and (2) by producing kynurenic acid, xanthurenic acid, anthranilic acid, nicotinic acid, and others through the kynurenine pathway catalyzed by tryptophan 2,3-dioxygenase (TDO) in the liver or indoleamine 2,3-dioxygenase (IDO) in the extrahepatic tissues , 28.Many studies have reported the antioxidant properties of Trp metabolites, especially melatonin. Melatonin can directly remove free radicals and enhance antioxidation capacity by enhancing the expression or activity of antioxidant enzymes . Richterin vivo and in vitro. Weiss et al. found that 3-hydroxyanthranilic acid can effectively remove reactive oxygen species and active chlorine [In the kynurenine pathway, kynurenine , kynurenchlorine .In addition to these kynurenine metabolites, the metabolic enzymes IDO and TDO in the kynurenine pathway are also considered “scavengers” of “free radicals” and important antioxidant enzymes. TDO and IDO use superoxide anion as a cofactor to catalyze the oxidation of Trp to kynurenines . The effThe expression of IDO is closely associated with the formation of the placenta and is thus found in its greatest abundance in the placenta . During in vitro. Expression increased after LPS stimulation [In preeclampsia patients, the mRNA and protein expression of IDO and Trp metabolic activity decreased in the placenta. The decrease extent of Trp metabolic activity was found to be associated with the severity of the disease , 40. Commulation .In terms of antioxidation, Trp plays an important regulatory role in restoring the body antioxidant system. In 2016, Jiang et al. found that the level of GSH and GPx in muscle tissue could be enhanced by feeding Trp to grass carp, verifying that some signal molecules in the Nrf2/ARE pathway and mTOR pathway were closely related to Trp-initiated increases in body antioxidant capacity .The nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) pathway is the most important endogenous antioxidant pathway in the body. It plays an important role in cellular redox homeostasis and cellular defense against oxidative stress –46. In tAt a resting state, Nrf2 and Keap1 molecules combine in the cytoplasm in a nonfree and constantly degraded inactive state. After being stimulated by signals, however, Keap1 and Nrf2 uncouple; Nrf2 transfers into the nucleus from the cytoplasm and heterodimerizes with members of the small musculoaponeurotic fibrosarcoma (Maf) family of transcription factors that bind with the antioxidant response elements (AREs), activating the expression of downstream antioxidant proteins and detoxifying enzymes . ResearcIn the process of Nrf2 activation, the dissociation of Nrf2-Keap1 is a key step . The disThe mTOR signaling pathway mainly comprises mTOR  and other series of protein kinases. mTOR participates in the composition of two structurally and functionally distinct multiprotein complexes: mTORC1  and mTORC2  . mTORC1 The mTORC1/S6 kinase (S6K) pathway can be activated by amino acids' entry into cells via various signaling cascades. Numerous studies have suggested that a Trp sufficiency signal was associated with activated mTOR activity in mTORC1 , 57. In in vitro kinase assay [The signaling adaptor p62 is an integral component in the central gated channel of the nuclear pore protein complex. It is also a key factor in mediating cellular functions due to its ability to establish interactions with multiple signaling molecules , 59. These assay . This suse assay . In mousse assay , while aMeanwhile, p62 mediates the regulation of the Keap1-Nrf2-ARE signaling pathway via kinase phosphorylation and the uncoupling of the protein kinase . In mousp62 interacts with the Nrf2-binding site of Keap1, competitively inhibiting the interaction between Keap1 and Nrf2 and then activating the expression of numerous genes to encode antioxidant proteins and anti-inflammatory enzymes . MeanwhiNumerous studies have shown that the activation of mTORC1 can lead to the activation of the Keap-Nrf2-ARE pathway in a p62-dependent manner , 66. IchAccordingly, combined with the aforementioned details that Trp could enhance the expression of numerous signal molecules in the Keap-Nrf2-ARE and mTOR pathways, we suggest that in extrahepatic tissue, Trp could activate the phosphorylation of p62 after activation of mTORC1. Phosphorylated p62 would then uncouple the interaction between Nrf2 and Keap1, with activated Nrf2 then entering the nucleus to induce expressions of antioxidant proteins, thus improving the cell's antioxidation capacity .Studies have indicated that the placenta was the most enriched site for indoleamine 2,3-dioxygenase (IDO) in the kynurenine pathway and that the expression of IDO is closely related to placental formation . Kudo deγ and LPS [κB, which affects the development of diseases such as vascular injury and aortic atherosclerotic inflammation [In the extrahepatic tissue, 3-hydroxyanthranilic acid (HA), one of Trp metabolites in the kynurenine pathway, is an effective antioxidant  Figure . HA not  and LPS . HA dose and LPS . In the ammation .Therefore, besides Trp activates the expressions of antioxidant proteins through activating the mTORC1 and Nrf2/ARE pathway, we also predict that HA could induce the expression of antioxidant proteins (such as HO-1) to enhance the cellular antioxidant capacity in the extrahepatic tissues. HA, a Trp metabolite formed along the kynurenine pathway catalyzed by IDO and kynureninase, could modify and change conformation of Keap1, induce the dissociation between Nrf2 and Keap1 after which activated Nrf2 enters into the nucleus, and induce the expression of antioxidant proteins . These tOverall, although Trp is suggested to be an effective antioxidant and has been used as a functional food additive, the antioxidant efficiency and antioxidant mechanism of Trp in the organism required clarification. Meanwhile, we still lack understanding of the effects of Trp, its metabolites, and metabolic enzymes on the antioxidant properties of extrahepatic tissues. This review summarized early experiments associated with the effect of Trp's antioxidant properties  to speculate on the antioxidation mechanism of Trp in the placenta. These predicted pathways pave the way for further research on Trp's antioxidant ability in the placenta and are beneficial for further exploring its antioxidation mechanism. Future findings will be useful for recommendations for Trp additives in food as an antioxidant amino acid. The present review should provide theoretical support for practical applications: to relieve oxidative stress and thus improve the reproductive outcomes for female mammals.


Hyalomma ticks.Crimean-Congo haemorrhagic fever virus (CCHFV) is considered an emerging infectious disease threat in the European Union. Since 2000, the incidence and geographic range of confirmed CCHF cases have markedly increased, following changes in the distribution of its main vector, To review scientific literature and collect experts’ opinion to analyse relevant aspects of the laboratory management of human CCHF cases and any exposed contacts, as well as identify areas for advancement of international collaborative preparedness and laboratory response plans.We conducted a literature review on CCHF molecular diagnostics through an online search. Further, we obtained expert opinions on the key laboratory aspects of CCHF diagnosis. Consulted experts were members of two European projects, EMERGE (Efficient response to highly dangerous and emerging pathogens at EU level) and EVD-LabNet .Consensus was reached on relevant and controversial aspects of CCHF disease with implications for laboratory management of human CCHF cases, including biosafety, diagnostic algorithm and advice to improve lab capabilities. Knowledge on the diffusion of CCHF can be obtained by promoting syndromic approach to infectious diseases diagnosis and by including CCHFV infection in the diagnostic algorithm of severe fevers of unknown origin.No effective vaccine and/or therapeutics are available at present so outbreak response relies on rapid identification and appropriate infection control measures. Frontline hospitals and reference laboratories have a crucial role in the response to a CCHF outbreak, which should integrate laboratory, clinical and public health responses. Crimean-Congo haemorrhagic fever virus (CCHFV) is a tick-borne pathogen that causes a frequently lethal disease in humans and is considered to be a major emerging infectious disease threat spreading to and within Europe -3.The severity of the disease, the presence of domestic and wild animal reservoirs and/or vectors, a large population of susceptible humans, limited diagnostic capacities and resources for epidemiological/ecological investigation, as well as the absence of effective prophylaxis and treatment render CCHFV a pathogen with outbreak potential .Hyalomma, specifically the Hyalomma marginatum species [Since 2000, the incidence and geographic range of CCHF cases have markedly increased ,6 follow species ,7,8. In  species . CCHF is species , In Bulg species . In the  species . Importe species , United  species , Greece  species  and Germ species . A detai species .Public health systems (including diagnostic laboratories) should be prepared to respond to the increased circulation of the virus in endemic EU countries, the potential for importation of human CCHF cases or the emergence of virus circulation in new areas e.g. Spain .The objectives of this study were to amalgamate the expertise of two EU expert networks (i) EMERGE (Efficient response to highly dangerous and emerging pathogens at EU level)  and (ii)We carried out an on line research of published paper related to CCHFV molecular detection methods. References were obtained by an online search in PubMed using an intentionally wide search-query to ensure that a large number of papers was retrieved also for a rare disease such as CCHF.https://www.ncbi.nlm.nih.gov/nucleotide), using ‘txid1980519(Organism)’ as term of query. All analyses have been focused only on CCHFV S-segment, because it resulted as the most conserved gene across CCHFVs [The query produced a large number of papers, 20% of them were discarded after a narrative review, as they did not contain a detailed description