This review illustrates how far we have come since the emergence of GE technologies and how they could be applied to obtain superior and sustainable crop production.The main challenges of today’s agriculture are maintaining and raising productivity, reducing its negative impact on the environment, and adapting to climate change. Efficient plant breeding can generate elite varieties that will rapidly replace obsolete ones and address ongoing challenges in an efficient and sustainable manner. Site-specific genome editing in plants is a rapidly evolving field with tangible results. The technology is equipped with a powerful toolbox of molecular scissors to cut DNA at a pre-determined site with different efficiencies for designing an approach that best suits the objectives of each plant breeding strategy. Genome editing (GE) not only revolutionizes plant biology, but provides the means to solve challenges related to plant architecture, food security, nutrient content, adaptation to the environment, resistance to diseases and production of plant-based materials. This review illustrates how far we have come since the emergence of these technologies and how these technologies could be applied to obtain superior, safe and sustainable crop production. Synergies of genome editing with other technological platforms that are gaining significance in plants lead to an exciting new, post-genomic era for plant research and production. In previous months, we have seen what global changes might arise from one new virus, reminding us of what drastic effects such events could have on food production. This demonstrates how important science, technology, and tools are to meet the current time and the future. Plant GE can make a real difference to future sustainable food production to the benefit of both mankind and our environment. Conventional breeding has enabled breeders to produce improved varieties of many crops and has led to increased food security and crops with higher yield and tolerance to biotic and abiotic stress, as well as increased nutrient content. However, in the era of the changing climate and greater consumer demands, breeders are still facing increasing challenges expected to be overcome. Projected changes in climate are expected to have far-reaching impacts on agricultural production, affecting future food production. The rising temperatures, droughts or floods in a certain geographical area, as well as new pests and diseases, will stress plants and demand new varieties and changed production systems differently in different geographic regions. Current global food systems are based on a few cereal crops and elite varieties feeding both animals and humans. Although seemingly efficient, it is not resilient to sudden changes in yield shocks posed by environmental changes or changed trading due to changed demands or changed financial market balance. By 2050, the FAO estimates that the food demand will have increased over 60% and it will have needed 50% more energy and 40% more water to feed the 10 billion people on the planet Earth. Furthermore, climate changes could result in a global temperature rise with detrimental effects which might be associated with disease outbreaks threatening the crop production and compromising the quality of the harvested products  and the studies on the mechanisms of DNA double-strand breaks (DSBs) repair have allowed plant scientists to develop tools for targeted mutagenesis in plants. One of the first tools in the GE toolbox was the zinc finger nucleases (ZFNs). ZFNs are chimeric proteins with a DNA cleavage domain of FokI restriction endonuclease  and an array of three or four zinc fingers that were originally identified in sequence-specific eukaryotic transcription factors. The first eukaryotic sequence-specific transcription factor to be characterized was found to have zinc-binding repeats in its DNA-binding domain , is composed of Transcription Activator-Like Effectors (TALEs) and the FokI endonuclease. TALE proteins are expressed by plant bacterial pathogens to manipulate host gene transcription and promote successful infection. Like ZFNs, TALENs function as pairs in a similar way to create a break at a specific DNA sequence recognized by the TALE domain. TALENs for DNA recognition use a tandem array of 16 (or more) nearly identical protein modules, each of which targets one nucleotide at the DNA target site, making thus TALENs highly specific  that occur only once in any given genome and for this reason are rare-cutting enzymes. They generate DSBs, and the site-specific I-SceI is the prototypical meganuclease that has been used as a tool for genome engineering. For genome editing purposes, thousands of meganucleases have been redesigned and mutants created with new specificities , crRNA, and tracrRNA. The combination of the crRNA and tracrRNA into a single synthetic single guide RNA (sgRNA) has resulted in a simplified two-component reagent that is now widely used to introduce targeted double-stranded breaks in genomic DNA. The sgRNA contains approximately 20-base-long sequence complementary to the target DNA, which guides Cas9 to the right genomic location.CRISPR/Cas  is an easier and more efficient genome editing tool than the engineered ZFNs and TALENs. It is based on the adaptive immune system in bacteria and archaea, enabling organisms to respond and eliminate invading viruses and plasmids , using RNA-guided Cas9 endonuclease  OsSWEET family of putative sugar transporter genes, OsSWEET14 and OsSWEET11, conferring tolerance against bacterial blight  gene. In grape, a Botrytis cinerea-responsive WRKY52 transcription factor has been targeted by the CRISPR/Cas9 system and the transgenic plants showed biallelic mutations less sensitive than the monoallelic mutants  gene, which is a suppressor of the immune system in Theobroma cacao, has led to improved resistance to Phytophthora tropicalis  transcription factor generated through a transient expression of ZFNs in seeds revealed increased soluble solids content, which is of prime importance in tomato fruit and a breeding target affecting flavour and nutritional value. Mutant lines enriched in β-carotene and antioxidants, ascorbic acid or succinic acid, were produced. Notably, the reduced content of the anti-nutrient oxalic acid in several mutant fruits suggests that L1L4 gene regulates the accumulation of this compound in tomato during fruit development  levels have been produced by targeting two glutamate decarboxylase (GAD) genes, GAD2 and GAD3, encoding a key enzyme in GABA biosynthesis. In this case, the CRISPR/Cas9 system created plants that produced tomatoes with 1.5- to tenfold higher GABA content, an amino acid that enhances the blood pressure-lowering function of tomato fruit  varieties with undetectable levels of reducing sugars and reduced levels of acrylamide (a carcinogen) in processed chips was achieved though transient expression of TALENs designed to target the vacuolar acid invertase gene (Vlnv)  grains, storage proteins called kafirins form protein bodies with poor digestibility. Kafirins are mostly composed of α-kafirins and encoded by the k1C family of highly similar genes. Li et al.  is the staple food for over half of the world's population and nutritional improvements of this species may have great impact on the human population. Utilization of ZFNs in this crop induced mutations in starch synthase IVa gene (SSIVa) that encodes a soluble starch synthase involved in starch biosynthesis pathway. Generation of transgenic plants revealed low starch contents and dwarf phenotypes  that activates zein gene promoters  oil is used in applications ranging from cooking and frying to industrial products. Change of individual fatty acids content in soybean oil could contribute to its increased shelf-life and frying stability, as well as improvement of its nutritional value. Soybean varieties with high oleic acid were created using TALEN technology to target the genes coding fatty acid desaturase 2 enzyme, FAD2-1A and FAD2-1B. The oil profile of the mutant seeds meets the soybean industry’s demand as the oleic acid increased fourfold (from 20 to 80%), while linoleic acid decreased from 50 to less than 4% , mutant lines with a 0.5–twofold increase in the oleic acid content were produced by targeted mutagenesis in the conserved coding sequence of FAD2 gene by TALENs indicating that TALEN-mediated targeted mutagenesis can be used to increase the oleic acid content in edible peanut oil  gene, which controls parthenocarpy, the production of seedless fruit without prior fertilization  RIPENING-INHIBITOR (RIN) gene, which encodes a MADS-box transcription factor regulating fruit ripening led to RIN-protein defective mutants with incomplete-ripening of fruits and reduced red colour pigmentation compared to wild-type fruit  gene mutagenesis in the presence of the homologous repair template. The resulting mutants produced tomatoes with prolonged shelf life  genes encode transcription factors involved in fruit ripening, and affect ripening in many fruit species, either climacteric or non-climacteric  gene, which is involved in both chlorogenic acid and lignin biosynthesis. In contrast to the RNAi-derived C3′H-knockdown mutants, the CRISPR-derived knockouts were severely dwarfed and sterile. The results of the study clearly indicated the impacts of C3′H suppression on lignin composition and on the assembly of other cell wall components in rice. Such structural alterations in rice were reported to be highly useful for enhancements in biomass digestibility and saccharification. The same research group investigated the suppression effect of coniferaldehyde 5‐hydroxylase (OsCAld5H1) gene, which modulates syringyl (S)/guaiacyl (G) lignin composition ratio, on lignin structure of rice. Loss-of-function mutants clearly demonstrated alteration of S/G subunit in lignin composition of rice cell wall , which encodes a key enzyme in lignin biosynthesis, were successfully created in rice with the same GE technique. Cell wall analysis of the CAD2 mutants demonstrated altered lignification in rice and synergistic increase in saccharification efficiency via loss of function mutation (Matsumoto OsAT3) and acyltransferase 4 (OsAT4). The result of both mutants indicated the effect of gene suppressions on pectin methyltransferase activity by a significant reduction in conversion of monolignols into corresponding lignin conjugates. CRISPR efficiency was also tested and successfully optimized for other forage crops such as maize  to reduce lignin with a concurrent increase in cellulose content    and BRANCHED 2 (BRC1-2) transcription factors, which are important centres of signals controlling the ability of a bud to grow out, revealed altered shoot architecture and increased bud outgrowth  and to create multi-allelic mutagenesis in lignin biosynthesis and generate lines exhibiting a 29–32% reduction in lignin content compared to controls with significantly reduced S subunit content and elevated hemicellulose content. In another study, these TALEN-mediated COMT mutants were grown on field to test their survival and biomass production performance. The results of the study revealed 20% reduction in lignin content and S/G ratio, which