Cobalamin is a cofactor present in essential metabolic pathways in animals and one of the water-soluble vitamins. It is a complex compound synthesized solely by prokaryotes. Cobalamin dependence is scattered across the tree of life. In particular, fungi and plants were deemed devoid of cobalamin. We demonstrate that cobalamin is utilized by all non-Dikarya fungi lineages. This observation is supported by the genomic presence of both B12-dependent enzymes and cobalamin modifying enzymes. Fungal cobalamin-dependent enzymes are highly similar to their animal homologs. Phylogenetic analyses support a scenario of vertical inheritance of the cobalamin usage with several losses. Cobalamin usage was probably lost in Mucorinae and at the base of Dikarya which groups most of the model organisms and which hindered B12-dependent metabolism discovery in fungi. Our results indicate that cobalamin dependence was a widely distributed trait at least in Opisthokonta, across diverse microbial eukaryotes and was likely present in the LECA. Cobalamin dependence is scattered across the tree of life. In particular, fungi and plants have been deemed devoid of cobalamin. We demonstrate that cobalamin is utilized by all phyla of non-Dikarya fungal lineages. This observation is supported by the identification of B12-dependent enzymes and cobalamin modifying enzymes in published fungal genomes. We found that cobalamin dependence was a widely distributed trait at least in Opisthokonta, across diverse microbial eukaryotes and likely in the LECA (last eukaryotic common ancestor). Moreover, the genes identified are actively transcribed in many taxa.Cobalamin, also known as vitamin B12, is the most common cobalt-containing compound in nature and one of eight known water-soluble vitamins grouped into B class. Cobalamin is an organometallic complex compound that contains a cobalt atom placed within a corrin ring. Vitamin B12 is derived from uroporphyrinogen III, which is also the first macrocyclic intermediate in a common pathway of heme and chlorophyll biosynthesis . In animPorphyridium purpureum and Amphidinium operculatum, can obtain the cobalamin cofactor from associated bacteria  takes a part in DNA replication and repair  and proper myelin synthesis. Only seven enzymes from the above pathways seem to be uniquely present in B12-dependent organisms. They either modify cobalamin or use it as a cofactor. The former group contains methylmalonyl Co-A mutase-associated GTPase Cob (MeaB), cob(I)yrinic acid a, c-diamide adenosyltransferase (CblAdo transferase), cyanocobalamin reductase (CblC), and cobalamin trafficking protein D (CblD) proteins, whereas the latter includes methionine synthase (MetH), methylmalonyl-CoA epimerase (MM-CoA epimerase), and methylmalonyl-CoA mutase (MM-CoA mutase) . All thed repair . For conPhytophthora spp. uses cobalamin-dependent class II RNR.RNR is an enzyme that catalyzes the formation of deoxyribonucleotides from ribonucleotides. It plays a pivotal role in the synthesis, reparation, and regulation of the total rate of DNA synthesis . RNRs arMethionine synthetase (MetH) comes in two variants: cobalamin-dependent MetH (EC 2.1.1.13) and cobalamin-independent MetE (EC 2.1.1.14). MetH catalyzes the final step in the remethylation of homocysteine which explains increased levels of homocysteine upon vitamin B12 deficiency. In animals, this may lead to blindness, neurological symptoms, and birth defects . MetH reCblAdo transferase, cob(I)yrinic acid a, c-diamide adenosyltransferase, converts cobalamin into adenosylcobalamin (AdoCbl). AdoCbl is a cofactor of multiple enzymes that catalyze unusual rearrangement or elimination reactions. Some of them are restricted to prokaryotes, for example, lysine-5,6-aminomutase, isobutyryl-CoA mutase, and glutamate mutase. Others are present also in Eukaryotes, for example, methylmalonyl-CoA mutase .Methylobacterium sp. showed an inability to convert methylmalonyl-CoA to succinyl-CoA caused by an inactive form of methylmalonyl-CoA mutase -methylmalonyl-CoA to the (R) form and uses a vitamin B12 cofactor . MM-CoA A mutase .Kingdom Fungi comprises several lineages of non-Dikarya which, in the order of divergence, are classified into Chytrydiomycota  and Blas. Seven Mortierellomycotina genomes, which were not included in our data sets, have also MetE copies. This might suggest that the metabolic pathways which first required B12 are still conserved among fungi, but have become independent of cobalamin for some organisms.In order to ensure that all housekeeping functions provided by RNR class II, MM-CoA mutase, and MetH pathways are maintained in all of the studied isolates, even those devoid of B12-dependent enzymes, we searched for cobalamin-independent alternatives. We looked for MetE which can substitute MetH , RNR claSince most of the identified homologs of eight B12-dependent enzymes are annotated as hypothetical unknown proteins without experimental characterization, we performed TBlastN searches on them against the NCBI EST database. This served as intermediate evidence that the predicted B12-related proteins in non-Dikarya fungi originate from active genes. TBlastN search results allow also to expect that genes encoding all identified proteins will be expressed.To trace the evolution of the studied proteins, phylogenetic trees for each of the eight protein families were inferred using Bayesian (BA) and Maximum Likelihood (ML) approaches  online, Syncephalis pseudoplumigaleata  which displayed a very high sequence identity reaching 100% with Afipia alphaproteobacteria which might indicate sample contamination. Notably, misannotated Dikarya sequences, like other Dikarya representatives, could not be found in Eukaryotic clades for the analyzed enzymes.We noticed single bacterial sequences misannotated as fungal due to likely bacterial contamination of the fungal DNA samples. We also noticed single fungal sequences grouping within their bacterial relatives. In most cases these were proteins homologous to our enzyme yet with other function, for example, MeaB is similar to other GTPases (KAA6408927.1). Non-Dikarya fungal sequences rarely grouped with bacterial sequences with the exception of MM-CoA mutase from Exclusively fungal clades can be observed in five protein families . For theFonticula alba (Holomycota), the closest relative of fungi belonging to Nucleariida. This pattern was observed for class II with the following score of aLTR support of the fungal clade: MeaB—1.00, CblD—1.00, MetH—1.00, MM-CoA mutase—1.00, and RNR class II—0.98. In the case of CblAdo transferase, CblC and MM-CoA epimerase fungal sequences group together with either ancient Metazoa representatives (CblAdo transferase) or with microbial eukaryotes from Holomycota, Amoebozoa, and SAR groups (MM-CoA epimerase). Importantly, non-Dikarya fungal sequences are always sister to other Eukaryotic sequences which rules out bacterial contamination. Sequences from model organisms belonging to diverse lineages of microbial eukaryotes, not only Opisthokonta, were represented in these clades, including representatives from Polysphondylium pallidum and Dictyostelium spp. (Amebozoa), Thecamonas trahens (Apusozoa), Chlamydomonas reinhardtii (Chlorophyta), Stentor coeruleus (Alveolata), Emiliania huxleyi (Haptophyta), Thalassiosira pseudonana, and Blastocystis spp. (Heterokonta), Naegleria gruberi and Euglena gracilis (Excavata).For MeaB, CblD, MetH, MM-CoA mutase, and RNR class II, fungal sequences form a sister clade to Our discoveries contradict the current opinion that fungi neither synthesize nor use cobalamin  and do nIn Eukaryotes three main metabolic pathways use cobalamin—RNR class II, MM-CoA mutase, and MetH pathways. Functions provided by these pathways are needed for the independent functioning of a living cell and can be lost in parasites . TranspoWe found traces of all of these pathways among all lineages of early-diverging fungi. The distribution of the genes encoding the above-mentioned enzymes is not uniform across the analyzed organisms. For non-Dikarya fungi, it is common to either have two out of three pathways or to have them incomplete.Only Glomeromycotina and Blastocladiomycota have all three complete B12-dependent pathways. These two taxonomic groups are evolutionary and ecologically distant, they share only a few characteristics among others some of these fungi possess relatively big genomes. The latter may be a highlight of relaxed pressure on genome compactness.The least conserved among fungal lineages is the RNR class II pathway. Organisms missing this class use cobalamin-independent RNR class I, which is the RNR used by animals. It is worth noticing that organisms are not limited to having only one class of RNRs at once . CobalamThe best-conserved pathway in non-Dikarya fungi—MetH—can be substituted with a cobalamin-independent enzyme called MetE . We checBatrachochytrium and Mucor representatives showing no synteny. Moreover, no candidate dehydrogenases were found upstream or downstream of MCS and MCL genes. We assume that the function of MCD can be taken over by other dehydrogenases.MM-CoA mutase pathway is more or less conserved among early-diverging fungal lineages. Interestingly, all Mucorales members lack all three B12-dependent enzymes of that pathway. We checked for alternatives for this metabolism track and it turned up to be more complex than in the other two cases. In Dikarya propionate metabolism is carried out in the methylcitrate cycle (MCC). Three key enzymes for this track are methylcitrate synthase (MCS), methylcitrate dehydrogenase (MCD), and methylisocitrate lyase (MCL) . All of According to our results, we can speculate the best-conserved elements of cobalamin-dependent pathways are key enzymes. For example, in the MetH pathway, the best-conserved element is MetH protein. On the contrary, it is quite common to lose CblC and CblD proteins from the proteome  online. Batrachochytrium sp. and herbivorous mammals symbionts from class Neocallimastigomycetes. For these organisms, no cobalamin-dependent enzyme was found. These organisms may obtain the required resources from the host. However, our observations for plant-associated fungi are different. Mycorrhizal fungi from Glomeromycotina and Endogonales, despite maintaining extensive symbiotic relationships with 80% of plant species . Similarly, parasites of organisms without B12-dependent genes do not have any of these enzymes themselves (Caulochytrium protostelioides—Sordaria sp.). This remark needs further investigation because we do not have enough data to form a reliable conclusion. Our discovery challenges the current view that fungi can neither synthesize nor utilize cobalamin. We proved that non-Dikarya fungal proteomes contain three metabolic pathways utilizing vitamin B12. We speculate these organisms have the possibility to accumulate cobalamin. Yet, these genomic observations need to be tested experimentally. Our discoveries may open the way for the selection of B12 over accumulating strains of food fermenting fungi without the need for genetic material manipulation.During the analysis of obtained results, we tried to understand the evolution of cobalamin-dependent metabolic pathways among kingdom Fungi. To widen the picture we checked studied proteomes for cobalamin-independent alternative metabolic pathways, and we confirmed their occurrence. Based on current knowledge we hypothesized that B12-dependent pathways are replaced by B12-independent alternatives in course of the evolution, and finally disappear in Dikarya lineages. In fact, the ability to utilize cobalamin is either retained or lost independently from the time of phyla divergence. Surprisingly, we observed a correlation between the preservation of this ability and fungal ecology. As we observe, cobalamin-dependent pathways are more common in fungi associated with plants, than in species associated with animals and living as soil saprophytes. Correlation like this is unclear for mycoparasites. In our data set, we have three fungi representing such a lifestyle and they have different enzyme distribution. In this case, we observed a correlation that mycoparasites which have B12-dependent enzymes infect fungi that also possess such enzymes  and Fonticulida. These organisms retain a maximum of only six out of the eight enzymes, but it is worth noting they are not independent, free-living organisms.Dictyostelium discoideum with that vitamin  using PSI-BLAST  , all fungal hits were retained regardless of their sequence similarity. To get only homologs of a protein of our interest, there was a need to discard homologs from related protein families. To do this we visualized protein pairwise similarity using CLANS (Fifty-nine predicted non-Dikarya proteomes were downloaded from NCBI in October 2019  supplem online. ons = 3) . The datng CLANS  and seleIn the next step, sequences were aligned using local iterative mode in Mafft v. 3.7  . The ali2-based parametric branch supports. In the course of BA inference, four Markov chains were run for three runs from random starting trees for 107 generations, and trees were sampled every 2.5×102 generations. The first one-fourth of generations were discarded as burn-in. Then, we used the remaining samples to calculate the tree of maximum clade credibility.All alignments were trimmed with TrimAl (model = gappyout)  to removExpression of representatives of each of the protein sets (three randomly chosen homologs from each family) was confirmed by TBlastN  (defaultCreated phylogenetic trees were visualized and edited by iTOL v4 . Some baGenome Biology and Evolution online.evab043_Supplementary_DataClick here for additional data file.