of the detection methods employed including the nucleotide sequences of primers and/or probes. The search was done by one author and the results discussed among the authors. Papers related on non-previously retrieved molecular detection methods or to others relevant aspects discussed in this report have been directly provided by experts. For phylogenetic analysis all available CCHF virus genomes by 5 December 2017 were retrieved from GenBank  is tick-borne and is maintained in a tick-vertebrate-tick cycle with Hyalomma marginatum, the main vector species in Europe. Given the wide distribution of its vector, CCHFV has been detected over a wide geographic range: Africa, Europe, Asia and the Middle East [CCHFV  encodes the RNA-dependent RNA polymerase (L protein), the medium segment (M) encodes the glycoproteins GN and GC, while the small segment (S) encodes the nucleocapsid protein (N). Phylogenetic tree  was builMost of the isolates causing outbreaks in eastern Europe belong to clade V, whereas clade VI and VII include largely divergent strains isolated from ticks in Greece (including the prototype strain AP92) ,22 and RMoreover, isolates belonging to the African clade III were collected from infected ticks in 2010 and 2014, and recently  a virus For all strains analysed, the collection country was recorded and represented on the world map in Human infections are usually observed as single, sporadic cases when people in rural areas are bitten by ticks that have become infected by feeding on viraemic wild and domestic animals like hares, hedgehogs, horses, livestock and possibly birds . The infIn addition to tick exposure, CCHF infection can result from direct contact, especially through mucous membranes or skin wounds, with crushed infected ticks or the blood of infected animals . Person-to-person transmission can also occur through contact with virus-containing bodily fluids of patients during the first 7–10 days of illness . UnproteThe typical duration of viraemia ranges from 1– 9 days ,29, and 8 copies/mL is associated with fatal outcomes [Viral load is the most important prognostic factor: a value of viraemia higher than 10outcomes . Viraemioutcomes ,33.All CCHFV genotypes belong to one serogroup . Cross-rNucleocapside-directed IgM antibodies have been identified as the initial serological marker during infection, becoming detectable in a median of 2–3 days after disease onset, followed by glycoprotein precursor (GPC) directed IgM (4–6 days) and IgG antibodies (5–6 days) . In anotIgG seroconversion occurs 1–2 days after the IgM response  and IgG Antibody production against CCHFV is an important prognostic indicator for survival . PatientCCHFV is classified as a risk group 4 pathogen. The virus is stable under wet conditions for 7 hours at 37 °C, 11 days at 20 °C and 15 days at 4 °C [However, there is an ongoing debate about the absolute requirement of biosafety levels 4 (BSL4) for handling the virus . Many en2) or low pH (less than 6) [Like all lipid-enveloped viruses, CCHFV can be readily inactivated by common fixatives such as 2% glutaraldehyde , formali than 6) ,45. The  than 6) .Waltham, Massachusetts, USA) [2) [There is a general agreement among the consulted experts that a critical aspect for laboratory biosafety and operation is the proper and reliable inactivation of specimens before they can be removed from the high-level biocontainment environment for further diagnostic testing. Some of these inactivation methods include (i) chemical treatment   + 100% ethanol, SDS, 0.5% Tween-20  ,47 or (iUSA) [2) .It is opinion of the experts that further evaluation of inactivation procedures are needed, especially for their impact on other laboratory tests necessary for clinical evaluation and increased survival rates of patients. Although there is no direct evidence of its effects on CCHFV, it has been shown that Triton X-100 , without affecting the results of biochemical tests -52.General guidelines for suspected viral haemorrhagic fever infections apply for the transport of diagnostic samples from CCHF-suspected cases; these are listed under guidelines as Category A, Infectious Substances Affecting Humans UN 2814 and must be transported in packaging that meets the United Nations class 6.2 specifications and complies with Packing Instruction P620 .Additionally, as for other biological resources, the exchange of CCHF samples or virus strains needs to comply with the Nagoya protocol on access to genetic resources and the fair and equitable sharing of benefits arising from their utilisation, which regulates transnational exchange between countries .The choice of which CCHF detection assays should be used for diagnostics with maximum sensitivity and specificity depends on the stage of disease and the specimens available.Laboratory diagnosis of a patient with a clinical history compatible with CCHF is generally performed during the acute phase of the disease by viral RNA (RT-PCR) detection in blood . In addiThe available data, limited to very few patients, do not allow detailed comparisons of the sensitivity of RT-PCR detection methods performed on different sample types and, in particular, of urine and saliva vs serum samples. In two of six infected patients reported in a review of CCHF cases in Kosovo* , viraemind annual meeting, Rotterdam October 2017).During the small outbreak in Spain involving two patients in 2016, positive RT-PCR results were obtained via saliva and vaginal swab, but they became negative when viraemia was still detectable . Virus iThere is high genetic diversity within the different CCHFV strains , which cTherefore there is an agreement of experts that is advisable to perform more than one test to avoid exclusive reliance on a single assay and a single target, taking into account the travel history and the geographic distribution of the different strains.In published investigations, the methods employed for the detection of antibodies are indirect immunofluorescence assays (IFAs) and enzyme-linked immunosorbent assays (ELISAs) -57. SeveA CCHFV seroneutralisation test is not normally performed for diagnostic purposes; it requires work with an infectious virus, necessitating a BSL4 laboratory, and is difficult to perform. However, reverse genetic approaches employing non-infectious reporter viruses have been described recently , enablinViral isolation, i.e. from blood or organ for further characterisation or infectivity studies, is performed under BSL4 conditions on either LLC-MK2, Vero, BHK-21 or SW-13.4 cell lines and can be achieved in 2–10 days . CCHFV gIt is preferable that viral isolation is performed on samples collected during the first 5 days of infection, when the viraemia levels are high ,61.There is no official, agreed-upon case definition for CCHF in the EU, though several case definitions adopted by EU countries were reviewed by the European Centre for Disease Prevention and Control (ECDC) ,63. AccoBoxFor laboratory confirmation of a clinical CCHF diagnosis, the expert group opinion is that a CCHFV infection is laboratory confirmed when at least one criteria in the Box is fulfilled.• Detection by molecular tests of CCHFV RNA, in blood  or in other bodily fluids or tissues;• Detection of CCHFV IgM or relevant (fourfold) increase in CCHFV IgG titres between two serologic samples ;• CCHFV isolation and/or detection of CCHF viral antigens in blood .CCHFV: Crimean Congo haemorrhagic fever virus.Taking into account that laboratory screening is usually performed using molecular methods, we propose as expert opinion the following molecular diagnostic algorithm for patients with suspected CCHF infection . HoweverSupplementary material.Other relevant aspects related to management of CCHF patients as clinical manifestation and discharging criteria are reported in the CCHF is an important global health threat, as underlined by its inclusion in the list of priority diseases in the WHO document ‘An R & D blueprint for action to prevent epidemics’ .In Europe, two autochthonous cases in Spain in 2016  and the Prompt and accurate laboratory diagnosis during the first days of the disease is critical to improve patient management, guide infection control measures and reduce case fatality. Early detection of viral RNA in blood is considered the gold standard diagnostic approach in the acute phase of the disease . While tIn addition, appropriate biosafety measures must be in place when performing molecular testing.Measures to improve CCHFV laboratory preparedness and response should include: improving molecular tests to overcome the lack of sensitivity due to the high variability of the CCHFV genome; providing a positive control panel for molecular diagnostics, including the different CCHFV genotypes (including for serology testing) in order to support the improvement of diagnostic capability of clinical laboratories; enrolling diagnostic laboratories appointed for CCHFV diagnostics regularly in EQA programmes; improvement of diagnostic algorithms building on clinical experiences; validation of procedures to inactivate infectivity of clinical samples; and establishing of an international biorepository for the collection and storing of clinical samples, with the aim of validating new diagnostic tests and supporting pathogenicity studies. Some of these activities, including the evaluation of laboratory capability  and EQA There are knowledge gaps concerning the putative persistence of the CCHFV in various body compartments of survivors and the related consequences for infection transmission. Basic knowledge is needed to provide evidence to better inform hospital discharge guidelines and these issues need further research. Furthermore, a One Health approach is required for adequate public health preparedness for CCHF, and relevant measures should include vector and animal surveillance, focusing particularly on migratory birds ,9,69. GrUntil an effective vaccine and/or therapeutics have been developed, the CCHFV outbreak response will continue to rely on rapid identification and appropriate infection-control measures. Front-line hospitals, as well as reference laboratories, have a crucial role in the outbreak response, which should integrate laboratory, clinical and public health responses.


