Thus, cortical deficits may impair language ability in children with ASD by causing subcortical dysfunction at preschool age. These evidences support dysfunction of the auditory brainstem as a potential subtype of ASD. Besides, this subtype-based method may be useful for various clinical applications.Autism spectrum disorder (ASD) is very heterogeneous, particularly in language. Studies have suggested that language impairment is linked to auditory-brainstem dysfunction in ASD. However, not all ASD children have these deficits, which suggests potential subtypes of ASD. We classified ASD children into two subtypes according to their speech-evoked auditory brainstem response (speech-ABR) and explored the neural substrates for possible subtypes. Twenty-nine children with ASD and 25 typically developing (TD) peers were enrolled to undergo speech-ABR testing and structural magnetic resonance imaging (sMRI). There were significant differences between the ASD group and TD group in surface area, cortical volume and cortical thickness. According to speech-ABR results, ASD participants were divided into the ASD-typical (ASD-T) group and ASD-atypical (ASD-A) group. Compared with the ASD-T group, the ASD-A group had a lower score in language of the Gesell Developmental Diagnosis Scale (GDDS), increased left rostral middle frontal gyrus (lRMFG) area and decreased local gyrification index of the right superior temporal gyrus. GDDS-language and surface area of lRMFG were correlated to the wave-A amplitude in ASD. Surface area of lRMFG had an indirect effect on language performance  Autism spectrum disorder (ASD) is a neurodevelopmental disorder involving deficits in social communication and restricted and repetitive behaviors, with an onset prior to 3 years of age . The hetOver the past several decades, researchers have made great efforts to find ways to categorize ASD heterogeneity . ClassifThere is an absence of neurobiological evidence for why individuals with ASD present symptoms of varying severity. However, paying attention to the phenotypic behavioral heterogeneity of language in early development is important . One impThe auditory brainstem response (ABR) is a far-field electric potential measured from the scalp reflecting the electrophysiological activity of the subcortical auditory pathway from the distal portion of the auditory nerve to higher midbrain structures . The speFunctional neuroimaging studies in the past decade have provided evidence that the cortico–subcortical network contributes to auditory, speech and language functions . AccordiIn the present study, we subcategorized participants with ASD by the speech-ABR. Then, we used structural magnetic resonance imaging (sMRI) to find the neural substrates for a possible subtype of the subcortical auditory function in ASD. Finally, we explored the interactive relationship among the cortex, subcortex, and language. We hypothesized that: (i) two distinct subtypes in a population of ASD cases aged 3–6 years would have significant differences in language scores; (ii) there would be significant differences in brain structure among ASD subtypes and typically developing (TD) children; and (iii) structural differences of ASD subgroups would interact with the function of the subcortex and language in ASD children . For ASD children who met the inclusion criteria, relevant researchers informed the parents of the research content, and invited them to participate in the present study. Written informed consent was obtained from the parents of children enrolled in our study.Twenty-five TD and thirty ASD children between 3 and 6 years of age were recruited from department of Child Psychiatry and Rehabilitation, Affiliated Shenzhen Maternity & Child Healthcare Hospital . All ASD participants were diagnosed by child psychiatrists with extensive experience at ASMCHH, and met the criteria for ASD using the Autism Diagnostic Observational Schedule (ADOS) and Diagnostic and Statistical Manual, Fifth Edition (DSM-5) of the All participants passed hearing screenings at birth and had good hearing sensitivity bilaterally . Besides, all participants were screened to ensure that they met the inclusion criteria: (i) met criteria of autistic disorder using both the ADOS and DSM-5 criteria; (ii) age of 3–6 years; (iii) no diseases of the acoustic meatus or hearing disorders; (iv) no history of epilepsy or head injury; and (v) no combined other neurodevelopmental disorders. Age-matched TD participants were screened with questionnaires. Individuals with a family history of any neuropsychiatric disorder  were excluded. All parents of participants were informed of the need to add a three-dimensional (3D) scan sequence, speech-ABR, and relevant clinical evaluation.The ADOS is a semi-structured, standardized interaction-and-observation tool that measures autism symptoms for individuals with possible autism or other pervasive developmental disorders . Each moThe Childhood Autism Rating Scale (CARS) is a behavior-observation instrument which differentiates people with ASD from those with other developmental disorders . The insThe Gesell Developmental Diagnosis Scale (GDDS) can be used to evaluate the developmental status of infants and young children from the age of 0 to 72 months . GDDS asThe stimulus comprised the five first formants of the syllable /da/, including consonant /d/ and vowel /a/. The /da/ was 40 ms in duration, and synthesized