resulted in 44% improved saccharification efficiency. Biomass production performance of COMT mutants did not differ significantly from the original cultivar under field conditions  in lignin biosynthesis with CRSPR/Cas system in switchgrass, a model biomass species characterized by high ploidy level. The results of the study revealed less lignin content and significantly increased glucose and xylose release in knockout plants compared to control. Furthermore, another CRISPR/Cas9 system introduced simultaneous mutations at three gene loci, teosinte branched 1(tb1)a, b and phosphoglycerate mutase (PGM), when stably transformed mesophyll protoplasts of switchgrass showing that the CRISPR/Cas9 can be used for multiplex GE and produce homozygous mutant plants in T0 generation. Interestingly, plants with tb1 gene mutations had increased tiller numbers, which is a useful genetic material for breeding switchgrass cultivars with high biomass yield  is a non-food, second-generation bioenergy crop and a perennial C4 grass that grows well even in marginal lands. The biomass of Miscanthus has a carbohydrate content of more than 60% (w/w) regardless of the variety, in which approximately 40% is cellulose based on dry weight  represent a promising source of renewable biofuel. For most of these feedstocks, agronomic and crop production improvements are just beginning to be applied through GE approaches. Oilseed rape (Brassica napus) is an annual crop native to the Mediterranean region and Asia that produces seeds with an oil content of about 40–45%. The CRISPR/Cas9 system was used to target FAD2 gene, which encodes an enzyme that catalyses the desaturation of oleic acid, creating mutants with statistically significant increase in the oleic acid content compared to that present in wild-type seeds  is another promising plant for biodiesel production due to its high oil content in seeds. Several studies suggested that exogenous cytokinin treatment can significantly increase the total number of flowers per inflorescence, the female-to-male flower ratio, and the seed yield , an allohexaploid species of the Brassicaceae family and an oilseed crop for biofuel production, Cas9 and a sgRNA targeted all three diacylglycerol O-acyltransferase 1 (DGAT1) or phospholipid:diacylglycerol acyltransferase 1 (PDAT1) homeologs simultaneously, which are important genes for triacylglycerol biosynthesis. The resulting mutant lines reduced seed oil and altered fatty acid composition. This application demonstrated the ability of the technology to target all three homeologs simultaneously in this species  of triacylglycerols (TAGs) and short-chain alcohols. Another source of biodiesel that reduces its cost is waste vegetable oils and non-edible crude vegetable oils. Currently, non-edible oil yielding plants for the second-generation biodiesel production include GhMYB25 genes functional in lignocellulosic fibre formation and elongation. A similar system was also applied in cotton to mutate alanine-rich protein (ALARP) gene, which encodes an alanine-rich protein that is preferentially expressed in cotton fibres  without foreign DNA may help increase public acceptance of agricultural products and free them from regulatory monitoring in order to advance their use in plant breeding programs and commercial-scale production. Plant systems are relatively economical to maintain with short generation cycle and ease to be handled compared to animals, and hence, they can be particularly attractive for testing new concepts and develop new approaches in GE-related research that aims to improve accuracy, versatility, and efficiency of these molecular tools and apply them with ease to other systems. However, the lack of regulations and uncertainty about possible applications could affect implementation of GE for plant improvement. The researchers have knowledge and resources to apply these new tools for introduction of new traits into plants, but for the time being this research is generally put on hold, waiting for new regulations and proper assessments of GE methods used for plant improvement. Gene targeting in plants plays an important role in providing new information on genetic and genomic analyses, gene networks and single gene variant function, which in case of well-conserved genes across living species may advance our understanding on the cellular and molecular workings of advanced eukaryotic species and at the same time pave the way for development and implementation of new, more efficient breeding tools, and shorter time needed to transfer the knowledge from laboratory to field.


Genomic studies of tumor specimens are becoming standard of care in patients with gliomas to characterize druggable molecular features. Unfortunately, with the exception of IDH1-R132 mutation and MGMT promoter methylation, molecular markers have failed to predict clinical responses to drugs, and the impact of targeted therapies remains minimal. There is a need for a high-throughput, patient-specific, and significantly predictive method to inform a most effective personalized therapy. This pilot trial tests the safety and feasibility of drug-releasing microdevices which are temporarily implanted into the tumor during a standard craniotomy. They release microdoses of up to 20 drugs or drug combinations into surrounding tissue in a controlled spatial distribution. The devices, together with a cuff of surrounding tumor tissue, are removed at the end of surgery, and the tissue is analyzed for biological and molecular response markers allowing for in situ characterization of the drug efficacy. Four patients have been enrolled to date, out of a total planned of six. Two microdevices were implanted into each tumor . Average indwelling time in tumor tissue was 139 minutes. Eight devices (100%) were successfully retrieved, and all surgeries were completed without immediate (<24 hours) or delayed (<30 days) complications. Seven (87%) specimens were of adequate quality, allowing for planned histological and molecular studies. For all analyzed specimens, the intraoperative incubation time was sufficient to observe: 1) Drug concentration gradients; 2) Differential molecular signs of cell toxicity (DNA damage and Caspase 3 activation); 3) Whole genome transcriptional changes; 4) Tumor microenvironment composition; and 5) Preliminary evidence of concordance between the biological readout obtained from microdevice analysis and clinical response. Drug-releasing microdevices were well tolerated, seamlessly integrated in standard craniotomy workflow, and allowed for collection of a significant amount of data related to the differential efficacy of multiple drugs in a personalized manner.


Recent scientific reports have shown that these channels contribute to the increase in glioblastoma aggressiveness, with glioblastoma representing the ultimate level of glioma malignancy. The current review focuses on each type of TRP ion channel potentially involved in malignant glioma cell death, with the ultimate goal of identifying new therapeutic targets to clinically combat malignant gliomas. It thus appears that cannabidiol targeting the TRPV2 type could be such a potential target.Among the most biologically, thus clinically, aggressive primary brain tumors are found malignant gliomas. Despite recent advances in adjuvant therapies, which include targeted and immunotherapies, after surgery and radio/chemotherapy, the tumor is recurrent and always lethal. Malignant gliomas also contain a pool of initiating stem cells that are highly invasive and resistant to conventional treatment. Ion channels and transporters are markedly involved in cancer cell biology, including glioma cell biology. Transient receptor potential (TRP) ion channels are calcium-permeable channels implicated in Ca Among the most common malignant primary tumors are encountered malignant gliomas, which are associated with dismal prognosis. Precise statistics from the United States report for example 17,000 new diagnoses in 2017 . These tHeterogeneous populations of tumor-differentiated cells coexisting with subpopulations displaying stem cell properties are present in glioblastomas. The marked biological, thus clinical, aggressiveness of glioblastoma stem cells (GSCs) relates among others to their dramatic invasive nature into the brain parenchyma, high level of mobility into the brain parenchyma, and high resistance to both radio- and chemotherapy. GSCs have also the capacity to self-renew and are now known to be directly responsible for the recurrence and clinical relapse of glioblastomas .All grade II gliomas have the tendency to transform into more aggressive grade III (anaplastic) or even grade IV gliomas (secondary glioblastoma); likewise, grade III gliomas can similarly transform into grade IV (secondary glioblastoma).A glioblastoma is a mosaic of various cell populations associated with distinct dynamic cell states as recently revealed by genome-wide sequencing, and this dramatic cell heterogeneity within a given glioblastoma renders any type of treatment very difficult .Ion channels are classified by ion selectivity , gating mechanism , or localization (plasma membrane or intracellular) . These cSeveral ion channels are implicated in malignant glioma proliferation, migration, invasion, and cell death. For example, genome-wide analyses of glioblastoma revealed that, of 555 genes involved in potassium, sodium, chloride, calcium channels, and other ion transport, 55 mutations were detected, affecting 90% of the glioblastoma samples studied .It has been experimentally demonstrated already two decades ago that glioma cells invading the brain parenchyma must modify their shape and/or volume to perform their invasive journey . Shape-vIn gliomas, cell condensation is a hallmark of intrinsic and extrinsic apoptosis and requires the concerted activation of chloride- and calcium-activated potassium channels, leading to the loss of water . We prev2+ entry from extra- into intracellular compartments  calcium channels, which modulate the driving force for Caartments . For eacartments .Glioma, melanoma, non-small cell lung cancer, and esophageal cancer, among others, are resistant to proapoptotic stimuli and are typically associated with dismal prognoses  and dispIn the current review, the subchapter entitled therapeutic perspective aims to analyze the types of TRP-targeting drugs that could be of help in overcoming the resistance of glioma cancer cells and glioma stem cells to conventional therapies .2+-permeable channels, with diverse physiological functions and cellular distributions  appears to be important for cytokinesis in cell proliferation and migration  and angi2+-permeable TRPC1 channels as evidenced biophysically by in vivo evidence that loss of TRPC1 function impairs tumor growth in immunocompromised mice, suggesting that pharmacological inhibition of these channels may slow tumor growth  for survival with short OS glioblastoma patients, showing a lower TRPV1 variant 3 mRNA expression compared with long OS patients  of the glioblastomas analyzed by patients . TRPV1 iin vitro . TRPV1 s2+ responses. Endovanilloids directly stimulate the vanilloid receptor TRPV1  are thought to be the source of high-grade