Making frequent large-scale screenings for several diseases economically affordable would represent a real breakthrough in healthcare. One of the most promising routes to pursue such an objective is developing rapid, non-invasive, and cost-effective home-testing devices. As a first step toward a diagnostic revolution, glycemia self-monitoring represents a solid base to start exploring new diagnostic strategies. Glucose self-monitoring is improving people’s life quality in recent years; however, current approaches still present vast room for improvement. In most cases, they still involve invasive sampling processes , quite discomforting for frequent measurements, or implantable devices which are costly and commonly dedicated to selected chronic patients, thus precluding large-scale monitoring. Thanks to their unique physicochemical properties, nanoparticles hold great promises for the development of rapid colorimetric devices. Here, we overview and analyze the main instrument-free nanosensing strategies reported so far for glucose detection, highlighting their advantages/disadvantages in view of their implementation as cost-effective rapid home-testing devices, including the potential use of alternative non-invasive biofluids as samples sources. Early diagnostics and preventive medicine are increasingly recognized as essential tools to improve public healthcare, yet few clinical solutions are currently available for rapid biomarker analysis due to technological limitations in achieving reliable sensors for frequent screenings. Nevertheless, point-of-care (POC) devices and particularly home-testing kits have already shown their tremendous potential on public health, improving patients’ survival rate and people’s life quality ,2. In thDiabetes mellitus is a chronic, metabolic disease characterized by elevated levels of blood glucose and, according to the World Health Organization, it affects about 422 million people worldwide. Millions of diabetic patients already rely on self-monitoring of blood glucose , but theDuring the 1970s, the idea of a diabetic patient acquiring blood glucose data at home was contemplated; Anton Clemens developed the first blood glucometer combining dry chemistry test strips (Dextrostix) with reflectance photometry. During the 1980s the Dextrometer  was launched, paving the way for the extensive development of new POCs, and blood glucose monitoring became a standard . During However, most of the commercial glucometers still involve invasive sampling processes, which are quite discomforting for frequent measurements  or implantable devices that are not cost-effective ,7 and neTo reduce sampling-related discomfort and healthcare costs , the development of instrument-free rapid diagnostic devices is a fundamental step for a real breakthrough in the diagnostic field.The monitoring of health-related biomarkers by simple colorimetric in vitro tests or paper-based devices  ,9,10,11,The main advantage is that the test results do not need ad hoc reading systems, but they can be visualized by naked-eye (or with a smartphone), requiring no technical skills, and presenting the possibility of testing anywhere at any time with no need of medical personnel.This article reviews the state-of-the-art of instrument-free glucose detection with a particular focus on nanosensors and non-invasive biofluids. We describe different kinds of colorimetric sensing strategies present in the literature, including those attempting visual glucose detection in biological fluids alternative to blood. Interestingly, some of the approaches and methodologies here summarized can be potentially applied, with minimal changes, to several other biomarkers.Blood represents the most investigated biomarker source and the most employed biofluid for diagnostics, followed by urine, while saliva is only recently rising significant interest, e.g., in relation to rapid diagnostic tests for COVID19 . In addiDespite the higher glucose concentration, compared to the other biofluids, blood samples present fast coagulation, intense red color, and a very high concentration of biomolecules, leading to a higher possibility of interference-related problems or limited specificity. Therefore, in most cases, in vitro analyses are performed on serum samples, often diluted even up to 100 times.Similar to blood, urine, saliva, tears, sweat, and interstitial fluids contain a multitude of constituents (including proteins and metabolites) that reflect biological functions and are currently attracting increasing interest as non-invasive alternative biosources for rapid diagnostics ,17,18,19Non-invasive biofluids indeed present several advantages, including needle-less sampling, reduced physical discomfort and anxiety , no need for trained personnel, and consequently reduced healthcare costs, no risks related to bloodborne pathogens (safer approach), no rapid coagulation (higher stability).Furthermore, the simplicity of urine and saliva sampling (even for a relatively large amount) allows for specimen self-collection, ideal for home-testing.Glucose is typically not present in urine (or in very little concentration). However, in the case of elevated levels in the blood, its excretion into urine occurs. Commonly, when glucose concentration is above 50–100 mg/dL (2.77–5.55 mM), the urine test is considered positive for hyperglycemia .Salivary glucose concentration and its correlation with the blood one have been extensively investigated. Depending on the enrolled populations, the exclusion criteria, collection and analysis methods, different correlations have been found for salivary glucose concentration (from 10 to 100 times lower than the hematic one); however, to the best of our knowledge, the most accredited physiological glucose concentration is 1.5–4 mg/dL (0.08–0.22 mM), while values above 4–6 mg/dL (0.22–0.33 mM) represent pathological conditions .Several studies described a relationship between blood and tear glucose levels, and an increase in tear glucose level in diabetic patients was observed after oral glucose load, while no significant rise was found in healthy subjects ,23,24. DIn recent years, thanks to the improvement in sampling techniques for the extraction of biological fluids by absorbent patches and microneedles, sweat and ISF are also emerging as promising alternatives for glucose detection. Sweat glucose has been reported to correlate well with blood level, with a concentration in the 1–4 mg/dL (0.06–0.22 mM) range , while bTo cope with the low glucose concentration in non-invasive fluids and the ultrahigh sensitivity needed for rapid naked-eye detection, the use of nanoparticles (NPs) represents a great asset to the development of cost-effective kits.Nanoparticles (NPs), in particular noble metal NPs, hold enormous potential for a new generation of highly sensitive instrument-free colorimetric biosensors ,33,34,35Clearly, given the LSPR sensitivity to the dispersion state and shape variation in in vitro colorimetric tests, the chemical and colloidal stability of plasmonic NPs is of utmost importance.3O, and others) and carbon-based NPs  have also emerged as promising candidates to substitute the natural enzymes commonly used in the diagnostic assay . A. A2O2 th2O2 formation, which reacts with HRP generating hydroxyl radicals ·OH, which promotes oxidative etching . F. F2 two-2 nanozymes was performed both in vitro and using a plasma separation pad. In this work, blood samples were analyzed without pre-treatments or dilutions. Linking the GOx to the nanozyme (acting as peroxidase), the system performed an enzymatic tandem reaction in one single step. The consequent reaction with a chromogenic substrate (TMB) led to visual detection of glucose in a few minutes (LOD = 0.17 mM)  propyl trimethoxy silane (AEPTMS) and octyl trimethoxy silane (OTMS) were employed to coat a glass plate, obtaining a pH-responsive substrate for glucose detection. Depending on the pH, the prepared substrate can switch from superhydrophilic to superhydrophobic in virtue of protonation/deprotonation of the NPs functional group responsible for the wettability .A drop of the sample was deposited on the assay’s substrate after incubation with GOx. When the glucose concentration increases, the GOx produces more gluconic acid, decreasing the pH, hence decreasing the drop’s contact angle and resulting in a visible change of the drop shape  with LOD = 0.32 nM. This original visual detection assay presents the advantage of being suitable for color-blind subjects. The method was validated on saliva (urine and sweat) samples from nine healthy donors and nine diabetic subjects. Nevertheless, the prolonged assay time (60 min) might represent a limit for POC applicability .As seen for the other biofluids, a relatively reliable colorimetric approach is based on artificial nanozymes combined with GOx and TMB chromogenic catalyzed oxidation reaction. In a recent work, polyethyleneimine-stabilized PtNPs presenting notable peroxidase-like activity were employed for salivary glucose detection (LOD = 0.15 mM) . PolyethA similar colorimetric strategy was employed exploiting nanoporous MOF nanozymes oxidizing TMB . The MOF2 quantum dots decorated AuNPs [2-QDs surface sulfur atoms allow for strong Au-S bond formation, increasing the stability of AuNPs and promoting the charge transfer, enhancing the catalytic activity. All reagents were embedded in agarose gel as a portable kit. The colorimetric assay was validated for glucose detection on pre-treated  and diluted saliva (tears and serum) samples. The reported assay time was 40 min.Another GOx-nanozyme-TMB sensing approach was developed by using MoSed AuNPs . MoS2-QDRecently, a paper-based device involving plasmonic AuNPs was shown to be effective on whole saliva samples. Unlike the previously discussed etching-based approaches, accompanied by loss of color intensity due to significant nanostructure corrosion needed to obtain naked-eye appreciable color change, this example relies on an optical density conservative reshaping process .2O2 and halogen ions , undergo rapid structural reorganization, leading to nanospheres, passing from blue to red color in 10–15 min  were investigated as a fluorescent sensor. Firstly, AuNPs were synthesized with activated dextran as a reducing and stabilizing agent and conjugated with aminophenyl boronic acid (ABA), forming a Schiff base. Then, the fluorophore was chemisorbed on AuNPs surface via hydrogen bonding. RBITC presents intense fluorescence properties in the visible range but is quenched by AuNP proximity. Glucose can form covalent bonds with boronic acid, displacing the fluorophore that, once free, can emit the light signal. This strategy was applied on real tear samples of five female volunteers with the onion-induced method. However, despite the fluorescence in the visible range and the potential for visual/smartphone detection, the naked-eye detection was not investigated in this study .On the contrary, a similar strategy exploiting graphene quantum dots (GQDs) was shown to give a visually appreciable response . GQDs ar2O2 from glucose. The visual detection was achieved via nanozyme-mediated catalytic oxidation of TMB, leading to a blue color solution with a glucose concentration-dependent intensity. In vitro testing was performed on real tear samples containing glucose spikes by the addition of the nanohybrid system solution (plus GOx and TMB), and the color change