The primary function of mitochondria is respiration, where catabolism of substrates is coupled to adenosine triphosphate (ATP) synthesis via oxidative phosphorylation (OxPhos). Organic acids such as pyruvate and malate produced in the cytosol are oxidised in mitochondria by the tricarboxylic acid (TCA) cycle and subsequently by the electron transport chain (ETC). Energy released by this oxidation is used to synthesise ATP, which is then exported to the cytosol for use in biosynthesis and growth. In plants, mitochondrial composition is relatively complex and flexible and has specific pathways to enable continuous survival during abiotic stress exposure and to support photosynthetic processes in illuminated leaves.Plant mitochondria are double-membrane organelles where the inner membrane is invaginated to form folds known as cristae to increase the surface area of the membrane. The outer membrane contains relatively few proteins (<100) and is permeable to most small compounds (<Mr = 5 kDa) due to the presence of the pore-forming protein VDAC (voltage dependent anion channel), which is a member of the porin family of ion channels. The inner membrane is the main permeability barrier of the organelle and controls the movement of molecules by means of a series of carrier proteins, many of which are members of mitochondrial substrate carrier family (MSCF). The inner membrane also houses the large complexes that carry out electron transfer in two inter-connected pathways that finish with two terminal oxidases. It is also the site of oxidative phosphorylation (OxPhos) and contains a non-phosphorylating bypass of the classical ETC. The inner membrane also encloses the soluble matrix which contains the enzymes of the TCA cycle and many other soluble proteins involved in a myriad of mitochondrial functions.Mitochondria are semi-autonomous organelles with their own DNA, protein synthesis, and degradation machinery. The proteins encoded by the mitochondrial genome undergo a range of post-transcriptional and post-translational processing during their synthesis. The mitochondrial genome also encodes a number of pollen abortion related genes involved in controlling plant fertility in a process known as cytoplasmic male sterility (CMS). These CMS plants are used to produce hybrids that benefit from hybrid vigor or heterosis, producing greater biomass and yield. However, the mitochondrial genome encodes only a small portion of the proteins which make up the mitochondrion; the rest are encoded by nuclear genes and synthesised in the cytosol. These proteins are then transported into the mitochondrion by the protein import machinery and assembled with the mitochondrially synthesised subunits to form the large respiratory complexes and other proteins.Stress tolerance is a very complex trait, involving a multitude of developmental, physiological, and biochemical processes. Compared to other organelles, plant mitochondria are disproportionately involved in stress tolerance, probably because they are a convergence point between metabolism, signaling, and cell fate . MitochoThe number of mitochondria per cell varies with tissue type, with more active cells with high energy demands, such as those in growing meristems, generally equipped with larger numbers of mitochondria per unit cell volume and typically these show faster respiration rates. Research on plant mitochondria has rapidly developed in the last few decades with the availability of genome sequences for a wide range of model and crop plants. Recent prominent themes in the plant mitochondrial research include linking mitochondrial composition to environmental stress responses and how this oxidative stress impacts upon mitochondrial function. Similarly, interest in the signaling capacity of mitochondria  has revealed the transcriptional changes of stress responsive genes as a framework to define specific signals emanating to and from the mitochondrion. There has also been considerable interest in RNA metabolic processes in plant mitochondria including RNA transcription, RNA editing, the splicing of group I and group II introns, and RNA degradation and translation. Despite their identification more than 100 years ago plant mitochondria remain a significant area of research in the plant sciences. In this Special Issue, “Plant Mitochondria”, a total of 19 articles were accepted with 15 original research articles and 4 review articles broadly covering the field of plant mitochondrial research . ManuscrELM1 paralogue (ELM2) seemed to have only a limited role in mitochondrial fission in an elm1 mutant, suggesting that Arabidopsis has a unique, cold induced mitochondrial fission that involves only DRP3A to control the size and shape of mitochondria. The mitochondrial transcription termination factors (mTERFs) which are involved in the control of organellar gene expression (OGE) with mutations in some characterized mTERFs  suggesting a role for these proteins in abiotic stress tolerance. Here Robles et al. [mterf6-2 was hypersensitive to NaCl and mannitol during seedling establishment, while mterf6-5 showed a greater sensitivity to heat later in development. Rurek et al. presented a pair of research papers that used physiological, proteomic, and transcript analysis approaches to examine the thermal (hot and cold) and drought responses of cauliflower mitochondria [Brassica genus.A number of research articles in this Special Issue focused on the responses of mitochondria to abiotic stress, with studies that examined thermal stress (both hot and cold), salinity, and drought. Arimura et al.  demonstrs et al.  showed tchondria ,10. In tatp9 in the male sterility of CMS PET2 in sunflower. Here they showed that CMS PET2, which has the potential to become an alternative CMS source for commercial breeding, has a duplicated atp9 with a 271-bp-insertion in the 5’ region of one of the atp9 genes which results in two unique open reading frames (orf288 and orf231). The reduced anther-specific co-transcription of these open reading frames in fertility-restored hybrids supports their involvement in male sterility in CMS PET2.Male sterility refers to the inability of a plant to make viable pollen. It can be mediated through nuclear genes leading to genic male sterility (GMS) or through mitochondrial proteins interacting with nuclear genes, leading to cytoplasmic male sterility (CMS). Both GMS and CMS are widely used in agricultural production for the production of hybrid crops that benefit from heterosis. In this Special Issue Štorchová  presentsOlea europaea cv. Galega Vulgar (European olive). Podgórska et al. [fro1 (frostbite 1) that has a point mutation in the 8 kDa Fe-S subunit NDUFS4 grown on different nitrogen sources. When these plants were grown on NO3- they showed a carbon flux towards nitrogen assimilation and energy production, whereas cellulose integration into the cell wall was restricted. In contrast they showed improved growth on NH4+ and not the expected ammonium toxicity syndrome. Similarly, Podgórska et al. [4+ treatment and had milder oxidative stress symptoms with lower ROS accumulation and induction of glutathione peroxidase-like enzymes and peroxiredoxins antoxidants. Mansilla et al. provided a comprehensive review of the composition and biogenesis of the terminal oxygen acceptor cytochome c oxidase (Complex IV) in yeast, mammals, and plants. This revealed that while plants retain many biogenesis features common to other organisms, they have also developed plant specific features.A total of five papers we submitted examining OxPhos, with two of these focused on identifying non-phosphorylating bypasses of the classical ETC. Wanniarachchi et al.  identifia et al.  examineda et al.  showed tAs the majority of proteins that function in mitochondria are imported from nuclear encoded cytosolic synthesized proteins, studies understanding the process of how mitochondrial protein import is controlled and regulated is vital to alter mitochondrial functions. Here Zhao et al.  examinedftsh4 mutant on meristem proliferation [nmat2/pmh2 mutant-line. Together this suggests that these proteins serve as components of a proto-spliceosomal complex in plant mitochondria. Robles et al. [Two papers examined the mitochondrial protease FTSH4, one looking at the impact of a feration , and anoferation  showed ts et al.  provides2O2 scavenging ability in mitochondria. Accompanying this were higher activities of CAT, GR, MDHAR, and DHAR in the AsA-GSH scavenging system, enhanced TCA cycle-related enzymes , and activated alternative pathways.Mao et al.  examinedhttps://www.icpmb2019.com/).Overall, the 19 contributions published in this special issue illustrate the advances in the field of plant mitochondria and I look forward to catching up with the plant mitochondrial community at the next biannual meeting in Ein Gedi, Israel (


Organic acidurias and aminoacidopathies are groups of frequent inborn errors ofmetabolism (IEMs), which are caused by mutations in specific genes that lead toloss of protein/enzyme or transport function with important deleterious effectsto cell metabolism. Since a considerable number of such disorders arepotentially treatable when diagnosed at an early stage of life, diagnosis iscrucial for the patients. In the present report, we describe symptomaticindividuals referred to our service that were diagnosed with these disordersfrom 2006 to 2016. We used blood and urine samples from 21,800 patientssuspected of aminoacidopathies or organic acidemias that were processed by theanalytical techniques reverse phase high-performance liquid chromatography foramino acid quantification and gas chromatography coupled to mass spectrometryfor organic acid detection. Analysis of dried blood spots by liquidchromatography-tandem mass spectrometry was used in some cases. We detected 258cases of organic acidurias, and 117 patients with aminoacidopathies werediagnosed. Once diagnosis was performed, patients were promptly submitted to theavailable treatments with clear reduction of mortality and morbidity. Theobtained data may help pediatricians and metabolic geneticists to become awareof these diseases and possibly expand newborn screening programs in thefuture. Inborn errors of metabolism (IEM) are phenotypically and genetically heterogeneousdisorders caused by defects in specific proteins, mostly enzymes, which compromisecellular metabolism. Although any given IEM is rare when taken individually, theyare collectively numerous, causing significant morbidity and mortality  of physiological levels of organic acids andamino acids, respectively in the body and biological fluids that were referred to our service, which is a regional and national reference centerfor the diagnosis of these diseases. Our objective was to find out the mostprevalent organic acid and amino acid disorders in a high-risk Brazilian populationand to promote the awareness of these disorders within the medical community asimportant and prevalent groups of genetic diseases.The methodology for the diagnosis of organic acidurias and aminoacidopathies wasset up in 1994 at the Medical Genetics Service of the Hospital de Clínicas dePorto Alegre (HCPA), in the city of Porto Alegre, Southern Brazil. We aimed toprovide an adequate treatment for a considerable number of these disorders,justifying therefore their detection in patients without established diagnosis.Plasma (1 mL) and fresh random urine specimens (2–10 mL) without preservativewere collected and stored frozen within 2–3 h under –30 °C. Sometimescerebrospinal fluid (CSF) was collected and frozen under the same conditions ordried whole blood spots in filter paper we used for amino acid detection. Thisstudy was approved by the Ethical Committee of Hospital de Clínicas de PortoAlegre. Patients who participated in this study or their parents and/orguardians signed informed consent forms for collection of the biologicalsamples.Samples were collected from symptomatic 21,800 children (52% boys and 48% girls)with suspected IEM or disorders of amino acid (plasma amino acid determinationwas requested in 48 % of samples) or organic acid metabolism (organic aciddetection was requested in 52 % of urine samples), at the age varying from 24 hto 23 years from 17 Brazilian states (17/26 – 65.4%) from public and privatehospitals and outpatient clinics. Children less than 3 years old were 66% of thesample, and less than 12 months old were 40%. Patients’ clinical information wasobtained from a questionnaire filled by health professionals. Neurologicalfeatures (81%), feeding refuse (21%), hepatomegaly (14%), dysmorphias (13%),vomiting (11%), and failure to thrive (5%) were the most frequent clinicalmanifestations, whereas hypoglycemia (15%), persistent or recurrent metabolicacidosis (13%), and lactic acidemia (6%) were the most common laboratoryfindings. Major neurological signs were unexplained psychomotordevelopment/mental retardation (39%), persistent seizures (32%), abnormalmuscular tone (22%), macrocephaly (3%), cerebral atrophy (2%), lethargy/coma(1%), and encephalopathic crises (1%).The free amino acids in plasma, urine, or CSF were determined by reverse phaseHPLC according to Organic acids were determined in urine by GC/MS according to Amino acids and acylcarnitines were analyzed in dried blood spot samples byliquid chromatography coupled to mass spectrometry in Tandem (LC/MS/MS)according to Our lab participates in the ERNDIM (European Research Network for InheritedDisorders of Metabolism) schemes for qualitative organic acid, quantitativeamino acid, qualitative blood spot acylcarnitine as an external quality controlprogram for accuracy evaluation.Samples from 65% of referred patients came from six states of the southeast and southof Brazil that corresponds to the most prosperous Brazilian regions. We identified258 cases of organic acid disorders and 117 patients with aminoacidopathies, makinga positive diagnostic yield of about 1.72%. Rare disorders are neglected in most of developing countries due to other commoninfectious and other more prevalent diseases associated with malnutrition. However,rare disorders need