astrocytomas which are much more frequent in adults than in children. or TRPV1 . Howeveror TRPV1 , suggest2+ influx, increases chemotherapeutic drug uptake and synergizes with cytotoxic agents  to induce in vitro apoptosis in glioma cells but not in normal human astrocytes . A link between TRPML channel physiology and tumor biology has been suggested, and we focus here on glioma biology.Endosome/lysosome Ca2+ and Fe2+. Chaperone-mediated autophagy-related proteins [for example the heat shock cognate protein Hsc70 and the 40-kDa heat shock protein (Hsp40)] interact with the large TRPML1 intraluminal loop  TRPM8 signaling is involved in cell cycle regulation and represses apoptotic cell death; (ii) clinically compatible ionizing radiation doses for treating glioblastoma patients induce upregulation of TRPM8 function; and (iii) elevated TRPM8 function, in turn, confers radioresistance -tetrahydrocannabinol (Δ9-THC) on human glioblastoma cell survival and proliferation  and found an enhanced antineoplastic effect  . This stol group . In anotol group . The autol group .2+, and it makes sense to target a common Ca2+ source, such as specific TRP channels, which are heavily involved in glioma cell biology.Multiple ion channels rely on intracellular CaWe are only at the beginning of our understanding of the precise roles of various TRP channels in glioma cell biology, and further studies are required to truly understand the physiopathological roles of TRP channels in glioma progression. However, some promising data from the literature, even if still scarce, already point to the very high promise of targeting TRPV2 by means of cannabidiol, a cannabinoid that lacks unwanted psychotropic liability.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We hypothesized that NRF2 is a key regulator after burn and inhalation injury, and activation of NRF2 can limit the severity of burn and inhalation injury.Severe burn injury leads to many systemic stresses that can seriously impact multiple systems throughout the body. These stresses are further exacerbated if there is a combined burn and inhalation injury, which leads to increased morbidity and mortality for many patients. Combined burn and inhalation injury causes an intense systemic inflammatory response and activation of the innate immune system which can lead to inflammatory complications, such as systemic inflammatory response syndrome and multiple organ failure. Nuclear Factor-Erythroid-2-Related Factor (NRF2) is a transcription factor that acts to downregulate overt damaging pro-inflammatory and oxidative responses and maintain immune homeostasis. This transcription factor remains bound to Kelch-like ECH-associated protein 1 (KEAP1) in the cytoplasm. Under oxidative stress, NRF2 dissociates from KEAP1 and translocates to the nucleus where it facilitates the transcription of anti-inflammatory and antioxidant genes. -/- knockout mice have higher mortality compared to wild-type (WT) mice and suffer from increased vascular permeability and lung edema, suggesting that this transcription factor is important for controlling morbidity and mortality. In WT mice, NRF2 is activated following burn and inhalation injury, however, based on immunohistochemical staining, it is not sufficiently induced following this insult since it remains in the cytoplasm and is unable to transcribe anti-inflammatory genes. Therefore, we treated WT mice with an intraperitoneal injection of bardoxolone-methyl microparticles, a NRF2 activator that separates KEAP1 from NRF2 in the cytoplasm, immediately after burn and inhalation injury in WT miceTo test this, we have developed a mouse model of combined cutaneous burn and woodsmoke inhalation injury. After burn and inhalation injury, we found that NRF2We observed significant decreases in mortality as well as reduced concentrations of certain pro-inflammatory cytokines in the blood and bronchoalveolar-lavage fluid of these mice. In the lungs of these mice, there was an upregulation in numerous pathways involved in the management of inflammation and immune response compared to mice that did not receive bardoxolone-methyl microparticles.In conclusion, treatment with bardoxolone-methyl microparticles immediately after burn and inhalation injury might be effective for reducing the severity of the inflammatory response and limiting inflammatory complications.


CBS inhibition results in accumulation of the cardiovascular risk factor homocysteine (Hcy) and evidence is emerging for plasma Hcy elevation in patients with acute hepatic porphyrias. Decreased CBS activity may also induce a proinflammatory state, inhibit expression of haem oxygenase and activate the extrahepatic kynurenine pathway (KP) thereby further contributing to the Hcy elevation. The hypothesis predicts likely changes in CBS activity and plasma Hcy levels in untreated hepatic porphyria patients and in those receiving hemin or certain gene-based therapies. In the present review, these aspects are discussed, means of testing the hypothesis in preclinical experimental settings and porphyric patients are suggested and potential nutritional and other therapies are proposed.The role of haem in the activity of cystathionine β-synthase (CBS) is reviewed and a hypothesis postulating multiple effects of haem on enzyme activity under conditions of haem excess or deficiency is proposed, with implications for some therapies of acute hepatic porphyrias. CBS utilises both haem and pyridoxal 5′-phosphate (PLP) as cofactors. Although haem does not participate directly in the catalytic process, it is vital for PLP binding to the enzyme and potentially also for CBS stability. Haem deficiency can therefore undermine CBS activity by impairing PLP binding and facilitating CBS degradation. Excess haem can also impair CBS activity by inhibiting it via CO resulting from haem induction of haem oxygenase 1 (HO 1), and by induction of a functional vitamin B Competition for PLP by the relevant enzymes of haem, Hcy and Trp metabolism will be discussed. Elevation of plasma Hcy levels by CBS inhibition in AIP will then be considered in relation to the potential harmful effects of this amino acid and the impact of certain AIP therapies. Next, the effects of haem will be summarised in a hypothesis predicting likely changes in CBS activity and plasma [Hcy] under basal conditions and following some AIP therapies. Finally, means of testing the hypothesis, and nutrition-based and other therapies countering the Hcy elevation and/or effects will be proposed.The present review discusses the role of haem in control of activity of cystathionine β-synthase  and advances a hypothesis postulating that this control is exerted by the haem cofactor at multiple levels. This hypothesis was prompted by the reported  homocysthydroxymethylbilane gene  deaminase (PBGD), the ane gene  7] and  and hydrne  gene, as in homocystinuria  can be increased by a nutritional or a functional B6 deficiency. The latter can be induced by drugs that bind and thus inactivate PLP, such as hydrazine compounds  was higher in symptomatic and biochemically characterised (by raised urinary 5-ALA and PBG levels), compared with asymptomatic, patients and was more pronounced in patients receiving haem therapy  is the most commonly used marker of the vitamin B6 status, it can be influenced by factors such as carbohydrate intake (causing a decrease), fasting (an increase), low albumin levels in critical patients and late pregnancy (a decrease), changes in non-specific alkaline phosphatase, as in hypophosphatasia (an increase) or rickets (a decrease), inflammation and renal dysfunction (a decrease)  [6 deficiency and/or its extent in liver may not be detectable simply by measuring plasma [PLP]. From the above account, it may be concluded that assessment of the B6 status should not rely solely on determination of plasma or erythrocyte [PLP], but should include additional measures. The liver is a major source of PLP formation and for the human liver PLP status that is most relevant to the present discussion, and in the absence of a justifiable liver biopsy, indirect methods should be used. The hepatic functional B6 status can be assessed indirectly by two tests based on the amino acids Met and Trp. The Met loading test (leading to elevated levels of urinary cystathionine in B6 deficiency) is, however, difficult to interpret under conditions of PLP deficiency, as the test does not currently have the means of distinguishing between impaired activity of CBS and also that of the cystathionase activity of CγL, both of which utilise PLP as cofactor (see below). A more clear-cut measure is that of assessment of PLP-dependent Trp metabolites of the KP   , thus fuf the KP .The KP is the major tryptophan-degradative pathway, accounting for ∼95% of dietary Trp metabolism, with the hepatic pathway handling ∼90% and other tissues including immune cells handling the remaining 5% . The patIn liver of humans, rats, mice and certain other species, TDO exists in both the haem-free inactive apoenzyme and haem-containing active holoenzyme in roughly equal proportions, such that saturation of the enzyme with haem is ∼50%, defined as the percentage haem saturation  or ∼1, defined as the haem-saturation ratio (holoenzyme activity/apoenzyme activity). Certain other species  do not possess the free apoenzyme and their TDO therefore exists only as the active holoenzyme that cannot be activated by the addition of haem. Unlike the TDO of rats, mice and humans, the TDO of these other species does not respond to glucocorticoid induction, but can process Trp relatively rapidly, as they are sensitive to Trp toxicity .This was developed largely by the group of the late R.R. Brown at Wisconsin, U.S.A., and involved measuring the urinary excretion of the various pathway metabolites before and after an oral Trp load, usually of 2 g . Increasing dose . A repreing dose . In gene6 status, with values <65 µmol/day being considered indicative of adequate pyridoxine intake  is the major index of the Be intake . The realevation , with silevation  and refeol/24 h) . Assumin6-deficient rats, Kyn monooxygenase activity is enhanced, but KAT B inhibition can be ruled out in view of the great elevation of [XA]  can be increased by enhanced formation from Kyn by Kyn monooxygenase, decreased hydrolysis by kynase or decreased transamination by KAT. In B of [XA] . Kynase  of rats  and pigs of rats . Kynase  of rats ,54, thus of rats .6 deficiency, despite the increase in [Kyn] may be explained by a KAT inhibition. KAT can be inhibited not only by a PLP deficiency, but by other means. As stated above, KAT exists in four isoforms. KAT I and KAT II (acting on Kyn) are highly expressed in liver and deletion of the KAT II gene results in dramatic decreases (>90%) in hepatic KAT activity and [KA] in mice across ages  in Boss ages . Lesser oss ages . In rat oss ages . In miceoss ages . Other ioss ages . Additiooss ages . While i6 status. The advantages of plasma over urine is that metabolite levels in the latter reflect total body metabolism. In particular, the kidney plays an important role in Kyn handling and disposition [6 status and reported strong negative correlations between the [3-HK]/[XA], [3-HK]/[3-HAA] and [3-HK]/[KA] ratios and decreasing B6 levels and that the first of these ratios shows the strongest correlation with PLP levels. A mathematical model based on literature data /[Trp] ratio having been used for many years as an indirect measure of IDO activity, though also of TDO activity and other determinants of Kyn metabolism . In a stture, we  observedture, we .6 deficiency by a number of mechanisms: (1) producing higher and more consistent levels of urinary Kyn metabolites than is the case under basal conditions, which show daily and individual variations due to nutritional and physiological confounders  is well below the Km of the enzyme (3.8–5.7 mM)  in patients and/or experimental animals over a 24-h period after a single haem infusion or for a longer duration with repeated dosing. The TDO status may also be subject to a dual effect of haem: initial activation by an increase, followed by subsequent inhibition by a decrease, in haem availability. The plasma [Kyn]/[Trp] ratio, a measure of TDO or IDO activity, was reported [Whereas a decrease in plasma [Hcy] by haem therapy is transient, it cannot be reconciled with the ability of haem to cause a CO-induced CBS inhibition. Perhaps, the normalisation of plasma [Hcy] by haem therapy reflects opposite actions by haem on CBS activity: an increase due to increased haem availability for PLP cofactor activity and a decrease due to TDO activation inducing a functional Breported  for ninereported ), 5/9 AI6 deficiency or PLP-responsive CBS variants can be reversed by pyridoxine supplementation, the same may not be true for the functional B6 deficiency in AIP, as suggested by the failure of pyridoxine to reverse the increases in urinary Kyn metabolites  in conditions associated with Hcy elevation is determined by the underlying mechanism(s). For example, in renal failure, folate is effective, whereas B6 is not [6 irrespective of the defective remethylation may indicate that other factors are involved. In experimental renal failure in rats, activities of the following KP enzymes are greatly enhanced: liver TDO, liver KAT, kidney Kynase and liver and kidney KMO, leading to several-fold increases in tissue concentrations of Kyn, KA, AA, 3-HK, XA and QA [6 supplementation may further enhance KAT and Kynase activities as long as their substrate availability is maintained through TDO activation or other mechanisms. This further suggests that TDO inhibition may be an effective therapeutic strategy to block PLP utilisation by KP enzymes. Similarly, acute pancreatitis in humans  is alsosuffered , decreassuffered .6 deficiency. Non-steroidal antiinflammatory drugs may be more suitable, with TDO inhibition having already been reported with salicylate, the active form of aspirin [Current therapies that act by inhibiting haem synthesis at the 5-ALAS 1 step, such as glucose, hemin or 5-ALAS 1 gene silencing with givosiran will most likely contribute to the Hcy elevation. As a proinflammatory environment is present in AIP and can be induced by Hcy, the potential usefulness of anti-inflammatory therapy should be explored. That the use of steroidal and non-steroidal antiinflammatory drugs is safe in the acute porphyrias is indicated in many published lists, e.g. that in . Example aspirin  and dicl aspirin . TDO2 ge aspirin  and a si aspirin  been pro6 deficiency underpinned by multiple effects of haem. It is hoped that this account has provided a platform for further investigation of the Hcy status and its actions in AIP and will stimulate researchers to explore the present hypothesis at the basic mechanistic and clinical levels. The present account has also highlighted important issues that could be profitably addressed in future AIP-related studies. These include whether decreased CBS activity contributes to the proinflammatory environment of AIP, do changes in KA and QA and their ratio play a role in the neurological features of acute attacks?, does an increase in KA promote PARP 1 expression by AhR activation to induce NAD+ depletion?, is there a potential occurrence of CBS and/or CγL variants in AIP?, does the functional B6 deficiency in AIP, particularly after givosiran or hemin therapy, modulate the Trp-metabolic activity in the GIT or resident microbiota to undermine their protective effects?Whereas mild elevation of plasma Hcy in AIP may not be associated with significant health consequences, stronger elevations are generally undesirable and justify intervention. The plasma Hcy elevation and its consequences are almost certainly caused by decreased CBS and CγL activities resulting from a functional vitamin B


GLA gene, emphasizing a need for an individualized approach to patient care. The experts reached consensus on the critical role of a high index of suspicion in symptomatic patients and screening of certain at-risk groups to reveal timely and accurate diagnosis of FD along with an increased awareness of the treating physician about the different kinds of pathogenic variants and their clinical implications. The experts emphasized the crucial role of timely recognition of FD with minimal delay from symptom onset to definite diagnosis in better management of FD patients, given the likelihood of changing the disease’s natural history, improving the patients’ quality of life and the prognosis after enzyme replacement therapy (ERT) administered through a coordinated, multidisciplinary care approach. In this regard, this consensus document is expected to increase awareness among physicians about unique characteristics of FD to assist clinicians in recognizing FD with a well-established clinical suspicion consistent with pathogenic variants and gender-based heterogeneous clinical manifestations of FD and in translating this information into their clinical practice for best practice in the management of patients with FD.This consensus statement by a panel of Fabry experts aimed to identify areas of consensus on conceptual, clinical and therapeutic aspects of Fabry disease (FD) and to provide guidance to healthcare providers on best practice in the management of pediatric and adult patients with FD. This consensus statement indicated the clinical heterogeneity of FD as well as a large number of pathogenic variants in the  Fabry disease  is an X-linked lysosomal storage disorder caused by deficiency in the lysosomal enzyme α-galactosidase A , and consequent accumulation of glycosphingolipids, such as globotriaosylceramide (Gb3) and its deacylated derivative globotriaosylsphingosine (lyso-Gb3) within tissues that progressively affect multiple organ systems  #301500), is a progressive X-linked lysosomal storage disorder, caused by deficiency of lysosomal enzyme α-Gal A (Enzyme Commission [EC] number 3.2.1.22) due to pathogenic variants in the encoding NC 4296) . This reNC 4296) .The primary disease process, through this continuous deposition, starts as early as during the fetal stage of development, whereas unlike to many other lysosomal storage diseases, most patients remain clinically asymptomatic during the very first years of life until occurrence of first symptoms at ages of 3 to 10 years, and generally a few years later in girls than in boys –22.With advancing age, significant lysosomal and cellular dysfunction probably trigger a cascade of events  and results in development of a multisystem disorder via progressive damage to the vascular endothelia, particularly of small vessels, affecting multiple vital organ systems including the heart, nervous system, and kidneys , 4, 23. GLA gene have been excluded  and cerebrovascular complications  that can cause premature death , 39, 42.Fabry Disease can have overlapping signs and symptoms with other disorders such as Familial Mediterranean Fever (FMF), juvenile systemic lupus erythematosus and celiac disease which are frequently seen in Turkey. Fabry Disease should also be considered for patients with a preliminary/confirmed diagnosis of such disorders as the real etiologic/co-existent disease –49.Fabry nephropathy is related to progressive decrease in renal function with onset of deposition of Gb3 in almost all renal cell types  as early as during fetal development , 50. IncWith progressive chronic kidney disease (CKD) including albuminuria and overt proteinuria in the second decade of life –54, renaThe dialysis is initiated due to ESRD among male patients with FD in the 4–5th decades of life  and kidnLaboratory investigations of kidney function that should be carried out in every patient include serum creatinine, cystatin C, estimated GFR (e-GFR), urinary protein, albumin and/or microalbumin excretion and urinary sodium excretion . AssessmLight microscopy in biopsy specimens from kidney shows the accumulation of sphingolipids as PAS-positive Sudan-positive intra-lysosomal inclusions viewed under polarized light . However-based ultrastructural studies of kidney biopsies can reveal lysosomal storage in a variety of kidney cellular types, based on identification of the inclusions as whorled layers of alternating dense and pale material (‘zebra bodies’ or myelin figures) ., 8.4, 8]However, asymmetric septal or apical hypertrophy has also been described, along with sub-aortic obstruction, which may mimic the phenotypical and clinical features of sarcomeric HCM –74. In sIndeed, cardiac MRI is becoming increasingly important for the diagnosis of cardiac involvement in FD, since it is able to identify patients with fibrosis as well as LVH more precisely and T1-mapping MRI and strain echocardiography might be more sensitive for the early detection of the cardiac manifestations of FD Fig.  72–74].