by a n = 15) and ASD-T group (n = 15). The BioMARK score was normal for all TD children. The ASD-T, ASD-A, and TD groups were matched on age. Relevant demographic information is shown in Biological Marker of Auditory Processing is a testing protocol developed by Auditory Neuroscience Laboratory of Northwestern University . The parameters evaluated for the speech-ABR were the: latency of wave-V; latency of wave-A; V/A slope; first formant frequencies; higher frequencies . The BioFor ASD participants who could cooperate to undergo MRI, 10% chloral hydrate  was used for sedation. All ASD children were sedated for MRI according to the sedation protocol set by the Department of Radiology of ASMCHH. The imaging data of TD children were collected during natural sleep at night without use of sedation. High-resolution T1-weighted MRI sequences were obtained on a 1.5-T Magnetom Symphony Maestro Class Syngo MR 2002B scanner  with echo time = 3.30 ms, repetition time = 10 ms, flip angle = 15°, 180 slices, and in-plane voxel size = 1 mm × 1 mm.1, the raw data from one ASD participant which were of insufficient quality were excluded. Hence, the final analysis comprised data from 29 ASD cases and 25 TD participants.Following visual assessment of MRI data and output from the FreeSurfer image analysis suiteT1-weighted images were processed using FreeSurfer v6.0.0. Cortical reconstruction for each participant was examined slice-by-slice to identify inaccuracies in surface placement. Inaccuracies were corrected or excluded by a single experienced FreeSurfer user. The details of these procedures are described elsewhere . Surfacepost hoc pairwise comparisons were conducted using the Bonferroni correction. The complete characterization and compare of speech-ABR among three group were reported in t-test was undertaken to detect differences in scores for the ADOS, CARS, GDDS, and speech-ABR between the ASD-A group and ASD-T group. A two-step general linear model was used to estimate the difference in surface area, cortical thickness, cortical volume and the local gyrification index between two groups, including ASD and TD, ASD-A and TD, ASD-T and TD, and ASD-A and ASD-T. Surface area, cortical thickness, and cortical volume were smoothed using a 10-mm full-width at half maximum (FWHM) 2D Gaussian kernel. The local gyrification index measure is intrinsically smooth, so data were smoothed at 5-mm FWHM. To provide stringent criteria to minimize false-positive results, all analyses were set at p < 0.01 (corrected for multiple comparisons using Monte Carlo stimulations)  with the diagnostic group as a three-level  factor adjusted for multiple comparisons. If the main effect of the group was significant, lations) . Clusterlations) . Based oX), language score of the GDDS as the outcome (Y), and the amplitude of wave-V as the moderator (M). Statistical tests were evaluated at p < 0.05 (two-tailed).According to the result of correlation analyses, we further explored whether the auditory brainstem function moderated the relationship between the brain structure and language ability of all ASD participants controlled for age and sex. The PROCESS macro program within SPSS  designed by t = 2.425, p = 0.025).As shown in p < 0.01) differences between the TD group and ASD group for cortical thickness, cortical volume and surface area . There was a significant positive association between the wave-V amplitude and GDDS-language . The bootstrap procedure revealed significant indirect effects between surface area of lRMFG and GDDS-language controlled for age and sex .This is the first study to classify ASD children into two subtypes based on function of auditory brainstem and to discover neural substrates for the possible subtypes.via the wave-V amplitude in ASD children.Our study elicited four main findings. First, the language score of the ASD-A group was lower than that of the ASD-T group. Second, compared with the TD group, the ASD subgroups showed significant differences in brain structure. Surface area of lRMFG in the ASD-A group was larger, and the local gyrification index of rSTG in the ASD-A group was decreased significantly, compared with those in the ASD-T group. Third, GDDS-language and surface area of lRMFG were correlated to the wave-A amplitude in ASD. Finally, analysis revealed surface area of lRMFG to have an indirect effect on language Autism spectrum disorder subgroups defined in the present study showed a significant difference in the speech-ABR at preschool age. The ASD-T group exhibited age-appropriate subcortical auditory processing function. In contrast, the ASD-A group showed the latency of speech-ABR waves to be longer and/or the amplitude to be smaller compared with those in the TD group and ASD-T group. Besides, ASD subtypes showed a significant difference in language but not in other aspects or symptom severity. Consistent with our previous research showing that the speech-ABR is related to language ability , childreWe found differences in brain structure not only between TD children and ASD children, but also among ASD subtypes. Consistent with other studies, we found an aberrant cortical structure in ASD children, including cortical thickness, surface area, and cortical volume . BesidesMoreover, we found a significant difference in brain structure between ASD subtypes, which may provide neuroimaging evidence for subtype classification. We found a larger regional