was obtained in 5 min. The assay was also tested in saliva but was better performing in tears. A translation of the colorimetric systems on a paper-based microfluidic device was also done, depositing all the reagents in line in physically separated spaces on the substrate. However, the paper-based system was only tested in buffer solution.Nickel and nitrogen-doped graphene nanotubes (GNTs) decorated with Pt-nanozymes were employed for visual tears  glucose detection. While nickel is supposed to improve the catalytic activity of PtNPs, nitrogen-doping was done to improve and promote the PtNP assembly with the nanotubes. Therefore, the PtNPs on the nanotube surface gained stability and enhanced the peroxidase catalytic activity . GOx wasTo perform glucose measurements in sweat, it is essential to employ wearable devices, like skin patches. Among the sweat glucose detection strategies, NPs-based electrochemical wearable sensors are the most explored ; howeverSaliva Glucose Nanosensors section. This device was validated with real sweat samples from nine healthy donors and nine diabetic subjects. However, the long assay time (60 min) could limit the sensor applicability.One previously mentioned example of naked-eye nanosensor performing in saliva and sweat involving a silica NPs-based pH-sensitive substrate  reacting2O2, resulting from the GOx oxidation of glucose, in the presence of Fe2+ (by Fenton reaction) transforms into hydroxyl radicals that quench the bidots fluorescence. However, the fluorescence quenching related to increasing glucose concentration was only estimated by spectroscopy, and the assay only tested on artificial sweat.The detection of glucose in sweat was also achieved using bioinspired fluorescent nanodots (bidots) non covalently conjugated with glucose oxidase . The H2O2O2 leads to the oxidation of PSi. The fast release of Si, further enhanced by the bimetallic NPs that increases the electron transition efficiency, allows for a change in the fluorescence from red to blue, shown to be easily distinguishable by naked-eye . The wearable skin pad fabrication was achieved by transferring the system on a transparent and biocompatible chitosan film. This system was tested in vivo on the back area of the neck of some volunteers before and after sleeping. The strategy presents limitations in clearly distinguish samples by naked-eyes because of the low difference in the amount of glucose in sweat. Nevertheless, the detection was achieved with a smartphone presenting UV integrated light source.Real sweat samples analysis was achieved with a wearable skin pad involving hybrid nanocomposites made of porous silicon nanoparticles (PSi), plasmonic nanoparticles, and CQDs . The redIn This work presented an overview of the main instrument-free NPs-based strategies employed to date for glucose detection in biological fluids and their potential (and limits) for developing cost-effective rapid POC devices.The advantages and drawbacks of non-invasive biofluids in this context were taken into account, including the impact of their biomolecular interferent, the different glucose concentrations, and the suitability for home-testing.In most cases, we believe that the real potential of the described colorimetric strategies as portable sensors would probably be enhanced relying on their translation from in-solution in vitro tests, often requiring multiple steps, to single-step paper-based devices. The examples based on such an approach were the most promising. Since most of the reported methods were designed for blood analysis but tested in diluted (1:10–1:100) serum to avoid interference, it would be interesting to adapt them and test their potential applicability in other non-invasive biological media and/or implement them with LFDs (exploiting rapid in-situ membrane-based filtration).The unique physical-chemical properties of NPs can allow overcoming the difficulties related to the detection of glucose content in non-invasive fluids, especially if aiming at rapid screening of hyperglycemia involving an ON-OFF approach for a set range of critical glucose concentrations. However, to the best of our knowledge, there are currently no instrument-free methods tested by statistically relevant clinical trials, able to perform quantitatively. Nevertheless, etching/reshaping of anisotropic plasmonic nanoparticles (presenting concentration-dependent multiple color change) and the nanozyme-colorimetric substrate systems held good promises for semi-quantitative naked-eye home-testing devices, even if the second one should probably rely on smartphone-assisted measurements (since mainly accompanied to color intensity changes).In conclusion, while we are not yet ready to get rid of instruments for glucose monitoring, we believe that shortly, with some further effort in this context, there will be several concrete possibilities for non-invasive tests based on colorimetric nanosensors, which could significantly impact and improve people’s life quality and healthcare costs.


The SARS-CoV-2 outbreak is the COVID-19 disease, which has caused massive health devastation, prompting the World Health Organization to declare a worldwide health emergency. The corona virus infected millions of people worldwide, and many died as a result of a lack of particular medications. The current emergency necessitates extensive therapy in order to stop the spread of the coronavirus. There are various vaccinations available, but no validated COVID-19 treatments. Since its outbreak, many therapeutics have been tested, including the use of repurposed medications, nucleoside inhibitors, protease inhibitors, broad spectrum antivirals, convalescence plasma therapies, immune-modulators, and monoclonal antibodies. However, these approaches have not yielded any outcomes and are mostly used to alleviate symptoms associated with potentially fatal adverse drug reactions. Nanoparticles, on the other hand, may prove to be an effective treatment for COVID-19. They can be designed to boost the efficacy of currently available antiviral medications or to trigger a rapid immune response against COVID-19. In the last decade, there has been significant progress in nanotechnology. This review focuses on the virus’s basic structure, pathogenesis, and current treatment options for COVID-19. This study addresses nanotechnology and its applications in diagnosis, prevention, treatment, and targeted vaccine delivery, laying the groundwork for a successful pandemic fight. This COVID-19 has quickly turned an epidemic into a pandemic. SARS-CoV-2 is a spherical encapsulated virus with a diameter of 60–140 nm that contains positive-sense single-stranded RNA [(+) ssRNA] [The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was responsible for the recent coronavirus 2019 outbreak (COVID-19) [) ssRNA] . COVID-1The [(+) ssRNA] interacts with N-protein to produce a helical nucleocapsid . Fever, SARS-CoV-2 is a crown-shaped, single-stranded RNA virus with active-sense single-stranded RNA. It belongs to the Coronaviridae family and has a diameter range of 600–1400 Aº . An elecBy creating a nucleocapsid, the N protein aids in the replication process in the host cell. The three transmembrane segments of the M protein are crucial for the virus’s shape, size, and replication. The E protein plays a vital part in the virus’s pathogenic processes and the various stages of replication in infected cells . In connThe pathogenesis of the COVID-19 virus was disseminated to the host body by attachment, penetration, biosynthesis, maturity, and release of virus are all steps in the process . The virCOVID-19 has yet to receive a clinically recognized therapy. So far, the therapeutic options currently used have been aimed at minimizing and managing symptoms. A few antiviral medicines are now being tested in clinical trials. These medications may operate as immunomodulators or as antiviral agents directly. Since the SARS-CoV-2 outbreak, this antiviral drug research has continued. COVID-19 control treatments are primarily focused on the disease’s pathogenesis. If targeting is done in essential viral proteins to upgrade virus internal structure and reproduction, medicines may be classified as virus targeting antivirals (VTA). If the target is a protein located inside the sick host, they may target antivirals (HTA) . The phaTo date, various standard diagnostic methods have been used to detect SARS-CoV-2 infection. RT-PCR has long been considered the gold standard for detecting RNA viruses. This methodology was developed by the Centers for Disease Control and Prevention (CDC) in the United States. The RT-PCR test examined sputum, bronchoalveolar lavage, nasopharyngeal swab, pharyngeal swab, and saliva samples . The eleIn comparison to RT-PCR, point-of-care is a revolutionary diagnostic procedure that requires relatively little time. It assists physicians and medical workers develop suitable quarantine procedures for positive patients, allowing them to receive urgent medical attention and prevent the disease from spreading further. Point-of-Care techniques are classified as either serological antigen or molecular detection methods, with the former detecting COVID-19 in blood via lateral flow immunoassay and colloidal gold immunochromatography, and the latter detecting COVID-19 in nasopharyngeal swab, throat swab, saliva, and nasal swab via PCR technique. WHO  now recommends using these new point-of-care immunodiagnostic tests solely in research settings, based on current evidence. They should not be utilized in any other situation, including clinical decision-making, unless there is sufficient evidence to justify their use for specific reasons .The FDA has approved the use of a lateral flow immunoassay-based point-of-care test to diagnose COVID-19 (EUA). One test, developed by Cellex Inc., is able to detect COVID-19 IgM/IgG antibodies to SARS-CoV-2. This method can be processed in very short time and used for the qualitative identification and discrimination of IgM and IgG antibodies of SARS-CoV-2 . The othThis is a quick and straightforward detection approach that is commonly utilized for illness diagnosis ,30. With(a)amplification of target nucleic acid,(b)amplification of probes that interacts with the target nucleic acid,(c)amplification of signals obtained from target nucleic acid .In a disease state, a nucleic acid-containing pathogen in samples is present in few numbers so amplification is needed to detect pathogens ,53. The (a)branched DNA technique,(b)tyramide signal amplification,(c)hybrid capture,(d)cleavage-based signal amplification .The amplification techniques are designed into four different groups:The principle involved in the branched DNA technique is detecting a signal from target nucleic acid after immobilization and then amplification with the present multiple branched and labeled probes. In case of tyramide signal amplification, the hybridization of target nucleic acid and a biotinylated probe take place. It further promotes the complex of the formation of nucleic acid- biotinylated probe. Streptavidin containing hydrogen peroxidase is incorporated into the medium which binds to nucleic acid-biotinylated probe complex. The addition of inactivated tyramide as a substrate resulted in the formation of activated tyramide and precipitation takes place, precipitate amount indicates signal amplification . The hybviz RT-PCR and RT-LAMP  [Molecular methods for detecting