–74.72–74Cardiac MRI also offers higher definition of ventricular structures and visualization of the extent of scarring and fibrosis and more reproducible quantification of LVH for serial within-patient assessments, which is important given that in female patients, cardiac fibrosis may be present before LVH Fig.  36, 77], 7736, 7As MRI is widely available in Turkey, an effort should be made to establish referral centers in several regions specialized for the MRI based cardiac evaluation of patients.The early peripheral neuropathic hallmarks of FD are frequently followed by cerebrovascular complications and autonomic dysfunction in adulthood .The first neurological symptoms of FD occur due to small-fibre peripheral neuropathy and involve a spectrum of manifestation including peripheral neuropathic pain, deficits of thermal sensation and of physiologic pain perception, impaired sweating, GI dysmotility, and other sensory deficits such as hearing loss . In FabrFabry-related neuropathic pain due to small-fibre neuropathy is often the earliest manifestation for children with classical FD 60–80%) that can occur as early as 3 years of age or less , usually% that caThe most common presentation of neuropathic pain among FD patients is Fabry pain crises characterized by agonizing burning pain originating in the extremities and radiating inwards to the limbs and other parts of the body, and are often precipitated by rising body temperature due to exercise, fever, or warm ambient environments , 82, 83.Pain may wane in adulthood and it is very important to search for a medical history of Fabry crises in childhood during the first examination of a newly diagnosed adult patient .55 and in adults with chronic pain, celiac disease and multiple sclerosis are the most often-cited differential diagnoses, particularly in females. ..GLA pathGLA gene is important to the diagnosis of FD, other questions have emerged regarding how to elucidate VUS and how to explore potential genotype–phenotype relationships to perform correct risk stratification  and lifestyle modifications , whereas dialysis or renal transplantation are available for patients experiencing ESRD Table  1, 23, , 180.TabThe general therapeutic goal for optimizing patient management in FD should be to optimize both disease-specific and nonspecific adjunctive treatments to prevent or minimize effects of organ damage (e.g. kidney dysfunction) and prevent clinical events (e.g. stroke) as well as reduce symptoms, such as neuropathic pain .The management of FD should be carried out by an experienced multidisciplinary team based on an individualized approach to patient care consistent with the natural history of the specific disease phenotype and should involve early ERT initiation after comprehensive evaluation of disease involvement, routine monitoring for evidence of organ involvement in late onset asymptomatic patients and response to therapy in treated patients to regularly re-evaluate and appraise the therapeutic goals and use of adjuvant treatments to assist in the management of organ-specific complications , 39.There is growing evidence that early initiation of ERT optimizes treatment benefits, and can potentially prevent or delay progression to permanent organ damage . AccordiFor male patients with a classic Fabry pathogenic variants, ERT should be initiated promptly when there are clinical manifestations, but should be considered for males older than 10 years of age even in the absence of clinical signs or symptoms of organ involvement. In addition, female patients with classical pathogenic variants or patients with later onset Fabry pathogenic variants should be treated as soon as possible ERT if they present with symptoms suggesting major organ  involvement or laboratory, histological, or imaging evidence of injury to the major organs.Tissue-based assessment of FD pathology may assist the decision to initiate ERT and may occasionally be helpful in assessing disease progression and response to treatment during follow-up. The treatment should not be withheld from patients with severe renal insufficiency or with renal transplantation, from those on dialysis, or those with cognitive decline , 39.Given the rising number of pregnant women with FD, it should also be noted that the agalsidase alfa  and agalStrategies to optimize the management FD patients involves timely use of ERT along with appropriate non-specific adjunctive therapies through an individualized optimal treatment strategy suiting patient’s clinical signs and symptoms and improving QoL which is often impaired even in early-stage disease because of neuropathic pain, whereas a need for further investigation is obvious that would address the potential invasive or non-invasive markers of disease progression as well as the impact of seroconversion and IgG antibody production on treatment response among FD patients .While the advent of potential alternative therapeutic agents hold potential promise for FD patients, their combined use should also be investigated in future studies in terms of potential to enable better outcomes and treatment of organ-specific sequelae of FD should be continued via standard-of-care adjunctive therapies .Being the only approved treatment option in Turkey, enzyme replacement therapy stays as the first choice for the etiology targeted treatment in indicated patients.Given its multi-systemic nature, setting up a medical care plan for FD should ensure interdisciplinary communication across the specialties managing different organ complications , 36. AwaGLA gene, emphasizing a need for an individualized approach to patient care in accordance with genotype, gender, family history, phenotype, and specific clinical symptom severity of a given patient. The experts reached consensus on the critical role of a high index of suspicion in symptomatic patients and screening of certain at risk groups to reveal timely and accurate diagnosis of FD along with consideration of various causes of small-fiber neuropathy, cryptogenic young stroke and sarcomeric HCM in the differential diagnosis. The expert panel emphasized the need for increased awareness of the treating physician about the different kinds of pathogenic variants s and their clinical implications as well as necessity of further clinical, biochemical, or histopathological evidence of FD to determine the pathogenicity of VUS variants in GLA gene. The expert panel agreed on the diagnostic and prognostic role of histological changes on renal biopsy as become evident before clinical and laboratory indicators of disease, the role of MRI in differential diagnosis of Fabry-specific cardiac and cerebrovascular involvement and higher specificity of ocular manifestations with higher opportunity of ophthalmologists to identify FD patients at early period. The expert panel emphasized a need for a coordinated, multidisciplinary care approach in the management of FD with early start of treatment, tailoring the treatment to the needs of individual patients and regular assessment of disease progression in all patients to obtain the therapeutic goals for improving QoL and reducing progression of the disease or to stabilize end organ structure and function. The experts emphasized the crucial role of timely recognition of FD with minimal delay from symptom onset to definite diagnosis in better management of FD patients, given the likelihood of changing the disease’s natural history, improving the patients’ QoL and the prognosis via ERT once a diagnosis is made. In this regard, this consensus document is expected to increase awareness among physicians about unique characteristics of FD to assist clinicians in recognizing FD with a well-established clinical suspicion consistent with pathogenic variants and gender-based heterogeneous clinical manifestations of FD and in translating this information into their clinical practice for best practice in the management of patients with FD.This consensus statement indicated the clinical heterogeneity of FD as well as a large number of pathogenic variants in the


The crosstalk between tumor cells and the tumor microenvironment (TME), triggers a variety of critical signaling pathways and promotes the malignant progression of cancer. The success rate of cancer therapy through targeting single molecule of this crosstalk may be extremely low, whereas co-targeting multiple components could be complicated design and likely to have more side effects. The six members of cellular communication network (CCN) family proteins are scaffolding proteins that may govern the TME, and several studies have shown targeted therapy of CCN family proteins may be effective for the treatment of cancer. CCN protein family shares similar structures, and they mutually reinforce and neutralize each other to serve various roles that are tightly regulated in a spatiotemporal manner by the TME. Here, we review the current knowledge on the structures and roles of CCN proteins in different types of cancer. We also analyze CCN mRNA expression, and reasons for its diverse relationship to prognosis in different cancers. In this review, we conclude that the discrepant functions of CCN proteins in different types of cancer are attributed to diverse TME and CCN truncated isoforms, and speculate that targeting CCN proteins to rebalance the TME could be a potent anti-cancer strategy. Cancer is the second leading cause of death in the United States and is becoming a major public health problem and central focus of modern medical research in China . AlthougCellular communication network (CCN) family are scaffolding proteins that may govern and balance the interconnection among individual signaling pathways. CCN proteins, first described in 1993, are a six-member family of cysteine-rich regulatory proteins that exist only in vertebrates, including CCN1 , CCN2 , CCN3 , CCN4 , CCN5 (WISP-2), and CCN6 (WISP-3). CCN proteins do not behave like individual cytokines in that they do not perform a single function but instead coordinate in various functions of extracellular and intracellular proteins . All CCNAlthough CCN proteins were discovered three decades ago, they have not received widespread interest, and their roles and modes of action in human cancers are still ambiguous. CCN protein members always appear to have paradoxical effects across different types of cancer  and evenCCN proteins are secreted proteins, with full-length CCN proteins consisting of a signal peptide for extracellular release followed by four structural domains (with CCN5 lacking the CT domain): IGFBP, VWC, TSP-1, and CT . PrototyCCN proteins are multifunctional regulatory molecules in the TME that are involved in many vital biological functions, including angiogenesis, fibrosis, tissue regeneration and repair, and cancer . The divThe insulin-like growth factor-binding protein (IGFBP) domain of CCN proteins is found in every CCN family members, and shares strong sequence homology to the N-terminal domain of traditional IGFBPs, which bind to and influence the actions of IGFs . AlthougThe Von Willebrand factor type C (VWC) domain is also found in every CCN family member, and the VWC domain most commonly binds to bone morphogenic proteins (BMPs) , TGF-β , and divThe thrombospondin type 1 repeat (TSP-1) domain is another common domain in CCN proteins and plays strong roles in some biological functions of tumor, primarily through interactions with lipoprotein-related receptors , vasculaThe carboxyterminal (CT) domain is thought to mediate key functions in several CCN proteins (except CCN5), because it also acts as a dimerization module in a manner analogous to domains in other molecules, such as nerve growth factor (NGF), TGF-β, VEGF, BMPs, platelet-derived growth factor (PDGF) and diverse integrins. In addition, many biological functions of cytokines arise through their interactions with heparin . InteresOne fascinating aspect of TME that adds to the complexity of tumor progression. CCN proteins can be potential therapeutic targets that can be manipulated to rebalance the TME. Recently, Tao et al. proved that CCN4 was preferentially secreted by glioma stem cells (GSCs), and which played critical roles in maintaining GSCs and tumor-supportive macrophage . Jia et Recently, CCN members also play direct roles in tumor progression through diverse signaling pathways. CCN1 has been shown to promotes cell adhesion and migration as a mediator of Notch1 signaling in breast cancer . OverexpCCN proteins lacking one or more of the functional domains can be produced by alternative splicing  or post-Although all CCN family members (except CCN5) have four highly conserved functional domains, they have different roles within particular types of cancer. Some CCN proteins have established associations with cancer malignancy progression and are considered as prognostic markers and therapeutic targets