surface area (lRMFG) in the ASD-A group compared with that in the ASD-T group and TD group. This result indicated that an increase in surface area of lRMFG was a characteristic structural change in the ASD-A subtype. The RMFG, as part of the dorsolateral prefrontal cortex (DLPFC), is related to the function of language, as discovered in various lesion studies , 1998. PFurther analyses revealed that surface area of lRMFG had an indirect effect on language by altering the wave-V amplitude in ASD children. Combined with the previous findings showing that the neural generators of waves V and A may be the inferior colliculus , one parUsing subtypes may be the most useful method for specific intervention in ASD. Experience-dependent mechanisms drive the plasticity of the auditory brainstem, which can be improved by auditory training  and langIn interpreting our results, some limitations should be considered. First, our study had a relatively small sample size. There were only two female participants in this study, and both were in the ASD-A. This study does not exclude the potential of gender factors for subtypes. Our future studies will recruit more children with ASD who meet the criteria for an appropriate gender ratio. Second, MRI was done on a 1.5-T system, but several recent studies have used 3-T MRI scanners. MRI scanners working at 3 T have a higher field strength and increased signal-to-noise ratio compared with those using a 1.5-T MRI scanner. In future work, we will use a 3.0-T MRI scanner to acquire higher spatial resolution data. Finally, intelligence and language ability are highly correlated. Although there were no significant differences in overall developmental levels between the two subtypes. It is possible that there were significant differences in intelligence levels. Our future studies will investigate whether intellectual factors affect subcortical auditory processing in ASD.This is first study to distinguish ASD children by auditory brainstem function and to explore neural evidence for identification of potential subtypes. There were significant differences between the ASD group and TD group in surface area, cortical volume, and cortical thickness. Compared with the ASD-T group, the ASD-A group showed a lower score in GDDS-language, increased surface area of lRMFG, and decreased local gyrification index of rSTG. GDDS-language and surface area of lRMFG were correlated with the wave-A amplitude. Surface area of lRMFG had an indirect effect on the performance of language by altering the wave-V amplitude. Thus, cortical deficits may impair language ability in children with ASD by causing subcortical dysfunction at preschool age. These evidences support dysfunction of the auditory brainstem as a potential subtype of ASD. Besides, this subtype-based method may be useful for various clinical applications .The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Research Ethics Board of Affiliated Shenzhen Maternity & Child Healthcare Hospital. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.JC, ZP, and GW conceived the study, prepared the data, analyzed the data, and drafted and revised the manuscript. BL, JG, and MH helped to analyze the data and helped to draft the manuscript. XK and CL participated in the study design and helped to draft and revise the manuscript. All authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Over the last few years, CAR-T cells have arisen as one of the most promising immunotherapies against relapsed or refractory hematological cancers. Despite their good results in clinical trials, there are some limitations to overcome, such as undesirable side-effects or the restraints of an autologous treatment. Therefore, CAR-NK cells have emerged as a good alternative for these kinds of treatments. This review discusses the advantages of CAR-NK cells compared to CAR-T cells, as well as the different sources and strategies in order to obtain these CAR-NK cells.Acute lymphoblastic leukemia (ALL) and Chronic lymphocytic leukemia (CLL) are the most common leukemias in children and elderly people, respectively. Standard therapies, such as chemotherapy, are only effective in 40% of ALL adult patients with a five-year survival rate and therefore new alternatives need to be used, such as immunotherapy targeting specific receptors of malignant cells. Among all the options, CAR (Chimeric antigen receptor)-based therapy has arisen as a new opportunity for refractory or relapsed hematological cancer patients. CARs were designed to be used along with T lymphocytes, creating CAR-T cells, but they are presenting such encouraging results that they are already in use as drugs. Nonetheless, their side-effects and the fact that it is not possible to infuse an allogenic CAR-T product without causing graft-versus-host-disease, have meant using a different cell source to solve these problems, such as Natural Killer (NK) cells. Although CAR-based treatment is a high-speed race led by CAR-T cells, CAR-NK cells are slowly (but surely) consolidating their position; their demonstrated efficacy and the lack of undesirable side-effects is opening a new door for CAR-based treatments. CAR-NKs are now in the field to stay. B cell leukemias, such as Acute lymphoblastic leukemia (ALL) and Chronic lymphocytic leukemia (CLL), are hematological cancers affecting young and elderly patients . In a pa9/L clonal B-cells in the peripheral blood, which