SARS-CoV-2 are mainly based on the proteomic and genomic composition of the virus. Two types of nucleic acid composition are currently available ication) . Three mication) .The loop-based isothermal amplification was created to obtain a test result that was quick, accurate, and cost-effective. Instead of the thermal cycle used in RT-PCR, amplification of the target site occurs without changing the temperature reaction . The advThe molecular methods are effective and sensitive for the diagnosis of COVID-19. However, it has some limitations like sampling failures, complex protocols, and expensive equipment, and these methods may sometimes give false negative results. Immunological assays are preferred to mitigate these limitations, while this technique requires comparatively more straightforward sampling with fewer technical experts and inexpensive equipment. Besides this, immunological assays are also used for antigen detection as an alternative for RT-PCR . ImmunolAfter the virus infection has subsided, the body develops an immune response by manufacturing specific antibodies in reaction to the harmful organism. Antibody-based assays help diagnose this immune response, as well as detecting the earlier SARS-CoV-2 infection. Since the body takes time to elicit a reaction, antibody tests are rarely used to detect acute infections in their early stages because the body has not yet fully adapted to the associated antigen, which will require time to detect by establishing an immune response. As a result, antibody testing will yield false-negative findings at that time due to the lack of antibodies despite the presence of disease. Due to their short-lived nature, IgM antibodies were the first antibodies generated after a virus attack, and their identification necessitates a likely active or recent infection. Aside from that, IgG is the primary antibody that gives long-term protection against re-infection with the same virus  As a resEarly and precise diagnosis of the disease and adequate quarantine conditions for both symptomatic and asymptomatic individuals are key factors in limiting the COVID-19 pandemic . Since aAt present, nanotechnology has gained much attention for detecting the novel coronavirus disease due to some known problems with the current diagnostic techniques available . The curInstrumental RT-PCR diagnostic approaches are currently limited due to procedures that use isothermal conditions for nucleic acid amplification. Loop-mediated isothermal amplification is one of them, and it is thought to be a reasonably sensitive technique for detection ,68.−1 with this approach [Chen et al. investigated magnetic nanoparticles by using RT-PCR procedures to isolate SARS-CoV-2 viral RNA. For collecting RNA on its surface with carboxyl groups, these nanoparticles use poly(amino ester). For RT-PCR diagnosis, the carboxyl group is used in a complex of RNA magnetic nanoparticles. This procedure takes very little time and poses very little danger. Magnetic nanoparticles, rather than extraction, were utilized to isolate viral particles in another investigation. Because of the functionalized target receptors, the virus particles get sturdily linked to the nanoparticles, according to the findings. SPIONs (Superparamagnetic iron oxide nanoparticle) were used for this, as their external magnet aids in the isolation of virus particles. The other techniques such as quantitative Reverse transcription—polymerase chain reaction (qRT—PCR) assays, cell triggering assays, and immunochromatographic strip tests can be used to diagnose the virus . Roh et approach .Because it is a quick testing approach that eliminates the need to transfer samples to a laboratory, point-of-care testing is commonly employed in remote regions with limited facilities and infrastructure. The biosensors utilized are known as colorimetric biosensors because they detect color changes ,72. Acco2O3 nanowire sensors and fibronectin-based antibodies. In comparison to antibodies and aptamers, antibodies resemble proteins with a higher binding capacity. The virus is detected by changes in the electric current, which are noticed as a signal [Biosensors based on electrochemistry are thought to be one of the utmost effective tools for detecting the COVID-19 virus. A modified electrochemical sensing technique with gold nanoparticles maintaining the biomolecule-based functional moiety was used in this approach . Layqah a signal .It has been reported that Chiral biosensors are utmost valuable technologies for diagnosing SARS and COVID-19 virus. According to a recent report, the COVID-19 virus was diagnosed using opened chiral zirconium quantum dots. In this concern the Zirconium quantum dots and magnetic nanoparticles interact with the virus in this manner, resulting in a magnetoplasmonic glow where the virus is present. The magneto plasmonic-fluorescent nanohybrids were then isolated using external magnets, and the virus was detected by measuring the fluorescence intensity. It has a 79.15 EID/50 L detection limit . A chiraFor COVID-19 treatment, nanotechnology-based treatment has applications in membrane fusion, inhibition of virus-cell interaction, transcription, translation, viral replication, cell internalization, and activating such mechanism that causes irreversible damage to viruses. Nanotechnology-based treatment has drawn attention because of its properties. Some of the nanoparticles are discussed below. The respiratory chain can be harmed by silver nanoparticles (AgNPs), and the electron chain enzymes particularly useful in combating several viruses . AgNPs’ In in vivo and in vitro investigations, AuNPs with long linkages of sulfonate undecanesulfonic acid (MUS) and mercaptoethanesulfonic acid (MES) demonstrated irreversible deformation in various viruses, including respiratory syncytial virus. AuNPs combined with MUS, which is present as a multivalent binding with the virus, are exposed in a mechanistic interaction. It also causes the capsid’s structure to collapse. As a result, this multivalent binding provides an engrossing course of action for COVID-19 therapy. AuNP was also employed against an RNA virus in one investigation. Another study discovered that the nanoparticle technology reduced membrane fusion caused by MERS-CoV. It also demonstrates the possibility of curing COVID-19 .The Mesoporous silica nanoparticles (MsNPs) have different pore sizes that enable molecules to accommodate outside and inside for co-delivery. The dual nature provides an outstanding manifesto for treating COVID-19 infections. These nanoparticles interact with the various ligands to stop the viral uptake to the host cell and the target-specific liberation of those ligands, it also prevents replication of the virus. Lee et al. documented significant dual delivery characteristics of the MsNPs and showed improved antiviral activity .Antiviral properties of iron oxide nanoparticles (IONPs) are particularly depending on the alteration of the surface with sulfonates or other compounds. Bromberg et al. formulated core-shell silica magnetic nanoparticles containing poly (hexamethylene biguanide) functionalization (PHMBG) for improved antiviral activity. On different strains of the virus, NPs had a differential virucidal effect. It happened as a result of the genetic composition’s amino alkylation. It also prevents gene replication, resulting in viral deactivation. Because many cases of COVID-19 have demonstrated iron dysregulation , IONPs nCarbon-based nanoparticles (CNPs), such as graphenes, carbon nanotubes (CNTs), and fullerenes nanoparticles, have intriguing physicochemical features that can be used for innovative industrial and scientific applications . When itDendrimers represent a significant advancement in nanotechnology since they can improve the efficacy of bioactive substances and medications. Dendrimers are synthetic nanoarchitects that are well-defined and highly branching . It has Because of their excellent biocompatibility and biodegradability, lipid-based nanoparticles have a large potential to be used in COVID-19 clinical situations . Many liPolymeric nanoparticles are used to combat virus-borne illnesses, because they can be arranged to travel to specific intracellular or extracellular targets and impede virus attachment to the host cell receptor. These features are essential to stop viruses from causing diseases and to eradicate their negative consequences . The useSince the breakout of the new coronavirus, hand hygiene has been an essential tool in the fight against it. While the virus is transferred directly by coughing and sneezing, a study indicated that poor hand hygiene is responsible for 42% of infections. Hand hygiene is especially crucial in hospitals, where 40% of nosocomial infections are caused by poor hand hygiene . As a reSimilarly, a company named FN Nano Inc. in the United States has developed a titanium-based photo-catalytic coating that, when exposed to light, can destroy viruses by disrupting the viral membrane . NanomatVaccines are biological substances that stimulate the body’s immune system to produce antibodies and memory cells to resist foreign antigen particles. Immune cells generate a cascade that kills microorganisms with identical antigens, protecting the body from infection in the future. Nanoparticle-based vaccinations have diverse functionalities that can be tailored to specific challenges presented by microbes by boosting cellular immunity ,82,117.® unique recombinant nanoparticle vaccination technology with the spike protein and identical to immunogenic viruses [Sub-unit vaccinations are composed of structural components of the COVID-19 virus combined with molecular adjuvants given to boost immunogenicity by stimulating the immune system . Spike p viruses . In Bris viruses . The nan viruses .The antigen-antibody reaction occurs after the virus has triggered infection. After infection, the antigen encoded by the several unique nucleic acids is expressed in the host cell. The nucleic acid vaccine, a very effective vaccination technique, was developed based on the stated principle. To elicit an effective immune response, the vaccine employs chemically generated nucleic acid. Messenger ribonucleic acid (mRNA) vaccines have improved immunogenic characteristics because they can replicate the infectious process. The effects are maximized by mixing different mRNA in a single vaccination ,126. TheSeveral investigations have shown that nanotechnology is being used to improve vaccine efficacy. In recent studies, virus-like particles are appropriate for vaccine and treatment procedure development. Nano-sized virus-like particles having viral character can be effectively delivered through lymph and capillaries ,130. By COVID-19 caused a viral pandemic by impacting the global economy, health system, transportation, and all elements of human life. The capacity to detect COVID-19 infections early via a non-invasive approach can function as an epidemic tool for control. Shan et al. developed a non-invasive intelligent nanomaterial-based hybrid sensor array with multiplexed capabilities for the detection and monitoring of COVID-19-specific volatile organic compounds (VOC) mixtures from exhaled breath. The potency of the device was investigated in the case control clinical research by the healthcare agencies. The sensor of the device contains various gold nanoparticles that are coupled to organic ligands. VOC exposure causes changes