for certain types of cancer . HoweverCCN1 exhibits varying mRNA levels and associations with prognosis across different types of cancer. Comparisons of CCN1 mRNA levels among 32 human cancer types and adjacent normal tissue using GEPIA2 revealed significantly upregulated CCN1 expression in four types of cancer  and significantly downregulated expression in 14 types of cancer . To evaluate the association between CCN1 mRNA expression and prognosis, we also examined 32 human cancers using GEPIA2. The relationship between CCN1 expression and prognosis varied across different types of cancer. High expression of CCN1 was associated with shorter overall survival (OS) in five types of cancer  and longer OS only in SKCM, suggesting its role as a tumor suppressor. These bioinformatics results revealed the heterogeneous expression and functions of CCN1 in different types of cancer .in vitro, studies showed that CCN1 serves as a tumor promoter in most cancers but can also acts as a tumor suppressor in some cancers. Thus, to resolve the discrepant roles of CCN1 in different types of cancer, future studies should take diverse TMEs and different isoforms into consideration.Several previous studies reported that CCN1 participates in cancer development and can serve as both a tumor suppressor and promoter . In mostCCN2 mRNA levels and their association with prognosis also vary across different types of cancer. Comparison of CCN2 mRNA levels among different cancer tissues and their adjacent normal tissues revealed significantly higher CCN2 expression in five types of cancer  and significantly lower expression in 11 types of cancer . When we evaluated associations between CCN2 mRNA levels and prognosis, we found that high expression of CCN2 was associated with shorter OS in STAD and THCA and longer OS only in SKCM, suggesting that it acts as a tumor suppressor. Thus, these bioinformatics results further revealed the heterogeneous expression and function of CCN2 in different types of cancer .After reviewing the current studies on CCN proteins. In gastric cancer, high CCN2 expression correlates with more lymph node metastases, more peritoneal dissemination, and poorer 5-year survival . After CComparison of CCN3 mRNA levels among different types of cancer tissues and their adjacent normal tissues revealed that CCN3 expression was significantly upregulated in two types of cancer [ovarian serous cystadenocarcinoma (OV) and PAAD] and significantly downregulated in six types of cancer . When we further evaluated the association between CCN3 expression and prognosis in pan-cancer, we found that high CCN3 expression was associated with shorter OS in seven types of cancer  and longer OS in two types of cancer . When evaluating the association between CCN4 expression and prognosis in pan-cancer, we found that high CCN4 expression was associated with shorter OS in five types of cancer . The results of these bioinformatics analyses suggest that CCN4 mainly acts as a tumor promoter .The participation of CCN4 in cancer development has been reported by many previous studies, which showed that CCN4 serves as a tumor promoter in colorectal , breast CCN5 mRNA levels also vary across different types of cancer. Comparison of CCN5 mRNA levels across different cancer types and adjacent normal tissue revealed significantly lower expression of CCN5 in 16 types of cancer . Increased expression of CCN5 was not observed in any type of cancer. High CCN5 expression was associated with shorter OS in four types of cancer  and longer OS only in SARC, suggesting that CCN5 acts as an anti-oncogene. The results of these bioinformatics analyses suggest that CCN5 expression and function vary across different types of cancer, perhaps due to differences in its structure compared with other CCN family members .As CCN5 lacks a CT domain, this striking difference in structure compared with other CCN family members may allow it to have unique functional roles. Like its family members, however, previous studies reported inconsistent roles of CCN5 in carcinogenesis. CCN5 is downregulated in human leiomyoma , pancreaCCN6 mRNA levels and prognostic value also vary depending on the type of cancer. Comparison of CCN6 mRNA levels among diverse cancer types and adjacent normal tissue revealed that CCN6 expression was significantly downregulated in four types of cancer  and significantly upregulated only in OV. When evaluating the association between CCN6 expression and prognosis, we found that high CCN6 expression was associated with shorter OS only in LIHC. These bioinformatics analyses further suggest that the expression and functions of CCN6 are inconsistent across cancer types .CCN6 has received much attention in the last few years due to its involvement in many cancer-related processes, including EMT, cell death, invasion, and metastasis, and its function as a tumor suppressor . HoweverThe six members of CCN proteins have established associations with cancer malignancy progression and are considered as prognostic markers and therapeutic targets for several types of cancer. However, CCN proteins always appear to have contradictory roles in different types of cancer. After a retrospective analysis of the literature, we come to the conclusions . CellulaTargeting CCN protein expression or signaling pathways holds promise in the development of diagnostics and therapeutics for cancers, and the cocktail containing CCN proteins in different combinations should be a potential antitumor approach. Since the current literature has certain limitations in clarifying the exact role of CCN proteins, continued studies are still needed to reveal the exact roles of CCN proteins in cancer.QJ contributed to the conceptualization, literature search, writing, review, and editing. BX contributed to the literature search and editing. YZ contributed to the methodology and visualization. AA contributed to language proofreading. XL contributed to the critical review and editing. All authors have read and approved the final manuscript.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.


Early mammal embryogenesis starts with oocyte fertilization, giving rise to the zygote. The events that the newly formed zygote surpasses are crucial to the embryo developmental success. Shortly after activation of its genome, cells of the embryo segregate into the inner cell mass (ICM) or the trophectoderm (TE). The first will give rise to the embryo while the latter will become the placenta. This first segregation involves cellular and molecular processes that include cell polarity linked to intracellular pathway activation, which will regulate the transcription of trophectoderm-related genes. Then, cells of the ICM undergo the second event of mammalian cell differentiation, which consists of the separation between epiblast (EPI) and hypoblast or primitive endoderm (PrE). This second segregation involves paracrine signaling, leading to differential expression of key genes that will dictate the fate of the cell. Although these processes are described in detail in the mouse, recent studies suggest that the bovine embryo could also be an interesting model for early development, since there are differences to the mouse and similarities with early human embryogenesis. In this review, we gathered the main data available in the literature upon bovine and mouse early development events, suggesting that both models should be analyzed and studied in a complementary way, to better model early events occurring in human development. In mammals, the formation of the zygote initiates a series of events, known as early embryogenesis. The success of this process depends mainly on the first cell cleavages and the differentiation of the first cell lineages, mechanisms that are essential for the embryo implantation at the uterus and further fetal development  begins. From this moment on, embryos can develop independently of the maternal genome, until the formation of a blastocyst composed of three distinct cell lineages: the trophectoderm (TE), an outer epithelium made of polarized cells that surrounds a group of nonpolar cells, called the inner cell mass (ICM). The ICM will later differentiate into two distinct cell types, the epiblast (EPI) and the primitive endoderm (PrE) cells .The TE is responsible for giving rise to the placental trophoblasts . On the The first cell differentiation of the embryo is widely studied in mice, an animal model that provides the largest source of information about the theme. Although there are some crucial differences in the regulation of early developmental events, some evidence shows that bovine embryos can also be great models for understanding human embryonic development . StudiesConsidering the importance of the correct specialization of these cell lines for embryo developmental success, this study aims to gather the main data available in the current literature regarding the events of cell differentiation during pre-implantation development, proposing a comparative analysis between the early development of mouse and bovine embryos.In mammals, the newly fertilized zygote undergoes symmetrical cell divisions, called cleavages, which is a relatively conserved mechanism between species. In the early stages, the blastomeres are relatively spherical, similar to each other and they become smaller with each cleavage round . The evezonula adherent epithelial junction (ZA) characteristic proteins, forms the contact region of basolateral membrane domain between epithelial cells  present blastocyst formation and normal TE cell differentiation and expansion. Unlike in mice, TEAD4 knockdown did not influence the gene expression of OCT4, NANOG, CDX2, and GATA3, suggesting that TEAD4 is not essential for TE formation in the bovine embryo (ormation . Bovine e embryo .Yap1 and Wwtr1 (TAZ), a functional homolog of YAP1, led to the death of mouse embryos before reaching the morula stage. By contrast, overexpression of Yap1 and Taz promotes the expression of Cdx2, confirming the hypothesis that YAP1 together with TAZ and TEAD4 regulates the expression of Cdx2 , Sox2, and Nanog, are initially expressed  . In mousby NANOG  and, in by NANOG  (Figure by NANOG .Fgf4 deletion showed NANOG and GATA6 labeling in most ICM cells, from the morula stage to early blastocysts, suggesting that the initial expression of these proteins does not depend on the signaling triggered by FGF4. However, with further development of blastocysts, these embryos formed a predominantly NANOG-positive ICM, suggesting that FGF4 is necessary for the maintenance of GATA6 expression in ICM. The GATA4 and SOX17 expression was not detected in these Fgf4-/- embryos, indicating the need for an active PrE cell differentiation program (Mouse embryos with  program .Bovine embryos treated with exogenous FGF4 formed blastocysts with an ICM composed exclusively of GATA6-positive cells, demonstrating that FGF4 presence can induce gene expression pattern of the PrE precursors and block differentiation into EPI cells . StudiesFgfr1 expression, and Fgfr2 is preferentially expressed in PrE precursor cells only. Fgfr1 or Fgfr2 knockouts demonstrated that FGFR1 has a fundamental role in PrE differentiation, while FGFR2 played a secondary