to be investigated because a considerable number of patients inthese countries suffers from IEM. Due to the huge burden of IEM, reference centersdedicated to selective screenings are important even for developing nations for thescreening and diagnosis of the most prevalent IEMs such as organic acidurias andaminoacidopathies .Analyses of organic acids and amino acids by sophisticated analytical methods arerelatively expensive, especially considering the budget of developing countries.However, because of the relatively high prevalence of organic acidemias andaminoacidopathies among the known IEM , metabolic acidosis, hypoglycemia, and lacticacidosis. We incorporated in our laboratory 15 years ago an international qualitycontrol program for organic, amino acid and fatty acid determination that warrants abetter reliability of our analysis, implying a good diagnostic approach for thesedisorders.The use of a questionnaire containing a large spectrum of clinical and laboratoryfindings, as well as diet, medication, and age that was filled out by physiciansrequesting examinations for suspected patients helped us in the diagnosis of thesediseases. Thus, these data is utterly important for correct assessment ofchromatography findings on amino acids and especially organic acids, consideringthat there is a high variation on the level of these metabolites pending on age,clinical condition, and treatment at the time of sample collection. In our sample,no clinical and laboratory data were obtained from approximately 20% of thepatients.The average age of diagnosis (when patients were referred to our laboratory) was 3.8years for aminoacidopathies and 4.6 years for organic acidurias, which could beattributed to lack of awareness of these genetic diseases by the physicians, lack oflaboratories and expertise on the diagnosis of these disorders, high patientmortality for some disorders, or a combination of these factors. Nevertheless, thehighest proportion of patients was diagnosed in the first year of life, whereasdiagnosis of others disorders with lower mortality and less intense clinicalmanifestations such as L-2-hydroxyglutaric aciduria and glutaric acidemia type I hada late diagnosis.All cases were confirmed in additional plasma, urine and CSF (patients withnonketotic hyperglycinemia) samples collected on at least two different occasions.For some disorders, we needed additional tests that were carried out in otherlaboratories especially in Europe and USA. Tandem MS was also performed in driedblood in some patients suspected of having urea cycle defects since HPLC does notdiscriminate certain amino acids and this enabled us to diagnose another fifteencases of aminoacidopathies. Therefore, we strongly recommend this highly analyticalmethod (LC/MS/MS) for the diagnosis of urea cycle defects.et al., 1989; Regarding the relative frequency of organic acidemias in our population, aninteresting observation was the high number of glutaric aciduria type I (59patients) and 3-hydroxy-3-methylglutaric aciduria (40 patients) detected in ourstudy, with a similar prevalence to the other most prevalent organic acidurias, suchas methylmalonic acidemia . (Of the 64 cases of primary lactic acidemias diagnosed (high lactic aciduria andlactic acidemia) detected on three consecutive occasions, only a few of them had thebiochemical and/or the molecular defect defined , indicating the difficulty of establishingthe biochemical cause in this group of disorders. Furthermore, approximately half ofthese children died suddenly at the time of diagnosis or a few weeks later,reflecting the severity of these mitochondrial disorders associated with the lack ofeffective therapy. On the other hand, since most patients affected by organicacidurias lived in other states in Brazil, we did not have their follow up in orderto determine precisely the rate of mortality for these diseases. Nevertheless, alldiagnosed patients were immediately submitted to the available treatment mainlybased on restricted diets and supplementation of vitamins, amino acid mixture,glycine and/or L-carnitine, and liver transplantation for some those affected byaminoacidopathies as maple syrup urine disease and tyrosinemia type I after alltreatment efforts failed. Furthermore, the clinical symptomatology of our patientswith organic acidurias was often severe probably because of the late diagnosis ofwhich outcomes are much worse. This was particularly observed in glutaric aciduriatype I, in which striatum degeneration affected the vast majority of our patientsand represents an irreversible and crucial step towards spasticity and other markeddebilitating muscular symptoms.e.g., thefirst urine). Sampling after recovery, even if it is only partial, may result indecreased sensitivity and may not reveal diagnostic abnormalities detectable beforerecovery.Another aspect that seems worth mentioning is that specimens for selective organicacid screening need to be collected preferentially during a crisis of metabolicdecompensation usually caused by catabolic events triggered by intercurrentinfections, changes in nutrition, or prolonged fasting , nonketotic hyperglycinemia (17), and tyrosinemia type I (12),probably because of the severity of the clinical findings of these disorders, whichraises the awareness of pediatricians and neurologists. Of the urea cycle defectscases, only three were diagnosed by HPLC, and 15 additional patients with thesedisorders were diagnosed by LC/MS/MS. Thus, it is emphasized that althoughimportant, HPLC does not allow complete separation and/or identification of allamino acids that can be otherwise identified by amino acid analysis on LC/MS/MS,including arginine and citruline. Interestingly, 20 cases of PKU were referred toour laboratory for confirmation because of abnormal tests  in the first sample at private laboratories also dedicated to the NBSprogram, whereas we diagnosed four cases of PKU in mentally retarded children thatprobably escaped the NBS detection of this disorder, whose nationwide coverage isabout 86% in Brazilians babies.in loco and asearly as possible even in a developing country and particularly in severely illpatients because of the high prevalence of these diseases in these high-risk groupsof patients, and particularly because of the availability of therapy for many ofthem. Treatment monitoring of affected individuals by serial analysis of urine andblood metabolites is also important and justifies the setup of such facilitiesdespite extra costs. Our study shows that amino acids and organic acids disordersmay warrant the development of a nationwide screening program for the diagnosis ofthese disorders because of their high incidence in the neonatal period in Brazil.Thus, we propose that the NBS program should be expanded in Brazil to include thesedisorders, besides PKU, in neonates in order to avoid the high morbidity andmortality, ensuring easy and equal access to this preventive public healthinitiative and to increase the awareness and knowledge among the general public andespecially, health professionals. Further studies are needed to investigate thelong-term outcome and costs associated with the diagnosis and treatment of thesedisorders.Finally, we emphasize that some patients with organic acid disorders andaminoacidopathies were inadvertently confounded with septicemia, which also presentssevere symptoms such as acidosis and acute encephalopathy; these late diagnosesoften lead to death or permanent sequelae. Our results indicate the importance ofdiagnosing organic acidemias and aminoacidopathies


Drosophila melanogaster provides an extremely valid resource to investigate the mechanisms involved in organ formation and in the pathology of human diseases. Nearly 65% of human genes have orthologs in D. melanogaster, and nearly 75% of the genes involved in human disease have functional orthologs in flies  content, mitochondrial functionality, and chronological life span. They show that, during chronological aging, an altered expression of the specific mitochondrial NAD+ carriers deeply influences the metabolic reprogramming that enables cells to acquire features required to maintain viability during aging.Ohkuni et al. used yeast as a cellular model of neurodegenerative disorders such as Huntington’s disease (HD). They show that the SUMO-targeted ubiquitin ligase (STUbL) Slx5 reduces the toxicity and abnormal transcriptional activity associated with a mutant fragment of huntingtin (Htt) that induces aggregation, the causative agent of HD. Importantly, RNF4, the human ortholog of Slx5, limits the aberrant transcriptional activity of aggregation-prone Htt in yeast and in several cultured human cell lines. Thus, this study uncovers a conserved pathway that counteracts the accumulation of aggregating, transcriptionally active Htt, on chromatin in both yeast and in mammalian cells.Fraschini’s review is focused on the molecular pathways and proteins involved in the control mitotic spindle morphogenesis and function, which are highly conserved from yeast to humans and whose impairment is connected with the development of human diseases. Fraschini illustrates the processes of spindle formation and orientation in yeast and in humans and includes many examples of misregulation that lead to the development of cancer and other human diseases.Smurova and De Wulf report the role of centromeres and kinetochores in preserving genetic stability; in particular, they describe how centromere transcription contributes to faithful kinetochore function, how pericentromeric chromatin is silenced by RNA processing, and the transcriptional misregulation of (peri)centromeres during stress, natural aging, and disease.Bonetti et al. illustrate how DNA ends are processed in budding yeast in order to maintain genome stability. DNA double-strand breaks (DSBs) are dangerous lesions that can be repaired by homologous recombination (HR) that occurs after DNA ends are correctly processed by several nucleases by a process called DNA end resection. The same nucleases function also during DNA replication in the processing of replication fork structures. The authors describe current knowledge of the mechanism of resection at DNA DSBs and replication forks.Natali and Rancati describe the mutator phenotype in budding yeast. The mutator phenotype enhances genome instability and generation of phenotypic variation in a population of cells and increases the probability that some of these variations undergo selection and clonal expansion in challenging environments. This issue is particularly relevant for human health since cancer cells experience an increased mutational charge during early steps of carcinogenesis. The authors discuss the activity of the DNA replication and repair machineries and how mutations can confer increased genome instability. In addition, they describe recent clinical evidences in cancer biology indicating that these lessons can be applied to tumor development.Sollazzo et al. used Drosophila larval wing epithelium to investigate the impact of MYC upregulation on cells carrying mutations in neoplastic tumor suppressor genes (nTSGs). MYC overexpression confers to cells mutant for different nTSGs, the ability to initiate multifocal, three-dimensional growth, a hallmark of mammalian pre-cancerous fields.Anelli et al. demonstrate that six microRNAs increase following expression of a constitutively active HRASG12V allele. Two Ras-induced microRNAs, namely, miR-146a and miR93a, target the Jmjd6 gene, which encodes a JumonjiC domain protein. Results in this study show that Jmjd6 plays a critical role in zebrafish melanoma development and that miR-146a and miR93a function as tumor suppressors, antagonizing Jmjd6 activation.By using three different models of Ras induction and tumor formation in zebrafish, Mirzoyan et al. provide a comprehensive insight into the signaling pathways involved in tumorigenesis that are conserved in flies and highlight the ease to genetically manipulate these circuits to study cancer biology. In addition, they describe examples of Drosophila cancer models and their use to identify new therapeutic strategies.Merigliano et al. describe the link between vitamin B6, diabetes, and cancer. Although several data indicate that diabetes and cancer are correlated, the molecular mechanisms involved remain to be clarified. Recent results obtained in Drosophila indicate that vitamin B6 deficiency causes hyperglycemia and increases DNA damage. These data suggest that, in diabetic patients, high PLP levels should increase the frequency of DNA damage thus contributing to cancer formation and progression.Burla et al. (2018) demonstrate that mutant mice with progeroid traits, caused by reduced expression of the Ft1 gene (the ortholog of human AKTIP), display repeated seizures not linked to overt brain morphological alterations or severe neurodegeneration. However, Ft1 reduction is associated with the activation of the inflammatory markers IL-6 and TGF-β. Remarkably, reduction of the guardian of the genome p53 rescues the epileptic behavior and reverses back the expression of IL-6 and TGF-β in Ft1 mutant mice, suggesting an involvement of DNA damage response in these phenotypes.D. melanogaster for dissecting the molecular mechanisms underlying the neurological defects in inherited human diseases. Congenital disorders of glycosylation (CDGs) are multisystemic diseases caused by mutations in genes controlling the glycosylation pathways  is associated with intellectual disability, autism, hyperactivity and language delay, long face and large ears, macroorchidism, and irregular spermatids  syndrome, caused by mutations in the fragile-X mental retardation  and from the University of Milano Bicocca (FA). Financial support from the Italian Ministry of University and Research (MIUR) through grant “Dipartimenti di Eccellenza- 2017 “to University of Milano Bicocca, Department of Biotechnology and Biosciences is also acknowledged. Research in MG lab is supported by a grant from Fondazione AIRC per la ricerca sul Cancro (AIRC), grant IG 2017, Id 20779.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.