in the sensing layer, which might cause the organic ligand to expand or shrink. The inclusion of inorganic nanoparticles promotes electrical conductivity production. The changes in the electrical conductivity of the device indicate the presence of COVID-19 viral agents. The stated test could assist in rapid screening of large populations due to its simplicity and high sensitivity, and it could also be utilized as a valuable technique for COVID-19 screening . The invAs the spreading of the COVID-19 virus is increasing globally, various guidelines are being issued by the World Health Organization (WHO) for disease control and prevention ,141. It To create awareness among the general public posters and brochures prepared by many organizations related to preventing the COVID-19 virus should be widely used in all areas. The native should be asked to maintain social distancing and should stay at home as much as possible unless it is necessary to go out. In a public gathering maintaining 2 m (6 feet) distance from other people should be practiced. Such social distance is designed to bring down public interactions in a community. Individuals are suffering from infections and are yet to be identified and isolated ,143. By Proper handwashing should be done. We should remove the accessories such as wristwatches, finger rings before cleaning our hands. We are followed by wetting hands with water. Then, apply soap or hand washes and rub our hands thoroughly for at least 20 s and rinsing them well, followed by drying our hands with tissue paper or a clean towel and using the same tissue to turn off the faucet. Individuals are counseled to avoid touching their face, especially the nose, mouth, and eyes. We know that cleanliness is next to godliness. Here to prevent the infection, cleaning and disinfection play a vital role. High touching areas, including door handlers, window, bedside table, and switches, should be daily disinfected with surface sanitizers or household disinfectants containing dilute beach solution. The bathroom and toilet should be cleaned and disinfected regularly using a dilute bleach solution, having one part of bleach and nine parts of water. Our daily-wear clothes and hand and bathing towels should be cleaned using laundry soap or detergent. The wearing of disposable gloves while cleaning is also advised. All these measures should be taken to avoid the spread of the virus, such as wearing of mask in public, sanitizing of hands, maintaining social distancing etc. If any symptoms arise, immediately isolate yourself, consult a physician, check for temperature twice, and take proper care. Moreover, most importantly, “do not panic”.The use of nanoparticles has proven to be transformative in the accurate detection and effective in treating diseases. Nanoparticles with reduced toxicity, chemical flexibility, electrical charge, and compact design serve to alleviate several issues with generic medication administration routes. COVID-19 can be treated by focusing on the entry and life cycle treatment. The virus’s entry into the host cell can be inhibited by preventing membrane fusion, facilitated by the virus spike protein. We can use therapeutic nanoparticles to pre-block the entry of the COVID-19 virus by reducing the S protein’s interaction to the host cell. Viruses such as HIV1 and HIV2 ,145,146,Recent research has focused on creating innovative methods for safely and effectively delivering nanoparticles into the nasal cavity as a therapy for viral infection . BecauseCapsids composed consisting of structural proteins and adjuvants derived from viruses are known as virus-like nanoparticles. The capacity of virus-like nanoparticles to generate an immunogenic motif increases immunogenicity. Virus-like nanoparticles can also act as adjuvants, and changing the adjuvants can result in a far more potent immune response than viruses ,164,165.Exosomes and cell membrane-derived nanovesicles have previously been shown to have the ability to bind and eliminate bacterial toxins ,168,169.One of the benefits of administering medicine through the nasal cavity is that it affects the nasal cavity’s mucous membrane, where the infection is most prevalent. The lungs are the next target for COVID-19 infection treatment ,176,177.Many people have died due to the COVID-19 outbreak, with casualties coming from all corners of the world. The grievous nature of this pandemic has uncovered and necessitated new strategic approaches to curbing disease transmission. Because of the low efficacy and incidence of disastrous adverse drug reactions, novel COVID-19 therapies must be developed and implemented. Working on preventative and new technologies to combat infectious pandemics, which are equally as vital as the curative approach, is crucial when dealing with pandemics. The nano-based medicines are more effective compared to the conventional dosage forms. Despite these advantages, it has certain limitation such as stability, clinical study, primarily toxicity and large-scale production, all of which provide a challenge to manufacturers. Additionally, biocompatibility, nano-biointerface, safety, and regulatory issues are among the big obstacles for the manufacturers. As a result, research must be targeted in order to overcome these complications and produce better formulations. The different groups of scientists, particularly in nanomedicine, are working on their development using bioinformatics methods. Scientists have a lot of options when it comes to regulatory research. It has the potential to gain national and international exposure. The encouraging findings can also be used to patent the works in a major way.The diagnosis of SARS-CoV-2 infection is frequently confused as either influenza or seasonal upper respiratory system viral infections. The following trustworthy, high-sensitivity, simple, rapid, and cost-effective diagnostic tools, namely RT-PCR, point-of-care, lateral flow immunoassay, immunochromatography assay, signal amplification, immunological assays, antibody-based assays, and antigen-based assay are used for the confirmation of COVID-19 infection. Furthermore, the study proved the applicability of nanotechnology-based treatment of SARS-CoV-2 employing diverse nano carriers such as inorganic nanoparticles, organic nanoparticles, dendrimers, lipid-based nanoparticles, and polymeric nanoparticles in nano formulations of antiviral drugs. When compared to traditional dosage forms, these formulations show superior therapeutic efficacy and better pharmacokinetic attributes. Similarly, nanoparticle-based vaccines, theranostic nanoparticles, nano-based intranasal delivery therapy, virus-like nanoparticles, cell-derived vesicles, and pulmonary delivery using nanoparticles inhalation aerosols all boost therapeutic efficacy significantly. While nanomedicine is a promising tool in the battle against pandemics, but it faces substantial challenges in clinical implementation, primarily toxicity and large-scale production. This review allows researchers to do study and develop better formulations for treating COVID-19 in society.


Aortic blood pressure (aoBP) waveform-derived indexes could provide valuable (prognostic) information over and above cardiovascular risk factors (CRFs). To obtain aoBP waveform-characteristics, several (i) techniques, (ii) recording sites, (iii) pressure-only waveform analysis mathematical approaches , and (iv) indexes , were proposed. An accurate clinical use of these indexes requires knowing their physiological age-related profiles and the expected values for a specific subject. There are no works that have characterized waveform-derived indexes profiles in large populations considering: (i) as a continuous, data from different age stages , (ii) complementary indexes, (iii) data obtained from different techniques and approaches, and (iv) analyzing potential sex- and body height (BH)-related differences. In addition, (v) there is a lack of normative data  for waveform-derived indexes.(1) to evaluate the association and agreement between PWA- and/or WSA-derived indexes obtained with different techniques and approaches; (2) to determine the need for sex-, BH-, and/or age-specific RIs; (3) to define RIs for PWA- and WSA-derived indexes in a large cohort of healthy children, adolescents, and adults.3619 subjects (3–90 y) were included; 1688 healthy (2–84 y). AP, AIx, AIx@75, Pf, Pb, RM, and RIx were obtained . The association and agreement between indexes were analyzed . Mean and SD equations and sex-specific BH- and age-related profiles were obtained .vs. radial) and technique . The need for sex-, BH-, or age-specific RIs was dependent on the index and/or age considered. RIs were defined for each index considering differences between recording sites and techniques. Equations for waveform-derived indexes age-related profiles were included, enabling to determine for a specific subject, the expected values and potential data deviations.Waveform-derived indexes were not equivalent; for a specific index, there were systematic and proportional differences associated with the recording site (e.g., carotid  Central aortic blood pressure (aoBP) waveform contains valuable  information beyond and in addition to the obtained from its corresponding systolic, diastolic, and pulse pressure levels  . Severaln = 65  . Howevern = 65 , n = 267 d  to evaluate the association and/or agreement between PWA- and/or WSA-derived indexes obtained with different techniques and approaches, (2) to determine the need for sex-, body height (BH)-, and/or age-specific RIs, (3) to define RIs for PWA- and WSA-derived indexes in a large cohort of healthy children, adolescents, and adults from South America. As a secondary aim, non-linear equations for sex-, BH-, and/or age-specific percentiles were determined and included (text and spreadsheet formats); the information given enables to determine the expected value for a particular subject (and to assess any possible deviation from the anticipated).The study was carried out in the context of the CUiiDARTE project , a populAll procedures agree with the Declaration of Helsinki (1975 and reviewed in 1983). The study protocol was reviewed and approved by the Ethics Committee of Centro Hospitalario Pereira Rossell, Universidad de la República. Prior to the evaluation, the participants provided their written informed consent to participate in the study. In subjects under 18 y, parents’ written consent and children’s assent were obtained before the evaluations. Subjects or parents (in case of subjects aged < 18 y) provided informed written consent to have data from their medical records used in research.Before cardiovascular evaluation, a brief clinical interview together with the anthropometric and blood test results evaluation enabled to assess exposure to CRFs. Body weight and BH were measured with the participant wearing light clothing and no shoes. Standing BH was measured using a portable stadiometer and recorded to the nearest 0.1 cm. Body weight was measured with an electronic scale  and recorded to the nearest 0.1 kg. Body mass index (BMI) was calculated as body weight-to-squared BH ratio. In children and adolescents, z-scores for BMI were calculated using the WHO software  .2 (for subjects ≥ 18 y). Arterial hypertension was considered to be present, if it had been previously diagnosed in agreement with reference guidelines and/or use of blood pressure-lowering drugs