role (Studies indicate that the activity of the FGF/ERK pathway depends on the types of FGFR expressed in the cells. Studies in mice embryos have shown that both EPI and PrE precursors present ary role . There aary role . The locary role , but ICMary role  and mainary role .Grb2 in mouse embryos did not impair ICM compaction, but inhibited Gata6 and the expression of other PrE markers, resulting in the absence of PrE formation. However, these embryos maintained Nanog expression in all ICM cells, which developed into EPI, suggesting the role of the FGF/ERK pathway in PrE development (FGFRs activation causes the recruitment of adapter proteins that will connect FGF signaling and the ERK pathway . ERKs acelopment .Grb2 or Gata6 deletion. However, when the treatment was interrupted at the 16-cell stage, there were no changes observed in the expression pattern of ICM transcription factors, suggesting that the FGF/ERK signaling pathway activation occurs in the early blastocyst stages (Treatment of mouse embryos with FGFR or MEK inhibitors caused the same phenotype observed in blastocysts presenting t stages .Bovine embryos treated with MEK inhibitors had a significant decrease in TE and ICM cells numbers , as wellGata6 knockout embryos did not form PrE lineage, expressing NANOG in all ICM cells (Gata6 knockout embryos treated with exogenous FGF4 in the 8 cell stage, did not express NANOG, showing that the FGF/ERK pathway can inhibit NANOG independently of Gata6. However, when they were treated in later stages, as in the early blastocyst, NANOG expression was maintained (This differentiation process is essentially GATA6-dependent in mice since CM cells . Late exCM cells . Still, intained . CurrentThe main knowledge of early embryonic development in mammals derived mostly from studies in mouse models. Research in bovine embryos shows that the biological processes involved in early cell differentiation are not as conserved (summarized in


Fluorescence microscopy has become a critical tool for researchers to understand biological processes at the cellular level. Micrographs from fixed and live-cell imaging procedures feature in a plethora of scientific articles for the field of cell biology, but the complexities of fluorescence microscopy as an imaging tool can sometimes be overlooked or misunderstood. This review seeks to cover the three fundamental considerations when designing fluorescence microscopy experiments: (1) hardware availability; (2) amenability of biological models to fluorescence microscopy; and (3) suitability of imaging agents for intended applications. This review will help equip the reader to make judicious decisions when designing fluorescence microscopy experiments that deliver high-resolution and informative images for cell biology. Since the inception of fluorescence microscopes in the early 1900s, their use as a research tool for observing discrete subcellular structures and processes has grown immensely . The useThe rapid advances in the field of fluorescence microscopy have required a collaborative effort; from physicists and engineers developing microscopy hardware, to chemists that continually develop and refine novel fluorescent probes, and biologists who utilise these tools to investigate the biological functions of diverse specimens. A misunderstanding of some of these disciplines by researchers from other areas often leads to poor experimental design and research outcomes. This review aims to summarise the three main considerations when fluorescence microscopy experiments are employed: (1) current hardware availability; (2) the different biological models applicable for fluorescence microscopy; and (3) the plethora of fluorescent probes available. The advantages and limitations for each factor will be discussed to allow the reader to make informed decisions when contemplating experimental design. A helpful flowchart  is also Widefield epifluorescence microscopes, established during the early 20th century, represent the fundamental type of fluorescence technology . Many inConfocal microscopy represents a next step in fluorescence microscopy, offering higher spatial resolution, with the ability to visualise subcellular details, which are unachievable by conventional widefield systems. Invented by Minsky in 1955 , the conModifying the laser excitation source of a confocal microscope with an ultra-fast pulsed laser enables two- or three-photon microscopy . Ultra-fLaser scanning microscopes have been adapted to observe dynamic events in cellular function and exploit the properties of imaging probes, such as fluorescence lifetimes, and their interactions within and between cells. The three most common technologies employed are fluorescence lifetime imaging microscopy (FLIM) , fluoresAnother recent technique, used to image larger tissue samples quickly and efficiently, is light-sheet fluorescence microscopy (LSFM) . Light-sImprovements in laser scanning microscopes have enhanced imaging resolutions beyond Abbe’s limit (Equation (1)), the physical limitation due to the physics of diffraction, for example, a 200 nm resolution limit in air when using an excitation wavelength of 400 nm ; howeverd is the minimum distance that can be differentiated between two points, λ is the wavelength of light, n is the refractive index of the medium the light is traversing, θ is the angle at which the light is converging, and NA is the numerical aperture +[Ir(ppy)), can impart significant cellular uptake properties with H9c2 staining observed in the ER and mitochondria, respectively [3, Ir1 preferentially localised within the mitochondria (87% Pearson’s correlation coefficient with MitoTracker™), while the less hydrophobic Ir5 was observed in the lysosome (79% Pearson’s correlation coefficient with LysoTracker™) [2+ ions [The Massi group have also conducted a thorough investigation of Ir(III) complexes and how a subtle structural change, from a neutral tetrazole group  . These mracker™) . Other eracker™)  or labil[2+ ions . There a[2+ ions ,260 and [2+ ions ,262 meta[2+ ions  and thio[2+ ions  sensing Trivalent metal ions from the lanthanide series of f-block elements have also shown extensive application for cellular imaging. These systems nearly always rely on an antennae group to populate the metal excited state, which gives rise to the characteristic phosphorescence signal exhibited by the lanthanide ion ,265,266.ReMannose, 4, which contains a glucose moiety tethered by a polar TEG chain, exhibited cellular uptake through GLUT transporters in HeLa cells [Ir-CMYC) demonstrated co-staining with commercially nuclear stain Hoechst 33342 in fibroblast cells, exhibited near-IR emission, and was non-toxic [5), which interacts with DNA to provide an environment-dependent emission in both prokaryotic and eukaryotic cells. This probe does not contain any specific directing motif yet still exhibits good selectivity to DNA [The tracking of important biological molecules in live cells has also been achieved using metal ion complexes. Gillam and co-workers recently reported several neutral Re(I) complexes conjugated to sugar moieties , an overall positive charge typically leads to mitochondrial localisation (MitoTracker™ Red), and tertiary amines which become protonated in acidic environments are often employed for lysosomal uptake. In the case of fluorescent tags, the key functional group facilitates the actual linkage between the fluorophore and the target of choice. In the case of the Alexa Fluor series depicted in Organic fluorophores enjoy a privileged place as the most prominent class of compounds for fluorescence imaging. Owing to their ready accessibility, small size, wide variety, and excellent emissive properties, organic fluorophores are frequently used for imaging cellular components, visualising cellular processes and tagging larger molecules (including antibodies and drugs) for insights into their cellular activity. The advantages and applications of organic fluorophores have been thoroughly covered in many excellent reviews ,277,278,lles see , while tAlexa Fluor™ 350, 488, and 647). However, organic fluorophores often suffer from short lifetimes, photobleaching, cell toxicity, and poor solubility in aqueous environments. Nonetheless, organic fluorophores remain an indispensable tool for fluorescence microscopists for a range of applications.The chemistry of organic fluorophores has been well investigated and new classes continue to be reported in the literature. Design considerations to improve the internal charge transfer (ICT), quantum yield, lifetime, and cell viability are commonly considered by chemists synthesising novel probes. A major advantage of small organic fluorophores is their capacity to be attached to larger molecules for tagging purposes  is critical for the conversion of acetylcholine to choline and acetate; a vital component of the nervous system. Recently, two near-IR emitting cyanine probes were developed, which were conjugated to huprine derivatives to enable AChE binding. HupNIR2  demonstrcroscopy .7) for the visualisation of glycosidase activity, as demonstrated in cancerous cell lines [The ability of small organic fluorophores to act as drug-masks allows for the visualisation of drug delivery in real-time. For example, the Gunnlaugsson lab have reported a variety of chemosensors based on the 1,8-naphthalimide scaffold, including glycosylated derivatives (such as ll lines . These pll lines . This coll lines ,288.This review has briefly outlined the three fundamental factors that need to be considered when designing fluorescence microscopy experiments. The most financially expensive consideration is what hardware is available and whether the parameters of this instrumentation will be suitable for the desired result. Careful design of an appropriate biological model which is amenable to fluorescence microscopy is required. Finally, the most suitable imaging agent to provide the fluorescent signal needs to be selected, with an ever-growing range of commercially available and literature-reported examples on offer. These three considerations are not mutually exclusive, with each factor impacting on the other two and ultimately driven by the intended outcome of the experimental data required. Consequently, an understanding of all aspects of hardware, imaging compounds, and in vitro or in vivo models is important when designing fluorescence microscopy experiments. As highlighted by numerous examples here, confocal microscopy dominates as the fluorescence microscopy hardware setup of choice. This is likely due to accessible cost and availability of these microscopes and the broad applicability to a range of imaging probes and biological models. Greater uptake and more widespread use of the advanced imaging hardware discussed in this review is likely to accelerate in the near future, as biologists look to harness the power of these instruments to gain a deeper understanding of cellular mechanisms and biology. We believe that this review will be helpful for those not fully versed across fluorescence microscopy to steer them towards experimental conditions and parameters which will aid in generating high quality and informative images for their intended applications.