These findings suggest that simultaneous neuromonitoring of perfusion and metabolism could provide critical information regarding clinically significant hemodynamic events prior to the onset of brain injury.Preterm infants born with very low birth weights are at a high risk of brain injury, in part because the premature brain is believed to be prone to periods of low cerebral blood flow (CBF). Tissue damage is likely to occur if reduction in CBF is sufficient to impair cerebral energy metabolism for extended periods. Therefore, a neuromonitoring method that can detect reductions in CBF, large enough to affect metabolism, could alert the neonatal intensive care team before injury occurs. In this report, we present the development of an optical system that combines diffuse correlation spectroscopy (DCS) for monitoring CBF and broadband near-infrared spectroscopy (B-NIRS) for monitoring the oxidation state of cytochrome c oxidase (oxCCO) – a key biomarker of oxidative metabolism. The hybrid instrument includes a multiplexing system to enable concomitant DCS and B-NIRS measurements while avoiding crosstalk between the two subsystems. The ability of the instrument to monitor dynamic changes in CBF and oxCCO was demonstrated in a piglet model of neonatal hypoxia-ischemia (HI). Experiments conducted in eight animals, including two controls, showed that oxCCO exhibited a delayed response to ischemia while CBF and tissue oxygenation (S An estimated 15 million babies are born prematurely each year and those with very low birth weights (VLBW < 1500 g) are at a high risk of neurodevelopmental impairment ,3. AlthotO2), which has been used as a surrogate marker of CBF in the clinic  - Δ[Hb]) has been used as a surrogate marker of CBF, but its relationship to flow also depends on oxygen metabolism [Although the importance of monitoring both CBF and metabolism has been long recognized, Sn injury . The low effects ,33. HemoThe multiplexing approach presented here was necessary to avoid crosstalk between the B-NIRS and DCS techniques as the DCS laser would saturate the B-NIRS detector if they are operated simultaneously. As well, when the broadband source is used concurrently with DCS measurements, the autocorrelation curves are altered, particularly at short distances between the B-NIRS emission probe and DCS detection fiber. Even when a single-mode DCS detection fiber was used, to maximize the dynamic range of the correlation factor, we found that with a 10 mm distance between the B-NIRS source and the DCS detection, incoherent light excited additional modes in the DCS fiber. This manifested as a much lower β value in the autocorrelation curves  and resutO2, CBF, and oxCCO measurements every 14 s. This sampling rate could likely be increased to every 3-5 s using new technologies such as the DCS software correlator [The SNR can be further increased by reducing the sampling rate; lengthening the shutter cycle times allows for measures to be averaged over more acquisitions, but this would increase the time between successive measurements. In its current configuration, the hybrid B-NIRS/DCS device provided Srrelator . HowevertO2 was monitored by a combination of a spectral derivative fitting approach to determine baseline StO2 and a faster linear algorithm based on the Beer-Lambert law to track subsequent changes in HbO2 and Hb concentrations. The reproducibility of the baseline optical properties derived from the former was tested by conducting an error analysis involving Monte Carlo simulations. tO2 (64 ± 13%) was in good agreement with a previous study (StO2 = 69 ± 2%) involving the same animal model but using time-resolved NIRS to measure the optical properties [tO2 [Soperties . Furtheries [tO2 ; howeverSource-detector distances of 3 cm for B-NIRS and 2 cm for DCS were chosen to probe the piglet brain because it has been shown that in reflectance geometry, the penetration depth is approximately the square root of the source-detector distance . ConsidetO2, absolute blood flow, and oxCCO, which has not been previously reported. In terms of brain monitoring, a similar concept combining frequency-domain NIRS (FD-NIRS) and DCS [tO2, CBF, and CMRO2, and has been used to investigate neonatal hemodynamics. Instead of a 785-nm laser, a DCS laser emitting at 850 nm was used along with optical filters to prevent crosstalk between the two systems. An 850-nm laser is outside of the spectral range used for broadband analysis of hemoglobin concentration (680 to 845 nm), but more critically it is within the range of the CCO feature (780 to 900 nm). Although there are long coherence lasers operating at wavelengths lower than 680 nm, it is noteworthy that they have significantly lower power outputs. As well, above 900 nm, SPCM detectors commonly used in DCS systems have lower quantum efficiencies; both factors would reduce SNR. These limitations of current technology restrict the feasibility of using optical filters for concurrent monitoring of CBF and oxCCO.There have been a number of previous studies combining B-NIRS and DCS –40, in p and DCS  was recetO2, CBF, and oxCCO. More importantly, the experimental results clearly demonstrated the unique individual responses in oxygenation, flow, and metabolism (tO2 and hemoglobin concentrations allow for monitoring of hypoxia, CBF dynamics indicate ischemia, and changes in oxCCO relate to metabolic stress. Together, these different perspectives describe the hemodynamic and metabolic environment that surrounds and precedes brain injury.The HI piglet model produced rapid and dynamic changes in Stabolism  and 8. TtO2 reached their nadirs. A similar biphasic relationship between oxCCO and HbDiff was found in piglets experiencing anoxia [tO2 and a delayed oxCCO response. Reduced blood flow is likely met by increased oxygen extraction in order to maintain oxidative metabolism, which is consistent with previous reports between CBF and CMRO2 in piglets [In the 6 animals with HI injury, the decrease of oxCCO was delayed in comparison to CBF and continued to decline even after CBF and Sg anoxia . Group a piglets  and infa piglets . This coabsolute cerebral blood flow and changes in the oxidation state of cytochrome c oxidase − two sensitive biomarkers of brain health. The combination of the two subsystems was achieved through the use of a simple multiplexing approach based on electromechanical shutters and can be easily implemented elsewhere. In addition, the ability to simultaneously monitor CBF and oxCCO has led to a key finding; that is, the response of cerebral oxygen metabolism is delayed in hypoxia-ischemia while the CBF response is instantaneous. We anticipate that real-time monitoring of absolute CBF and oxCCO will provide greater insights into hemodynamic events that precede brain injury and could become valuable for guiding therapeutic interventions. Furthermore, because the hybrid B-NIRS/DCS system is safe and portable, it could be easily deployed in the NICU to monitor cerebral perfusion and metabolism at the bedside in early stages following birth, with the goal of detecting significant hemodynamic events before brain injury occurs.This article reports on the development of a novel optical system that combines DCS and B-NIRS to enable continuous monitoring of


Zinc performs many biochemical and physiological functions; however, toxicological studies demonstrate that Nano-zinc oxide has harmful effects on human health and environmental species in high concentrations. The aim of this study was to investigate the toxicity of zinc oxide nanoparticles on reproductive tissues of female rat.Eighty female Wistar adult rats weighing 180–200 gr, divided into eight groups (n= 10 in each group) including control, sham , and six groups injected with different doses of zinc oxide nanoparticle with 10–30 nanometer size  twice a week for four weeks. At the end of the study, the rats were bled and slaughtered; the Ovary and Uterus were taken for histopathology studies and blood samples were transferred to the laboratory for biochemical analysis.Microscopic diagnoses in ovary tissue were included; increase in the corpus luteum, follicular cysts, inflammatory cells infiltration and fibrosis. Histopathological changes in ovary in a dose-dependent manner. In uterus tissue the lesions consisted; epithelial destruction, hyperplasia of endometrial glands. The Estrogen and Progesterone level in the serum of rats increased in low doses and reduced in a dose-dependent manner at high doses.The results of the current study proved the toxicity of zinc oxide nanoparticles on the ovary and uterus organs at high concentrations, so further investigation is needed to reduce these effects. Heavy metals are relatively dense materials that exist in natural constituents of earth crust (1). In recent years, human activities such as industrial chemicals production and mining have increased the amounts of heavy metals into the environment and there has been increasing concern about their effects (1). In particular, zinc oxide (ZnO) nanoparticles (NPs) are commonly used to manufacturing products such as cosmetics, ceramics, plastics, glass, cement, rubber, grease, paints, pomade, glue and drug (2). The nanoparticle distribution in the environment depends on the properties of the metal including particle size, shape, surface characteristics, and various environmental factors (3). The evaluation of nanomaterials safety is of great importance for the industrial applications (4). It is also generally accepted that some of these metals are essentially required to retain various biochemical and physiological functions for living organisms in low amount, but toxic when they exceed their threshold concentrations (5). Many researchers investigated the potential side effect of Nano-sized particles and reported that prolonged exposure to environmental pollutants such as zinc can cause adverse health effects and harmful impacts on sexual function . Exposure ZnO NPs during the organogenesis stage indicated poor embryonic development in mice. (7).Also ZnO dissolution has been shown to be contributing to the acute or chronic toxicity of aquatic organisms (8). Heavy metals can be combined through food ingestion and inhalation and can influence the hypothalamic-pituitary-ovarian axis and play a role in the human fertility as a certain environmental factor (9). Mice treated with silica and titanium dioxide NPs had small uterus and fetuses (7). Jo and colleagues showed that exposure to Exposure to ZnO NPs prior to and during gestation could compromise the health of pregnant females and their fetuses. (8).In addition heavy metals accumulation reduce progesterone and estradiol output. Pregnancies that occur despite a high heavy metal body load are at a greater risk of abortion, fetal deformity, premature birth and placental insufficiency (10).The aim of this study was to investigate toxicity of ZnO NPs on reproductive tissues of female rat.Eighty female Wistar rats of 8 weeks in age weighting 200In this Experimental study, the animals were divided into eight different experimental groups of 10 animals each: control: normal and apparently healthy rats that did not take any type of foodstuff. All other animals were received intraperitoneally (IP) twice a week for four weeks  including: Group sham were given 10% normal saline body weight and the treatment group was given, respectively, 4, 8, 25, 50, 100, and 200 mg/kg body weight of ZnO nanoparticle Suspensions.0.9% Saline.ZnO Nano powder was bought from the Iranian Nanomaterials Pioneers Company, NANOSANY . ZnO nanoparticle milky white color and 10–30 nm, other specifications are reported in Table I and transmission electron micrograph and crystal specifications of ZnO NPs are shown in Figure 1. An existing suspension was read by suspending ZnO NPs in At the end of the fourth week, the rats were anesthetized with intraperitoneal injection of xylazine and ketamine . Blood samples (3–5 cc) were taken from the heart of all the rats. Serum Estrogen and Progesterone hormones level was measured by using Elisa kit protocol .From each animal, the sections of the ovaries and uterus were removed. The samples collected were fixed in 10% buffered formalin for 24 hr, and then routinely processed for histopathological examination and cut into 5-μm thick sections using a microtome . Finally, the sections were stained with hematoxylin and eosin (H&E)  and viewed under light microscope . A Tucsen TrueChrome Metrics camera and ISCapture software were used for histopathological evaluation.Ovary homogenates were prepared by mixing 0.5 g of ovary tissue with 5 ml of phosphate buffer saline and homogenized by homogenizer . The suspension was centrifuged at 12,000The experimental design adhered to the guidelines and was approved by the Animal Ethics Committee of Islamic Azad University, Babol branch, Babol, Iran.The data were analyzed using one-way analysis of variance, followed by post-hoc Duncan test by SPSS software version 22. A pHistopathological findings in ovary tissue included: Hyperemia, increase the corpus luteum, follicular cysts, inflammatory cells infiltration, and fibrosis . Histopathological findings in uterus tissue were epithelial destruction and hyperplasia of endometrial glands . Histopathological lesions in the ovary and uterus increased in a dose-dependent manner. The Estrogen and Progesterone level in the serum of rats after intraperitoneal injection of ZnO nanoparticle suspension are shown in Table III. The results showed that the exposure to ZnO nanoparticle dose-dependent increases the Estrogen level to 8 mg/kg and then reduces to 200 mg/kg . Destructive effects on different organs and systems can be caused by short- and long-term exposure of heavy metals such as zinc. In the present study, female rats injected IP with ZnO NPs have shown light microscopic changes in the ovary and uterus due to ZnO NPs treatment. Histopathological results indicated dose-dependent increases in lesions with different doses of ZnO NPs injection; in the histopathological examination, the ovary in moderate and high doses treated with ZnO NPs showed hyperemia, increased the corpus luteum, inflammatory cells infiltration and fibrosis, follicular cysts and the uterus in high doses showed epithelial destruction and hyperplasia of endometrial glands. Ovary and uterus tissue of control group did not show any macroscopic and microscopic lesion. In contrast to the 200 (mg/kg) ZnO NPs-treated rats, the ZnO NPs content in the ovary and uterus of other groups' rats significant lesions were identified. A study by Mehta and co-worker reported arsenic-induced lesions in the ovary and uterus and decreased folliculogenesis that lead to reproductive failures (13). Lead (Pb) accumulated and damaged in the ovary and also has been found in sheep uterine tissues. High-dose Pb administration caused ovarian follicular cysts and led to poor corpus luteum. (14). Degeneration of uterine and decrease in primary follicles of ovaries were observed after sodium arsenite treatment. (15). Another study reported that the ovaries of Ni NPs treatment rats after gavage were associated with the infiltration of inflammatory cells, congestion, and ovarian lymphocytosis (16). Jo and colleagues reported that the uterus of adult female rats orally exposed to ZnO nanomaterials (500 mg/kg) showed pathological lesions such as localized necrosis, inflammation, thrombosis, and histiocyte infiltration (8). Oxidative stress is one of the major mechanisms behind heavy metal toxicity as measured by MDA content in vivo lipid peroxidation (17). Statistical analysis showed that MDA levels were significantly higher in treated groups compared to the control group. Our results point out that ZnO NPs generate free radicals that lead to cell damage. Following exposure to heavy metals such as cadmium and lead, metal were accumulated in ovary and can induce oxidative stress. (18). Suhartono and colleagues reported that exposure to Cd caused an increase in Advanced Oxidation protein products' level in rat kidney and increased MDA level in ovarian rats (19). In a study reported that following administration, Cd, as and Hg, serum MDA levels increased time and dose dependent. (20).In the study of lipid peroxidation, MDA content in plasma, liver, and pancreas of exposed rats to Zinc was higher than that of ad libitum values (21). Chattopadhyay and co-worker showed that arsenic exposure led to a significant decrease of peroxidase activities in ovary and uterus. (15). A study showed that cadmium causes oxidative stress in kidney (19). The results of this study confirms that lipid peroxidation is one of the molecular mechanisms of cell injury in chronic ZnO NPs poisoning. Estrogen (E2) and Progesterone (P) are steroid hormones that have an important role in the reproductive tissues growth and differentiation, in the fertility, and maintain follicular development in general. These hormones are secreted of ovaries, so any destruction in ovary tissue can lead to their secretion disorder (22–24). In this study, Estrogen and Progesterone in serum were determined as well as ZnO concentrations in serum to investigate the effects of ZnO NPs. It has been reported that subchronic Aluminum exposure with medium and high doses causes a decrease in the levels of Estrogen and Progesterone in serum of the treated rats (25). In another study, Estrogen decreased after gavage administration of Ni NPs in adult rat (16). Low levels of ZnO NPs through stimulation of the glands cause increase in the Estrogen secretion. But high levels of ZnO NPs because of destructive effects on the ovary can cause decrease in the Estrogen secretion. It has been suggested that NPs have a greater risk of toxicity than larger particle (26), although the production and usage of these materials are increasing, the available information on the toxicological features about them are still incomplete.ZnO NPs can enter animal and human tissues via air inhalation or food and manual handling; and they get absorbed in the tissues such as ovary. So because of the various usage and the possible harmful effects of ZnO NPs, investigating its toxicity is critical especially in mammalian cells. Due to changes in Estrogen and Progesterone and in the absence of lesions, it seems that low doses have positive effects.The authors declare that they have no conflict of interest.