was reported. Cut-off values were: brachial systolic blood pressure (baSBP) ≥ 140 mmHg and/or diastolic blood pressure (baDBP) ≥ 90 mmHg (for subjects ≥ 18 y) and baSBP and baDBP > 95th percentile for sex, age, and BH (for subjects < 18 y). Personal and family histories of cardiovascular disease  were assessed. A family history of cardiovascular disease was defined by the presence of first-degree  and/or second-degree (for subjects ≤ 18 y) relatives with early  cardiovascular disease. History of dyslipidemia and diabetes were considered to be present if they had been previously diagnosed in agreement with reference guidelines and/or the use of lipid- or glucose-lowering drugs (respectively) was reported. Dyslipidemia was defined as total cholesterol > 240 mg/dL or high-density lipoprotein cholesterol for men < 40 mg/dL and for women < 46 mg/dL. In turn, diabetes diagnosis was based on plasma glucose levels (fasting plasma glucose ≥ 126 mg/dl). Regular (current) smokers, defined as usually smoking at least one cigarette/week, were identified.Obesity was defined as z-score for BMI ≥ 2.0 (for subjects < 18 y) or BMI > 30 Kg/mThe participants were asked to avoid exercise, tobacco, alcohol, caffeine, and food-intake 4 h before the evaluation. All hemodynamic measurements were performed in a temperature-controlled environment (21–23°C), with the subject in supine position and after resting for at least 10–15 min, which enabled reaching steady hemodynamic conditions. Using a validated oscillometric device , heart rate, baSBP, and baDBP were recorded in supine position simultaneously and/or immediately before or after each non-invasive arterial recording. Then, brachial artery pulse pressure  and mean BP  were calculated.Left and right common, internal, and external carotid arteries, vertebral artery, common femoral artery, and left brachial artery were examined . Transverse and longitudinal arterial views were obtained to assess the presence of atherosclerotic plaques  .Left and right brachial and tibial systolic and diastolic blood pressure levels were obtained (no fixed order) at 5 min intervals . At least five measurements were obtained from each recording site. The Ankle Brachial Index, an index of arterial permeability and central-peripheral blood pressure amplification, was calculated as tibial systolic blood pressure/baSBP . Right aCentral aoBP levels and waveforms were obtained (random order) using commercially available devices: SphygmoCor-CvMS  and Mobil-O-Graph PWA-monitor system .Using SCOR, aoBP waveforms were derived from, (i) radial artery  and (ii) carotid artery (directly) manual tonometric recordings . CarotidBoth devices (SCOR and MOG) enable PWA and WSA. A detailed (step-by-step) explanation of the method used for WSA based on recorded  and mathematically derived aortic waveforms (SCOR and MOG) was included as Using both devices (SCOR and MOG) and the three recording sites , we quantified: (i) AP, AIx, AIx@75 and (ii) Pf, Pb, RM, and RIx . Recordevs. Pf_Carotid_SCOR vs. Pf_Brachial_MOG).The variables were named based on the (i) waveform-derived index, (ii) recording site, and (iii) device  without any of the following  carollowing .vs. Pf_Carotid_SCOR vs. Pf_Brachial_MOG), we analyzed the degree of association and agreement between data by assessing: (i) concordance correlation coefficients and (ii) mean and proportional differences   . Figure n = 42), even in the case that sex-variable was significant, we defined RIs for the whole group .Fourth, we evaluated whether age-, BH-, and/or sex-specific RIs were necessary using multiple linear regression models that included interaction analysis  with Johnson-Neyman significance regions definition –10. Varip + c*Ageq + .,” where a, b, c, are the coefficients, and p and q are the powers, with numbers selected from the set  estimated from the regression for mean AIx@75_SCOR_Radial curve, and likewise from the regression for SD curve. Fractional polynomials with powers , that is, with p = 1 and q = 2, illustrate an equation with the form a + b*Age + c*Age2 . Then the differences varied in magnitude depending on the index values . The higvs. SCOR_Carotid vs. SCOR_Radial). For instance, Pb_MOG was independently associated with sex and BH, but not with age. In turn, Pb_Radial_SCOR and Pb_Carotid_SCOR showed independent associations with age and BH (but not with sex). AIx@75_MOG was independently associated with sex and age (and BH), without age-dependent changes in the association with sex (non-significant Johnson-Neyman regions).Age, BH, and/or sex-specific RIs for a given waveform-derived index  may (or may not) be required, depending on the approach used for its measurement  considered, RM and RIx were not independently associated with sex. However, for SCOR_Radial and SCOR_Carotid records, in subjects aged 17 years and older, sex moderated the relationship with age.Regardless of the measurement method, almost all waveform-derived indexes showed an independent association with age and BH (and/or an association through their interaction with sex). Consequently, both age- and BH-related RIs were necessary. In contrast, we identified waveform-derived indexes that: (i) required sex-specific RIs only from a certain age  or BH , (ii) required sex-specific RIs regardless of age or BH , (iii) did not require sex-specific RIs , or (iv) did not require sex-specific and/or BH-related RIs  –11.Year-by-year (for age) and decimeter-by-decimeter (for BH) RIs data can be found in Comparisons of data from this study , with data obtained by other authors , are shown in Considering their demonstrated value , there is growing interest in assessing central BP waveform-derived indexes in clinical practice. Their accurate clinical use requires knowing their physiological age-related profiles and the expected values for a specific subject. To our knowledge, this is the first time RIs for different waveform-derived indexes (obtained from different widely used measurement approaches) were defined (at the same time) in large population of healthy children, adolescents, and adults (2–84 y). The main findings can be summarized as follows:r ≥ 0.80)  showed mostly little  association with each other. Furthermore, in no case, the association was very strong ( ≥ 0.80) . Methodo ≥ 0.80) .Second, the need for sex-specific RIs relied on both physiological  and non-physiological factors  . Then, tThird, the population-based RIs for waveform-derived indexes were defined from data obtained in the same group of healthy children, adolescents, and adults –139. DefFourth, aiming at contributing with other groups and/or researchers, sex-specific BH- and age-related equations for mean value, SD, and percentile values were included in text and spreadsheet formats . Thus, tThe age-related profiles obtained for WSA-derived indexes showed similarities and differences with data reported by other authors , 4. HoweRegardless of the methodological approach considered, the Pf increased in childhood and adolescence, reaching a maximum at ∼20 y. Thereafter, it remained stable and finally showed a tendency to increase after 60–70 y . At leasTaking into account the above mentioned, the differences in Pf values among studies could be explained  by differences in the calibration methods, methodological approaches, and/or subjects considered.Similarly, Pb showed a significant rate of increase in the first decade of life, and then it continued to increase steadily throughout life. The age-related profiles obtained for adults are in agreement with data from other works. However, the age-related increase in Pb observed in this work was (apparently) smaller compared with data from other works. Furthermore, for young subjects , data from this and other works overlapped, but the maximum values reported by other authors for old subjects  were almost always higher than the observed in this work . The difJointly considering the above factors, it could be said that more flattened curves would be expected for both Pf and Pb in adults without cardiovascular disease and minimally exposed to traditional CRFs. Finally, the differences in the rate of Pf and Pb change observed in adults in this work are consistent with data from other studies , 2016.The age-related changes in RM (Pb/Pf) and RIx [Pb/(Pf + Pb)] showed great heterogeneity . Both RMData from multiple linear regression models  and age-The above data should be carefully analyzed. Whereas we (and other authors) analyzed Pf and Pb and absolute values, and other authors analyzed waveform components in terms relative to aoPP. In subjects younger than 60 y of age, Namasivayam et al. found that both incident and reflected waves showed a greater contribution to the age-related increase in aortic pressure in females than in males . Our curThe similarity observed in girls and boys agrees with previous works that reported no sex-related differences in structural and functional arterial parameters at prepubertal ages (4–8 y), but showed sex-related differences in adolescents (∼15 y) .Regardless of the methodological approach considered, and with independence of sex and age, Pf and Pb were positively associated with BH . In turnn = 65; 21–78 y; 43 male) evaluated with carotid tonometry. However, the results were alike when considering the association of BH with RM and RIx data obtained with SCOR_Carotid without adjusting for cofactors . This mathematical phenomenon could contribute to the “flattening” of age-AIx curve (described by other authors)  .Finally, it is to note that only few authors reported reference data or age-related profiles for AIx@75. The available works described an age-related increase, but whereas Gómez-Sánchez et al. showed a significant constant increase in AIx@75 with aging (like in this work), and Solanki et al. found a slight age-related increase .The augmentation pressure levels tended to be higher in females than in males , but theRegardless of BH, compared to males, females showed higher AIx and AIx@75 : (i) forWhen analyzing the (simple) association between AP and BH data, no clear trend was observed . HoweverOur results should be analyzed in the context of the work’s strengths and limitations. First, since this is a cross-sectional study, it provides no data on longitudinal age-related variations in waveform-derived indexes. Although useful for quantifying RIs, our data do not allow to determine with certainty the true impact of aging on arterial system properties . Second,Fifth, as a strength, in this study, waveform-derived indexes were obtained in a large population sample  that included subjects within a wide age-range , as a continuum. This would contribute to understand the arterial pulse propagations and their impact on waveform-derived indexes throughout life, providing important information for clinical diagnosis and cardiovascular research. To our knowledge, this is the first study of its sort in South Americans. Sixth, despite we previously demonstrated that aoBP and some waveform-derived indexes could vary depending on the calibration schema considered , in thisThis study adds to the knowledge of the physiological variations in waveform-derived indexes and arterial pulse propagative properties that would be expected during growth and aging, analyzing at the same time (and comparatively) the behavior of different indexes, obtained with three different approaches. Our data showed that the methods used to quantify aoBP waveform-derived indexes  showed little association with each other. Waveform-derived data from different approaches were not equivalent, but showed systematic and proportional errors. These results evidenced that the non-invasive methodological approach used is an important determinant of the results . Our study strongly emphasizes the need for consensus on non-invasive assessment of waveform-derived indexes.There were not uniform behaviors that standardize the need for sex-related RIs (normative data), but the need for sex-specific waveform-derived indexes RIs relied on the index and/or age considered. Population-based RIs for waveform-derived indexes were defined from data obtained in the same group of healthy children, adolescents, and adults. Aiming at contributing with other groups and/or researchers, sex-specific BH- and age-related equations for mean value, SD, and percentiles values were included in text and spreadsheet formats. Thus, the expected values for a given subject could be calculated for clinical and/or research purposes.