The excessive activation of the renin-angiotensin system in kidney disease leads to alteration of intracellular pathways which concur altogether to the induction of cardiovascular and renal remodeling, exposing these patients since the very beginning of the renal injury to chronic kidney disease and progression to end stage renal disease, a very harmful and life threatening clinical condition. Oxidative stress plays a pivotal role in the pathophysiology of renal injury and cardiovascular-renal remodeling, the long-term consequence of its effect. This review will examine the role of oxidative stress in the most significant pathways involved in cardiovascular and renal remodeling with a focus on the detrimental effects of oxidative stress-mediated renal abnormalities on the progression of the disease and of its complications. Food for thoughts on possible therapeutic target are proposed on the basis of experimental evidences. Oxidative stress arises when the physiologically-produced oxidant species overwhelm the endogenous antioxidant recovery capacity of the cells, leading to loss of intracellular redox homeostasis . As net The highly complex renal pathways involved in the induction of oxidative stress entail the activation of the renin-angiotensin system (RAS). This latter in fact, by regulating several components of the cardiovascular-renal system, influences also pathophysiological responses through hypertension, rapid progression of atherosclerosis, and vascular remodeling . ParticuThis review will explore the current knowledge on the RAS and the influence of oxidative stress in kidney disease with a focus on patients with chronic kidney disease stage 3–4 (CKD) and kidney failure with or without replacement therapy and on the possible available supportive therapeutic approach for these patients.Ang II signaling plays a pivotal role in the vasoconstriction of renal vascular smooth muscle cells (VSMCs) and in the sodium and water handling, involved in the pathophysiology of renal and cardiovascular remodeling. . The sigChronic Ang II stimulation induces elevation of blood pressure (BP) due to juxtaglomerular injury, hypoxia, tubular necrosis and interstitial fibrosis. Experimental evidence show in fact, that renal autoregulation is relatively ineffective in subjects with CKD, in hypertensive and in diabetic patients as afferent arteriolar vasoconstriction is not able to counteract the increased systemic BP and to prevent its spreading to the glomeruli . These eOverall, the RAS and, in particular, its major effector molecule Ang II, are principally involved in cardiovascular-renal remodeling in CKD driving oxidative stress, inflammation and immune responses .2•−), produced by the respiratory chain in the mitochondria and by several enzymes such as nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase, uncoupled nitric oxide synthase (NOS), xanthine oxidase, cytochromes P450, lipoxygenase (LOX), cyclooxygenase (COX).Oxidative stress is related to an imbalance in reactive oxygen species (ROS) that are generated during the sequential reduction of molecular oxygen. ROS usually refers to free radicals and other non-radical intermediates having the capacity to quickly react with all the surrounding molecules. The most representative reactive specie is superoxide (O2O2), a ROS with a greater stability able to cross membranes, which also undergoes catalytic decomposition to •OH, and acts as a signaling molecule together with superoxide itself. Further, superoxide favors the production of others extremely reactive and toxic compound such as radical nitric oxide (NO•) and peroxinitrites (ONOO−) . . 17]. As an example, permanent exposure of red blood cells to high oxygen concentration favors the production of ROS and the reduction of antioxidant defenses with ensuing oxidative injury to erythrocytes . The eryIn kidney disease many other cellular components are deeply impaired by ROS action in an irreversible fashion due to a “positive feedback” that spreads the damage to the surrounding structures and for the concomitant reduction of antioxidant defenses. phox in NOXs (with ensuing production of superoxide), but also for activation of other enzymes such as xanthine oxidase, lipoxygenase, cyclooxygenase and P450 monooxygenase and for release of ROS from endoplasmic reticulum and nuclear envelope [phox and oxidized LDL (oxLDL) levels correlated with the left ventricular mass [phox levels and oxLDL with concomitant significant reduction of the left ventricular mass providing clinical evidence for this hypothesis [A boost of mitochondrial respiratory chain is responsible for overexpressing p22envelope . Besidesenvelope . All theenvelope . We obselar mass . These elar mass . Interespothesis . The presence of oxidative stress influences the transcription of several factors involved in proliferation, fibrosis and atherosclerotic events as well. Plasminogen activator inhibitor (PAI)-1 is the inhibitor of both tissue plasminogen activator and urokinase, involved in fibrinolysis, and favors hypercoagulable state in diabetes patients . In addiOxidative stress in kidney failure with or without replacement therapy is present since the very early stages of the renal dysfunction, as it is demonstrated by the presence of 8-iso-PGF and C-reactive protein . As renaThe endogenous antioxidant responses against oxidative stress are multiple and involve the intervention of low molecular weight compounds such as NO, scavenger enzymatic mechanisms in terms of superoxide dismutases (SODs), glutathione peroxidases (GSH-Px) and more complex cellular responses such as autophagy that, depending on the entity of oxidative stress, can be either protective or harmful . A well-phox expression in addition to reduced antioxidant defenses such as HO-1 and total plasma antioxidant power when compared to normotensive transplanted recipients [Oxidative stress plays an harmful role also in patients receiving kidney transplant and it is strictly connected to post transplant hypertension ,49,50. Pcipients . Interescipients . The concipients . Taken tcipients ,50.Traditional risk factors such as smoke, diabetes and hypertension do not entirely justify the high incidence of cardiovascular morbidity and mortality observed in patients with CKD and kidney failure with or without replacement therapy. Other so-called “non- traditional” risk factors such as inflammation, endothelial dysfunction and in particular increased oxidative stress have been recognized as playing a major role in the induction of cardiovascular remodeling ,53,54.2+ handling [Left ventricular hypertrophy (LVH) is the most common myocardial structural abnormality associated with chronic kidney diseases . Among thandling  and induhandling . The inchandling . In addihandling .The activation of ROCK tracks not only with vascular contraction but also with the expression of proinflammatory cytokines and molecules of adhesion, with the expression of atherothrombogenic PAI-1, and with the regulation of cyclophilin A . ThroughAmong the implications of increased left ventricular mass, atrial fibrillation is the most frequent arrhythmia in patients with kidney failure with or without renal replacement therapy and is associated to marked MYPT-1 phosphorylation . CardiacAlongside specific pathways inducing fibrosis and electrical remodeling, CKD and dialysis patients exhibit also reduced bioavailability of antioxidant defenses such as NO which further influence the dysfunctional VSMCs migration toward intima. This migration causes intimal hyperplasia alongside with abnormal extracellular matrix deposition and hyaline material accumulation, vascular calcification with 0arteries stiffening and high pulse pressure ,72. The In a biochemical framework of increased oxidative stress and decreased endogenous antioxidant defenses, altered intracellular pathways leading to atherosclerosis, fibrosis and more in general to cardiac and renal remodeling, an efficient therapeutic measure could be the reduction of oxidative stress with pharmacological or nutritional options. Many clinical trials have been conducted targeting oxidative stress in attempt to reduce cardiovascular incidence and oxidative stress-related sequelae, however, most of them have failed to prove a significant clinical benefit in reduction of atherosclerosis and its complications . This ma2-microglobulin and fibroblast growth factor 23), parathyroid hormone, protein-bound solutes, AGEs, and trimethylamine N-oxide (TMAO) [Hemodialysis and hemofiltration are the main renal replacement therapies for subjects with end-stage renal diseases. Failure of kidney function leads to uremia and accumulation in the bloodstream of toxic uremic compounds such as low-molecular weight solutes , middle molecules (βe (TMAO) . Both ex2•− and H2O2 spreading high reactivity to surrounding molecules, [It is evident that during those renal replacement therapies blood cells are extremely forced against the membrane walls and against dialyzer/filter components. By leaving the vessels, blood loses the protective effect of the endothelium and by coming into contact with synthetic surfaces and by changing the geometry of the flow blood’s components activate leukocytes and increase oxidative stress . Activatlecules, . Furtherlecules, ,84.phox was significantly decreased already after six months of treatment and further declined after twelve months demonstrating a reduction of oxidative stress and blockade of leukocytes’ activation. In parallel, LDL oxidation and expression of PAI-1 were reduced as well as the phosphorylation state of ERK 1/2. In addition, the coating with vitamin E increased HO-1 expression further supporting an active role of vitamin E in the induction of antioxidant intracellular pathways through Nrf2 and Akt [Regarding extracorporeal dialysis, several types of membranes are available for dialyzers, spanning from high or low flux to high or low permeability and functionalized as needed ,85. Bein and Akt . phox gene and protein expression and of reduction of PAI-1 as well as LDL oxidation. In addition, also HFR promotes a significant increase of gene and protein expression of HO-1 [Another extracorporeal renal replacement procedure proven to be effective against oxidative stress is hemodiafiltration with online regeneration of ultrafiltrate (HFR). A double-chamber hemodialysis filter permit reinfusion of regenerated ultrafiltrate through a charcoal-resin cartridge. Due to its particular structure, the system combines diffusion, convection and absorbance: the ultrafiltrate is processed in a charcoal-resin component of the cartridge which has the peculiar characteristic to absorb pro-inflammatory cytokines. Subsequently, the regenerated ultrafiltrate is reinfused into the bloodstream before the diffusive section of the filter . This te of HO-1 ,89.phox, ROCK activity (MYPT1) and ferritin levels which all further increased after 6 months [In peritoneal dialysis, oxidative stress has been reported only in terms of increased AGEs, and other pro-oxidants derived by glucose . Very re6 months , therefoThe kidney is extremely vulnerable to damage caused by oxidative stress. The progression of kidney disease engenders biochemical mechanisms that alter cellular homeostasis and vascular structures, which in the long terms results in cardiovascular and renal remodeling with deleterious outcomes. It is now clear that oxidative stress plays a very important role in both the onset and progression of kidney diseases. Targeting oxidative stress and the related molecular mechanisms together with the availability of specific dialytic procedures which have provided high efficacy against oxidative stress are therefore a necessary objective to improve the prognosis of CKD and dialysis patients.