Dietary sulfur amino acid restriction, also referred to as methionine restriction, increases food intake and energy expenditure and alters body composition in rodents, resulting in improved metabolic health and a longer lifespan. Among the known nutrient-responsive signaling pathways, the evolutionary conserved integrated stress response (ISR) is a lesser-understood candidate in mediating the hormetic effects of dietary sulfur amino acid restriction (SAAR). A key feature of the ISR is the concept that a family of protein kinases phosphorylates eukaryotic initiation factor 2 (eIF2), dampening general protein synthesis to conserve cellular resources. This slowed translation simultaneously allows for preferential translation of genes with special sequence features in the 5′ leader. Among this class of mRNAs is activating transcription factor 4 (ATF4), an orchestrator of transcriptional control during nutrient stress. Several ATF4 gene targets help execute key processes affected by SAAR such as lipid metabolism, the transsulfuration pathway, and antioxidant defenses. Exploration of the canonical ISR demonstrates that eIF2 phosphorylation is not necessary for ATF4-driven changes in the transcriptome during SAAR. Additional research is needed to clarify the regulation of ATF4 and its gene targets during SAAR. Understanding how dietary protein quantity versus quality impacts growth and health has been studied and debated for over 100 years . The ideSubsequent investigations in rodent models indicate that the range of dietary methionine restriction which elicits leanness without protein wasting and food aversion is 0.12 to 0.25 g per 100 g diet, as compared to the 0.43 to 0.86 g per 100 g in complete rodent diets ,8,9. Indob/ob mice [Much of the recent interest paid to SAAR stems from the observation that rodents subjected to the restricted diet display increased healthspan. Health improvements induced by SAAR include attenuation of body weight and body fat gain. Investigation into the altered body composition seen in rodents fed SAAR points to increases in energy expenditure as well as heat increment of feeding . Dietary/ob mice ,14. Furt/ob mice .The reduced overall adiposity in SAA restricted rodents corresponds with reductions in fasting concentrations of insulin, glucose, thyroxine, insulin-like growth factor-1 and leptin, and increases in serum adiponectin ,14,16,17Other systemic effects of SAAR include delayed cataract development, downregulation of arrhythmogenic, hypertrophic, and cardiomyopathy signaling pathways in the heart, and attenuated cardiac response to beta adrenergic stimulation . On the At a glance, SAAR appears to recapitulate many of the beneficial effects attributed to caloric restriction; however, it is worth noting that SAAR elicits a transcriptional response in liver that partly differs from caloric restriction . FurtherWhile the currently available literature shows robust physiological improvements with SAAR in rodent models, the underlying mechanisms are only partly understood and are subject to ongoing research. Among the known nutrient-responsive signaling pathways, the evolutionary conserved integrated stress response (ISR) is a lesser-understood candidate in mediating leanness and/or longevity by SAAR. Therefore, the purpose of this review is to compile and delineate the current understanding of the involvement of the ISR in mediating pro-adaptive responses to SAAR in mammals. Throughout evolution, all living organisms have encountered periods of nutrient scarcity. In order to ensure survival during such periods, intricate and overlapping cellular processes have evolved to promote resilience and metabolic homeostasis. Many of these signaling networks are evolutionary well-conserved. Among these networks, the ISR is identified in all eukaryotic organisms as a means to allow for conservation of resources to adapt to environmental stress, ultimately improving survivability . A key feature of the classical or canonical ISR is the concept that a variety of cellular stresses are sensed by a family of protein kinases which together function as stress response regulators. These ISR regulators are: Protein Kinase R (PKR), which is stimulated by viral double stranded RNA; PKR-like endoplasmic reticulum kinase (PERK), which is activated by ER stress; heme regulated inhibitor (HRI), which modulates globin synthesis in response to heme deprivation; and general control nonderepressible 2 (GCN2), which senses amino acid insufficiency and cellular damage by UV light. Activation of these ISR regulators converge at the point of phosphorylation of the GTPase activating protein, eukaryotic initiation factor 2 (eIF2) at serine 51 of its α subunit. This covalent modification converts eIF2 into a competitive inhibitor of its guanine nucleotide exchange factor, eIF2B ,27. InhiAs one of the branches of the ISR, early detection of amino acid insufficiency by GCN2 functions to delay catastrophic depletion of the intracellular amino acid pool by reducing the bulk client load for protein synthesis . In brieAtf4 transcript contains two uORFs which together allows for precise control of its translation: a 5′-proximal uORF1 that facilitates downstream ribosome re-initiation and a slightly longer uORF2 that is overlapping and out-of-frame with the Atf4 coding sequence (CDS) [Atf4 transcript allows translation initiation at both uORFs, thus repressing synthesis of the CDS. In contrast, under conditions of cellular amino acid insufficiency there is reduced translation re-initiation efficiency and so scanning ribosomes bypass uORF2 in the Atf4 transcript and instead re-initiate translation at the CDS [While amino acid deprivation on the one hand triggers rapid reduction in global translation, it on the other hand leads to targeted increases in the translation of certain mRNAs which contain one or more upstream open reading frames (uORFs) in their 5′ leader or untranslated region (UTR) ,34,35. Wce (CDS) . Under w the CDS . Upon its translation, ATF4 translocates to the nucleus where it can form homodimers or heterodimers with other bZIP transcription factors and bind promoter sequence motifs referred to as CAAT enhancer binding proteinATF response elements (CAREs) or amino acid response elements (AAREs) . Such revia the ISR. As such, the interplay between SAAR and the ISR demands more attention and thus has been subject to several recent investigations, summarized below. In sum, the canonical ISR functions in the detection of amino acid insufficiency and contributes important information in decisions regarding cell fate. Adaptable stress such as that elicited by SAAR may be cytoprotective by promoting hormesis In several growth restricted rodent models with long life, reduced protein synthesis is a shared response ,43. Follin vivo, how this dietary change is detected is less clear. Interestingly, one study found that the global loss of Gcn2 did not prevent reductions in protein synthesis rates in both liver and skeletal muscle. Instead, Gcn2 deleted mice fed an SAA restricted diet retained more body fat as compared to intact mice. This finding differs from that reported in Gcn2 deleted mice fed a leucine-devoid diet which showed sustained liver protein synthesis at the expense of muscle mass and greater body weight loss as compared to intact mice [While SAAR is shown to reduce rates of protein synthesis in tissues act mice . These dvia the mechanistic target of rapamycin complex 1 pathway or in complimenting the action of other stress kinases such as AMP-activated protein kinase or the mitogen activated kinases MEK/ERK [Of interest, several reports collectively show that while GCN2 may be activated by SAAR, it is not the only sensor involved. ,46. Inst MEK/ERK . IrrespeAtf4 expression was elevated in mice after two days of feeding an SAA restricted diet even though levels of eIF2 phosphorylation and GDP/GTP exchange rates on eIF2 were unchanged relative to animals fed a control diet. These results indicate an uncoupling between eIF2 phosphorylation and ATF4 synthesis in liver [in vivo, such as altered protein stability. Additional research efforts are necessary to uncover the regulation of ATF4 by SAAR. A key component of the canonical ISR response to deprivation of most amino acids is increased ATF4 synthesis which activates the expression of cytoprotective genes ,48. Ratsin liver . Interesin vitro studies have revealed that the methionine-deprived transcriptional response is unique [As detailed above, the ATF4-mediated transcriptional response is tailored to the specific cellular stress. Indeed, individual amino acid stress responses are heterogeneous and s unique . Furthers unique . The timAmong the ATF4 gene target identified under conditions of protein dilution and amino acid deprivation is fibroblast growth factor 21 (FGF21), an endocrine member of the FGF superfamily ,54,55,56Fgf21 [Fgf21 promoter region [Gcn2. Interestingly, loss of Gcn2 did not completely abrogate the ATF4-mediated increase in FGF21, but rather caused a noticeable temporal delay. It was subsequently concluded that while GCN2/ATF4 signaling is imperative in the acute response to protein restriction, it is not essential; suggesting that upon loss of Gcn2, other regulators may facilitate ATF4-mediated induction of FGF21. Notably, this contrasts with drug-mediated amino acid starvation which requires GCN2 for hepatic expression of Fgf21 [Fgf21 expression during amino acid insufficiency. While various forms of amino acid insufficiency can induce the hepatic synthesis and secretion of FGF21 into the blood, the precise upstream mechanisms initially remained elusive. Involvement of the ISR in the physiological induction of FGF21 is supported by identification of up to three AAREs in the promoter region of Fgf21 ,59. Corrr region . Accordiof Fgf21 . In totaFgf21 prevents some of the metabolic improvements seen with SAAR [Fgf21 globally failed to increase energy expenditure as well as thermogenesis across adipose deposits. However, it was noted that mice lacking Fgf21 did maintain hepatic lipogenic signaling—similar to the response in genetically intact animals. Furthermore, mice lacking Fgf21 displayed normal hepatic ISR signaling, suggesting this signaling axis is dispensable.The first evidence connecting FGF21 to the physiological response to SAAR came from a study showing that global loss of ith SAAR . SpecifiGcn2 is dispensable in the SAAR-mediated induction of FGF21 and mice lacking Gcn2 show delayed increases in energy expenditure [Gcn2 could sustain hepatic phosphorylation of eIF2 to SAAR, presumably by another eIF2 kinase, PERK. Based on these observations, it has been proposed that reduced ER abundance of a cysteine metabolite, glutathione (GSH), during SAAR might contribute to the activation of PERK (further detailed below) [Gcn2 globally responded to acute SAAR by inducing hepatic Fgf21 transcript abundancy without increased phosphorylation of eIF2 nor reduced activity of eIF2B in liver [In agreement with previous reports, it was found that enditure . Of noted below) . In addiin liver . In summary, the currently available literature is supportive of FGF21 as a central mediator in the physiological response to SAAR. While the importance of FGF21 during SAAR is recognized, the upstream control remains elusive and appears complex in its nature, as pointed in a recent review . In reveAmong the more intriguing phenotypical changes observed in SAA restricted rodents is the increase in relative food intake. This SAAR-mediated increase in food consumption is reported on extensively in rodents ,16,63. NGcn2 in the anterior piriform cortex are unable to immediately sense and thus demonstrate delayed avoidance of the unbalanced diet [Gcn2 do reduce food intake when chronically consuming a leucine devoid diet, highlighting the early role of Gcn2 in instigating food aversion during exposure to amino acid devoid diets [In addition, FGF21 might, as discussed previously, play a role in altering the feeding behavior in response to SAAR . Recent ced diet . Indeed,id diets . Collectively, while there is ongoing investigation into the specific temporal dependency of GCN2, the overall importance of the ISR in changing feeding behavior in response to amino acid insufficiency remains an interesting area of research warranting additional exploration ,69.via the transsulfuration pathway. SAAR leads to observable changes