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Comité de Ética de Investigación, Centro Hospitalario Pereira Rossell, ASSE, Universidad de la República. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.YZ and DB contributed to conception and design of the study, performed the cardiovascular recordings, constructed and organized the database, performed the statistical analysis, wrote the first draft and final version of the manuscript, contributed to manuscript revision, read, and approved the submitted version. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Adaptive T-cell immunotherapy holds great promise for the successful treatment of leukemia, as well as other types of cancers. More recently, it was also shown to be an effective treatment option for chronic virus infections in immunosuppressed patients. Autologous or allogeneic T cells used for immunotherapy are usually genetically modified to express novel T-cell or chimeric antigen receptors. The production of such cells was significantly simplified with the CRISPR/Cas system, allowing for the deletion or insertion of novel genes at specific locations within the genome. In this review, we describe recent methodological breakthroughs that were important for the conduction of these genetic modifications, summarize crucial points to be considered when conducting such experiments, and highlight the potential pitfalls of these approaches. Its important parts are T cells characterized by the expression of a unique clone-specific cell-surface protein complex, the T-cell receptor (TCR). T-cell-mediated immunity is induced by TCR recognition of peptide antigens derived from intracellularly processed proteins and presented in the context of major histocompatibility complex molecules on the surface of antigen-presenting cells. Rare activated antigen-specific T cells initially differentiate and proliferate in the lymph nodes before traveling to the site of inflammation to exert their effector functions. There, cytotoxic CD8+ T cells. Immunodeficiency can also be a consequence of malignant tumors. Tumor cells, if they evade initial immune surveillance, create a microenvironment that obstructs and hinders antitumor immune responses [Not surprisingly, individuals with a compromised T-cell immunity, due to either primary immune defects or immunosuppression, are highly susceptible to a variety of microorganisms, which often induce severe disease. For example, human cytomegalovirus (HCMV) infection in healthy individuals is usually asymptomatic. However, in immunosuppressed organ-transplant patients, HCMV induces a severe disease, reducing transplant survival and leading to increased mortality ,2,3. Besesponses . To suppesponses ,6. Upregesponses ,6,7.Another promising approach for treating immunocompromised patients that lack functional T-cell responses has been the adoptive transfer of T cells. Transferred T cells provide protective immunity against harmful complications caused by infectious diseases or malignant tumors . For Of note, the first two CAR-T products approved by FDA were produced by lentiviral (Kymriah) or gamma-retroviral (Yescarta) transduction , vectorsFor precise manipulation of specific genomic sites, several genome-editing methods have been developed, including zinc-finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), and the CRISPR/Cas system  (reviewed in Reference ). ZFNs aSimilar to ZFN, TALENs contain the bacterial cleavage domain Fok1 to induce DSBs. However, in the case of TALENs, the DNA-recognition domain is obtained from a transcription factor produced by plant pathogenic bacteria ,38,39. IWhile both ZNFs  and TALEStreptococcus pyogenes [The CRISPR/Cas system originated from a defense mechanism originally found in bacteria and archaea to counteract infections with viruses known as bacteriophages ,46,47,48pyogenes . Cas9 wapyogenes . Furtherpyogenes . Cas9 ispyogenes , allowinTargeted by a crRNA to a specific location within the genome, Cas9 cuts the DNA 3 nucleotides (nt) upstream of the PAM sequence. The resulting Cas9-induced DSB initiates eukaryotic cell-repair mechanisms that prevent cell death due to the loss of genetic material (reviewed in detail in Reference ). Two maIf the Ku protein is not expressed, the resection machinery exposes extensive single-strand DNA (ss-DNA) on both sides of DSBs, allowing the cells to use microhomology-mediated end joining (MMEJ) or HDR for DNA repair. MMEJ is an alternative NHEJ mechanism that is initiated if the DNA on both sites of the DSB contains microhomologous sequences. Annealing of those sequences leads to the removal of heterologous overhangs, fill-in synthesis, and ligation (reviewed in Reference ). In conNHEJ ligates the blunt-end DNA fragments fast and independent of the cell cycle and is, therefore, the favored way for DSB-repair in mammalian cells ,73. In cFor successful application of CRISPR/Cas-mediated genome editing by using either the NHEJ or HDR pathway, several practical aspects appear to be critical.Unless stimulated, isolated T cells survive in vitro only for a few days. While human T cells can survive this period without any cytokines added to the medium, mouse T cells require IL-7, which upregulates the expression of the anti-apoptotic protein Bcl-2 and maintains their viability . During Unstimulated T cells are in a resting state, and many genes are not actively transcribed. It has been shown that Cas9 binding is affected by DNA packaging. Actively transcribed genes can be more efficiently edited than non-actively transcribed genes where the DNA is packaged in heterochromatin (reviewed in Reference ). SimilaGene editing via CRISPR/Cas-mediated HDR is considered not to be feasible in resting cells, which are in the G1 and not the S/G2 phase of the cell cycle ,78. HoweMost gene-editing experiments were performed on in vitro activated T cells, and a large number of divergent experimental protocols used to activate T cells in vitro have been reported in the literature. The most frequently used protocols involve non-specific T-cell activation, using anti-CD3 (and anti-CD28) antibodies coated on the surface of culture vessels or provided pre-bound to beads . PerformAnother point that needs to be taken into account when performing CRISPR/Cas experiments is the efficacy and specificity of the respective gRNA. To evaluate on-target efficacy, numerous computational tools for gRNA scoring have been developed and recently reviewed ,101. DueBesides efficacy, the off-target activity of the CRISPR/Cas9 system is still a great concern, especially regarding its clinical applications. Therefore, the detection of off-target effects is essential for the estimation of the precision of gene-editing, as well as for improvement. Off-target effects can be detected via different methods, including the T7 endonuclease I assay (can detect off-target mutation frequencies <1%) , deep seBesides selecting an in vitro protocol for T-cell activation, it is also important to consider in which form the CRISPR/Cas9 components get delivered into the target cells. In the case of gene-knockout experiments, gRNA and Cas9 can be delivered as plasmids, as mRNA encoding for Cas9 with separate delivery of gRNAs, or in the form of ribonucleoproteins (RNPs). Initially, Cas9 and the gRNA were incorporated into plasmids for cell delivery. However, delivery of high amounts of plasmid DNA can also have toxic effects , mostly Several current reports used preformed RNPs that allow for a faster onset of action and reduced off-target DNA cleavage without risk of integration into the genome ,114,115.Besides Cas9 and gRNA, an HDR template also has to be provided to the cells to incorporate genetic material by using the CRISPR/Cas system. HDR templates can be provided as a single-stranded DNA (ss-DNA), long linear or circular (plasmid) double-stranded DNA (ds-DNA), or AAV-incorporated DNA molecule . All thrExisting delivery approaches can broadly be divided into viral and non-viral strategies. Viral strategies use transduction of cells with viral vectors to deliver DNA, encoding the sequence of the Cas protein, as well as of the gRNA. The most frequently used vectors are derived from ADVs, AAVs, or lentiviruses. While lentiviral vectors for CRISPR/Cas components carry the oncogenic potential, the use of adenovirus (ADV) and adeno-associated virus (AAV) vectors is accompanied by concerns regarding specific ADV immunity in humans, which could compromise the engraftment of those cells in clinical settings (reviewed in Reference ). TherefThe literature reports an increasing arsenal of non-viral delivery strategies that successfully supply components of the CRISPR/Cas system into the cells In contrast, electroporation and nucleofection allow efficient delivery of different types of nucleic acids to T cells. Both techniques use electrical current to induce pores in the cell membrane that allow cargo entry into the cells by diffusion and/or movement along the electrical field . In contNucleofection (and electroporation) leads to a considerable loss of cell viability and also affects T-cell activation ,132. TheIn the absence of an HDR template, the cell relies almost exclusively on NHEJ to repair DSBs introduced by CRISPR/Cas9. As mentioned before, this can lead to indels in the coding region of a gene, resulting in frameshift mutations and disruption of protein function. This is why the CRISPR/Cas9 system is widely used in modern gene-editing research. It facilitates the creation of knockout cell lines or animals for investigation of disease mechanisms, as well as providing the basis for the development of therapeutic strategies.Itgb1, Itgb2, Itgb7, and Itgav). Since integrins form dimers on the cell surface, the simultaneous knockout of these four integrin genes prevents the expression of any integrin on immune cells. By using complete integrin-knockout CD4+ T cells or CD4+ T cells that lack the integrin-associated adaptor molecule Talin 1, we could establish that integrins contribute to T cells entry into the lymph node parenchyma and translocation to the T cell zone of the lymph node [Regarding T cells, CRISPR/Cas-induced knockouts have manifold applications in basic research, enabling elegant ways to study the role of individual molecules or complete signaling pathways. For example, we analyzed the mechanism of T-cell homing to the lymph nodes after intra lymphatic injection, which allows us to study the entry of T cells into lymph nodes that arrive via afferent lymph . Using amph node .+ T cells indicated that their effector functions are negatively regulated by transcription factor Fli [The CRISPR/Cas9 system can also be used as a large-scale loss-of-function screening method (reviewed in Reference ). For thctor Fli . These eIn addition to its impact on basic research, the NHEJ-mediated gene knockout by CRISPR/Cas system also opens up new possibilities for clinical applications, including cancer therapy. Since certain tumor cells express ligands for exactly these immune checkpoint molecules , the effFKBP12 with the CRISPR/Cas system [FKBP12−/− HCMV-specific T cells showed effector functions comparable to unmodified virus-specific T cells upon in vitro HCMV-peptide restimulation [DGK) to increase T-cell-receptor-mediated signaling in CAR T cells [DGK−/− CAR T cells were less sensitive to transforming growth factor beta (TGFβ) and other immunosuppressive factors, had enhanced effector functions, and were able to clear tumors in a xenograft mouse model [Knocking out genes via the CRISPR/Cas system can also be applied to render transferred T cells insensitive to immunosuppressive therapy. Immunosuppressive treatment after organ transplantation is essential for the prevention of graft rejection or destruction of the transplanted organ by the recipient’s T cells. One of the immunosuppressive drugs, tacrolimus, targets the T-cell adaptor protein FK506-binding protein 12 (FKBP12) and prevents cytokine release and T-cell activation. However, immunosuppressive treatment does not only specifically target the cells that are responsible for transplant rejection but leads to general immunosuppression. Hence, immunosuppressed patients are susceptible to a variety of pathogens, including HCMV. To overcome the problem that virus-specific T cells also become inhibited by tacrolimus upon adoptive transfer, Amini et al. created tacrolimus-resistant HCMV-specific T cells by knocking out s system . Importamulation . Therefo T cells . DGK−/− se model . In anotse model . The higse model .TRAC) gene on T cells, thus abrogating the expression of the endogenous T-cell receptor (TCR) on the cell surface [CD52 gene to render the transferred cells insensitive to anti-CD52-antibody depletion, a procedure frequently used to deplete hematopoietic leukemic cells. However, FACS analysis revealed that only 7–8% of cells completely lost the TCR and the CD52 molecule [The use of autologous T cells is often restricted by issues related to manufacturing time, scalability, and expenses caused by cell expansion. Therefore, the use of allogeneic T cells from healthy donors for adoptive T-cell therapy would offer several benefits. However, adoptive transfer of allogeneic cells causes graft-versus-host disease (GvHD), due to the recognition of host antigens by transferred T cells. Recently, Kamali et al. successfully used the CRISPR/Cas system to knock out the endogenous T-cell receptor alpha chain constant  coding gene immunodeficiency by NHEJ-mediated repair alone, possibly due to the introduction of small indels that restored an appropriate reading frame [B2m) gene can be repaired by HDR template-dependent and -independent mechanisms, as revealed by re-expression of major histocompatibility complex class I (MHC-I) on the cell surface  gene expression [F8 gene and were also fully functional in a mouse model of hemophilia [For precise gene editing, the HDR pathway can be exploited by providing an HDR template together with the gRNA and Cas9, offering an enormous potential in the field of gene therapy . Fopression . Furtherpression . Endothemophilia . Togethemophilia .TRAC gene, thus disrupting the expression of the endogenous TCR [HDR pathway can be used in T cells for research purposes, including the insertion of fluorescent protein genes (such as GPF) or bioluminescent tags . More imnous TCR ,160. ThiAs for gene knockout experiments, the most important parameter for the initiation of HDR seems to be the efficacy of the gRNA (or crRNA/tracrRNA) used . In addiFor effective HDR, it also seems to be crucial to select gRNAs that will guide Cas9 to PAM sites at the proper distance from the desired editing site . The gRNTRAC gene and replace it with a CD19 CAR with a knock-in efficiency of ≤40% [An HDR template can be in a form of ss-DNA, long linear or circular (plasmid) ds-DNA, or AAVs-incorporated DNA . Of note of ≤40% . On the  of ≤40% . However of ≤40% ,95. More of ≤40% . All of  of ≤40% .If ss-DNA are used as HDR templates, it is also important to consider whether it will be synthetized as the sequence of the genomic DNA strand containing the PAM  or as the sequence of genomic DNA strand that binds gRNA (target strand). Of note, while Cas9 globally dissociates from duplex DNA symmetrically, after cleavage, it locally first releases the non-target strand before the target strand . Hence, Irrespective of the HDR type used, it is crucial to properly design its sequences homologous to the specific sequence flanking the cut site (“homology arms”). If working with human material, special care should be taken to assure that these sequences are free of any polymorphisms that could prevent template incorporation. The length of the homology arms depends on the size of the desired change. Large insertions, such as those coding for GFP or a CAR, require templates with homology arms of a length ≥ 300 bp ,95. On tIn the case where ss-DNA templates are used, it is important to consider whether symmetric or asymmetric homology arms are used. The latter commonly have a shorter homology arm on the PAM-distal side and a longer homology arm on the PAM-proximal side of the break. The templates with asymmetric homology arms were reported to have enhanced HDR efficiency compared to their counterparts with symmetric homology arms ,165. HowRecently the group of from Alexander Marson showed that HDR efficiency can be two- to four-fold improved by adding the short truncated Cas9 target sequences (tCTS) to homology arms of ds-DNA templates. The increased HDR efficacy is a result of Cas9-mediated template shuttling into the nucleus, as RNPs can bind to, but not cleave, tCTS . SimilarIt is also important to incorporate mutations within the HDR template that prevent the re-cleavage by Cas9 once the template got integrated ,163,166.The delivery of high amounts of ds-DNA can have toxic effects, mostly due to the recognition of pathogen-associated molecular patterns within the plasmid DNA ,109. IntThe efficacy of HDR can be increased by tipping the choice for DSB repair away from NHEJ (reviewed in Reference ). This cCertain experimental protocol steps seem to be crucial for HDR-mediated CRISPR/Cas9 gene editing. First, it is important to provide the HDR template in the highest amount possible that does not induce toxic effects to the cells. This is especially important when ds-DNA templates are used and available data suggest a narrow range of 1–4 µg per reaction ,95,114. Appropriate timing of transfection or transduction after cell stimulation is an important parameter for successful template delivery into T cells. Several studies have shown that the best time is 2 to 4 days after activation, due to increased cell size and active TCR signaling ,95,115. Finally, although the expression of the integrated genetic information can be determined by using FACS or Western blot, DNA sequencing is of most importance, since some knocked-in genes resulted in protein products with different lengths that deviated from sequences coded by the template .CRISPR/Cas-mediated gene editing has revolutionized many areas of biology. In T-cell immunology, CRISPR/Cas-mediated modifications have facilitated crucial important insights into T-cell function. More importantly, they have also enabled the production of novel adoptive T-cell products for the treatment of malignant and chronic viral diseases. In recent years, we have witnessed constant optimization of experimental protocols for the delivery of CRISPR/Cas9 components to the T cells. Current protocols using nucleofection of Cas9 RNPS allow for efficacies of close to 100% for gene deletion and up to 60% for HDR template-mediated gene insertion ,128,129.


Traumatic brain injuries (TBIs) have contributed to approximately one million deaths in the United States over the last 2 decades ). Data on TBI-related deaths were stratified by state and principal mechanism of injury (cause). Injuries were grouped first by intent . Intentional injuries were further categorized as suicides or homicides. Unintentional injuries were categorized by mechanism of injury . Principal mechanism of injury was determined based on the CDC-recommended external cause of injury mortality matrix for ICD-10 codes  were based on U.S. bridged-race population estimates of the resident population . Overall, more than two in five (44.4%) TBI-related deaths were attributable to intentional causes (suicide and homicide). Suicide was the leading category or tied for the leading category of TBI-related deaths in 43 states  . Suicide was responsible for the highest number and the highest rate of TBI-related deaths for most states. The rates of TBI-related deaths due to homicide in some southern jurisdictions  were nearly double those in many states in the Northeast. A previous report found that nearly all TBI-related deaths from suicide had firearm injury as the underlying mechanism of injury  initiative includes resources and tools for health care providers that are designed to improve identification of patients at risk for a fall and support implementation of effective strategies to treat or reduce the risk for fall-related injuries, including TBI.The findings in this report are subject to at least three limitations. First, incomplete reporting or misclassification of cause of death  on death certificates might bias estimates of TBI-related deaths. For example, NVSS data might not include all TBI-related deaths, especially if a TBI was not explicitly documented in death records. Second, data were combined across multiple years; therefore, a trend analysis was not possible; different patterns have emerged over time. Finally, data presented in this report are from the years 2016–2018, and might not reflect more recent differences. The COVID-19 pandemic, in particular, might have had an impact on more recent estimates.Expanding evidence-based prevention strategies that address TBI-related deaths is warranted, especially among states with high rates attributable to suicide, unintentional falls, and motor vehicle crashes. States can employ a number of evidence-based strategies  to reduce the leading causes of TBI-related deaths, including those for suicide,Traumatic brain injuries (TBIs) have contributed to approximately one million U.S. deaths during the last 2 decades. Rates of TBIs vary by state.During 2016–2018, states in the Northeast had the lowest TBI–related death rates , whereas rates were highest in the South (19.2). Suicide and unintentional falls contributed the highest number of TBI-related deaths in most states. States with a large proportion of residents living in rural areas had higher TBI-related death rates.Expanding evidence-based prevention strategies that address TBI-related deaths is warranted, especially among states with high rates due to suicide, unintentional falls, and motor vehicle crashes.