in circulating levels of methionine metabolites. A series of publications shows that SAAR reduces levels of both total cysteine and cystathionine whereas total homocysteine levels increase [Cysteine is a dispensable amino acid under conditions of adequate methionine because it can be synthesized from the methionine metabolite cystathionine, generated increase ,8. BasedThe importance of both methionine and cysteine metabolites is further emphasized when returning to some of the original work which shows that SAAR-mediated lifespan extension coincides with a robust increase in circulating levels of the cysteine metabolite GSH . The majStudies note a negative correlation between advancing age and levels of circulating GSH . In agreGcn2 and corresponds with phosphorylation of PERK. These findings were corroborated in a study showing that SAAR altered GSH tissue distribution and induced activation of PERK and phosphorylation of eIF2 in the liver without inducing ER stress-related gene expression [In support of the notion that altered levels of GSH are part of the physiological response to SAAR, additional studies confirm that mice subjected to an SAA restricted diet display reduced levels of the antioxidant in liver ,44. Concpression . It is hypothesized that PERK activation by SAAR increases activation of the bZIP transcription factor nuclear factor erythroid 2 related factor 2 (NRF2), with the purpose of increasing GSH production during both oxidative and ER stress . Indeed,via NRF2 binding to antioxidant response elements (AREs) [Other studies postulate that the increased mitochondrial activity seen in various models of dietary restriction, including SAAR, is a function of increased ATF4 activity and might prompt increased antioxidant signaling s (AREs) ,75. Notas (AREs) . Furthers (AREs) . Atf4 to maintain homeostasis during GSH depletion, highlighting a role of the ISR in maintenance of adequate antioxidant defenses [Interestingly, earlier studies describe an increased cysteine requirement for mouse embryonic fibroblasts lacking defenses . This obdefenses . In addivia GCN2/ATF4-dependent signaling which in turn increases production of the proangiogenic gas, hydrogen sulfide (H2S) and promote skeletal muscle angiogenesis [2S production. Of interest, the production of H2S is mediated by the ATF4 target cystathionine-γ-lyase  and a recent report implicated the effects of H2S directly in the ISR. This study showed that the gas may cause a transient increase in eIF2 phosphorylation by inhibiting protein phosphatase 1cvia persulfidation [in vivo, they support the importance of the ISR in the response to SAAR and highlight the wide spectrum of physiological and metabolic effects induced by the restriction of methionine and cysteine.Reviewing the annotated effects of SAAR on oxidative signaling leads one to suppose that these effects may contribute to other alterations in physiology. For example, rodents fed an SAA-restricted diet demonstrate increases in vascular endothelial growth factor (VEGF) ogenesis . In thisfidation . While tThe ability of organisms to rapidly respond to changing environments, such as nutrient scarcity, is essential to survival and evolutionary fitness. However, recent decades have presented humans with a previously unencountered problem: chronic excess of dietary energy; leading to increased prevalence of metabolic ailments. Among the strategies employed to treat and prevent such metabolic ailments are various types of dietary restriction. The emergence of SAAR as a potential tool in improving metabolic health is intriguing, especially when considering the paradoxical nature of the response . Based on the numerous promising findings in animal models, there is emerging interest in translating SAAR to humans, however there is only a limited number of published reports on the topic ,83. NoneIn reviewing the current rodent SAAR literature, it is apparent that much still remains to be understood. Among the questions requiring immediate attention is the apparent sex dimorphic response to SAAR. Two recent studies showed that both restriction and short-term deprivation of methionine results in a noticeable sex dimorphic response, with males generally presenting a more robust response compared to females ,85. DrawDrosophila melanogaster have shown that while dietary restriction, such as SAAR, results in extension of lifespan, it concomitantly decreases fecundity [It will also be interesting to investigate the effects of the SAA restricted diet on reproduction. Indeed, studies in ecundity . Future ecundity ,89,90.Lastly, the involvement of the ISR in mediating lifespan extension in SAA restricted rodents needs additional investigation. With SAA restricted rodents living approximately 40% longer, there is an interest in understanding the underlying mechanisms and subsequently the translational aspects of this SAAR-mediated lifespan extension. To that end, it has recently been shown that progeroid mice fed an SAA-restricted diet display an extension of lifespan, indicative of possible therapeutic applications . Collect


Leishmania (Leishmania) infantum species [syn. L. (L.) infantum chagasi species in the Americas] which is transmitted by the bite of a female phlebotomine sand fly. This parasitosis is endemic and affect millions of dogs in Asia, the Americas and the Mediterranean basin. Domestic dogs are the main hosts and the main reservoir hosts for human zoonotic leishmaniosis. The outcome of infection is a consequence of intricate interactions between the protozoan and the immunological and genetic background of the host. Clinical manifestations can range from subclinical infection to very severe disease. Early detection of infected dogs, their close surveillance and treatment are essential to control the dissemination of the parasite among other dogs, being also a pivotal element for the control of human zoonotic leishmaniosis. Hence, the identification of biomarkers for the confirmation of Leishmania infection, disease and determination of an appropriate treatment would represent an important tool to assist clinicians in diagnosis, monitoring and in giving a realistic prognosis to subclinical infected and sick dogs. Here, we review the recent advances in the identification of Leishmania infantum biomarkers, focusing on those related to parasite exposure, susceptibility to infection and disease development. Markers related to the pathogenesis of the disease and to monitoring the evolution of leishmaniosis and treatment outcome are also summarized. Data emphasizes the complexity of parasite-host interactions and that a single biomarker cannot be used alone for CanL diagnosis or prognosis. Nevertheless, results are encouraging and future research to explore the potential clinical application of biomarkers is warranted.Canine leishmaniosis (CanL) is a vector-borne disease caused by the protozoan  Leishmania (Leishmania) infantum species  of canine neutrophils. The release of ROS from phagocytes present in inflammatory sites, leads to the consumption of antioxidant compounds  ratio have been associated with disease progression  has also been documented in diseased dogs Severe -1 cells producing interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and IL-2. Th1 is responsible for the activation of macrophages and concomitant intracellular killing of the parasites  by bone marrow monocytes was also significantly increased after treatment, probably reflecting a rise in the presentation of Leishmania antigens , together with decreased level of the cluster of differentiation (CD) CD21+ B cells and upregulation of MHCII+ molecules was observed  and CD8+ T cells and Foxp3+ regulatory T cells (Tregs) frequencies in mononuclear cells of cervical and mesenteric lymph nodes of naturally infected dogs have also been evaluated  and with the different sensitivity and specificity of methodologies to determine the presence of specific antibodies and/or of the parasite  from naturally and experimentally infected dogs was correlated with resistance to disease, asymptomatic status or mild disease  lymphocytes non-stimulated or stimulated with soluble + lymphocytes in resistance to CanL  donovani it was shown that cellular immunosuppression is mediated by failure of DC migration due to the decreased chemokine secretion by endothelium and to the reduced DCs CCR7 expression  infantum chagasi. The imunotherapy with F3 antigens prevented the migrating defect of DCs by restoring the expression of CCR7 receptors  infantum chagasi. Frequencies and expression of IL-10, IFN-γ, TGF-β, TNF-α, Foxp3+, CD4+, and CD8+ were higher in jejunum, while IL-4 expression was significantly higher in the colon. A positive correlation between CD4+ in the colon and between CD4+ Foxp3+ Tregs in jejunum and parasite load was found. Infected animals had reduced CD8+ expression in both intestinal compartments compared to controls.Figueiredo et al.  evaluateL. (L.) infantum chagasi infected dogs CD3+ T lymphocytes were found to be the major components of the inflammatory infiltrate at the choroid plexus and in the brain; according to the authors during the advanced stages of leishmaniosis leukocytes might participate in the pathogenesis of neurological disorders  and enzyme-linked immunosorbent assay (ELISA) or the qualitative rapid immunochromatographic tests (ICT) are available for CanL diagnosis  such as cytology, histology, immunohistochemistry, PCR or real-time PCR , which induces a delayed-type hypersensitivity response in dogs. Ex vivo tests to assess Leishmania-specific cell-mediated immunity include lymphocyte proliferation assay and assays measuring IFN-γ in circulating lymphocytes (such as IFN-γ cytophatic effect inhibition bioassay and IFN-γ release assay). Lymphocyte proliferation assay consists on the stimulation of PBMC with soluble Leishmania antigen (SLA) and a mitogen with non-stimulated cells representing the negative control. Cell proliferation is expressed as a stimulation index (SI), which is obtained by the ratios of stimulated cells to non-stimulated cells). SI ≥ 2 are considered positive  allows rapid screening of IFN-γ-secretion in whole blood challenged with SLA. It seems to be a useful tool to assess exposure to Leishmania amastigotes by apoptosis. Cytokines such as IFN-γ and TNF-α secreted by activated T cells have been found to induce nitric oxide synthase (iNOS) and NO production facilitating parasite control  infantum chagasi. NO enhanced the anti-leishmanial activity of macrophages alone or co-cultured with IFN-γ producing autologous lymphocytes  analyses. NO production by macrophages is the principal effector molecule mediating intracellular killing of o et al.  observedLeishmania infection in clinical settings should be of practical use to help monitoring CanL and treatment outcome (Maia and Campino, A practical and standardized assay to evaluate cellular immunity to L. infantum infection or disease, or a response to Leishmania treatment or vaccination would represent a major progress in the control of canine leishmaniosis. Data gathered from several studies have identified potential biomarkers but none of them provided a strong evidence of their practical applicability on diagnosis. In addition, no single marker seems sufficient to be a direct correlate of resistance or susceptibility to disease, as a complex network of regulatory and counter-regulatory interactions involving several cytokines, chemokines and cell populations are involved in mounting an effective immune response. Therefore, multiple laboratorial, immunological and parasite-specific biomarkers should be considered together to obtain a general view of L. infantum infection and disease outcome. The visceral tropism of the parasites makes sampling challenging, as well as owner compliance, especially when invasive procedures need to be repeated. Therefore, the identification of biomarkers obtained from non-invasive samples is warranted. Finally, the development of vaccines to prevent CanL represents an important step forward to control the disease but it has complicated its diagnosis, so, biomarkers able to discriminate naturally infected from vaccinated dogs are urgent.The identification of biological parameters that can be indicators of pathological processes related to CM writing and editing original draft. LC editing and review original draft.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.