usually express CD19, CD20, dim CD23, and CD5 [In adults, CLL is one of the most frequent types of leukemia, which usually evolves moderately slowly. It is a low-grade lymphoproliferative neoplasm with ≥5 × 10 and CD5 . It gene and CD5 .Over the last four decades, ALL treatment advances have principally been made in the children and adolescent populations, with less success reported in adults ,13 ALL pThere are two principal types of HSCT: allogenic stem cell transplants, in which the stem cells belong to a donor , which is the preferred transplant for ALL patients; and autologous stem cell transplants, in which the patient receives their own blood cells.A greater number of CLL patients have no symptoms at diagnosis, and all patients should be classified based on their risk. Patients classified as low-and intermediate-risk (~75% patients) should be checked for disease progression every half a year to a year, while those in the high and very high-risk group (~25% patients) should be checked every 3–6 months . For theRelapsed or refractory ALL or CLL patients after two or more lines of treatment may need alternative therapies to treat their disease. New therapies, especially immunotherapies, are emerging to treat these and other hematological cancers. Chimeric antigen receptor (CAR) based therapy is positioning itself as one of the most promising therapies for getting rid of the malignant cells. ALL and CLL biological and clinical milestones are summarized in Immunotherapy is a type of therapy that uses substances to stimulate or suppress the immune system to help the body fight cancer, infection, and other diseases ,27. The In ALL, complete remission (CR) rates after chemotherapy are low and range between 25 and 45% , with moAbout 30 to 50% of late pre-B cell lymphoblasts express CD20 and it is linked to a higher relapse and lower survival rate ,32. SeveCD22 is present on leukemic blasts in >90% of ALL patients . Mainly,CD19 is expressed in 90% of pre-B and mature ALL lymphoblasts, providing an interesting target for immunotherapy. A BiTE construct named blinatumomab binds CD19-positive B cells and CD3-positive cytotoxic T cells. The cytotoxic T cells are activated when binding to CD19 and induce cell death via direct tumor lysis . In OctoWith CLL, in most cases several immunotherapies are applied to the patients as a first-line therapy. These antibodies, such as Rituximab, Ofatumumab or Obinutuzumab, target the CD20 pathway , while aIn the 1980s, Israeli researchers expressed chimeric TCR genes including the TCR constant domains united to the variable domain from an antibody, which led to a hypothesis about CAR-T treatments ; in 1989As CARs seem to be a newer and more effective way to treat cancers in relapse or refractoriness, especially hematological cancers, there are various clinical trials going on. Most of them use T cells as a vehicle for the CARs, with more than 1.000 clinical trials worldwide, and CD19 is mostly used as the CAR antigen.Natural Killer cells, or NK cells, belong to the innate immune system, providing rapid responses against viral infections and tumors. Usually, the detection of the major histocompatibility complex (MHC) on the surface of the infected cells by the immune cells triggers cytokine release, causing lysis or apoptosis. NK cells have, in fact, a unique ability to recognize stressed cells lacking antibodies and MHC, accelerating an immune reaction. They owe their name “natural killers” to the initial perception that they do not need prior activation to kill cells with no “self” antigens of MHC class I. As malignant cells do not express MHC I markers, T cells cannot destroy them, so NK cells play a key role .NK cells and their functions were described more than 30 years ago, but for the first time in 1975 these cells were described as bigger lymphocytes than B cells and T cells, which contained distinctive cytoplasmic granules. NK cells were characterized as cells which showed a co-stimulatory independent spontaneous cytotoxic capacity, differentiating them functionally from B cells and T cells ,57.In the immune system, NK cells are the third major lymphocyte subset. These large granular cells constitute approximately 10–15% of lymphocytes in the blood . NK cellDuring the development process of NK cells, they express several surface markers progressively and in an orderly way, and they are classified into stage 1 , stage 2 , and stage 3 . Mature NK cells are phenotypically described as stage 4  and stage 5  .NK cell functions involve recognition of potential target cells by the initial binding interactions between activating and inhibitory receptors with ligands available on the target, and the integration of signals transmitted by these receptors, which determines whether the NK cell detaches and moves on or stays and responds. Therefore, the activating and inhibitory receptors are crucial for NK cell function regulation. NK cells express clonally distributed inhibitor receptors named killer cell immunoglobulin-like receptors (KIRs), that recognize allotypic determinants (KIR ligands) shared by particular groups of HLA class I alleles. The regulatory mechanism mediated by these receptors is thought to protect normal cells from autologous NK cell attack, while rendering cells for which class I expression is compromised  susceptible to NK-mediated killing . The absNK cell role during ALL or CLL may determine the prognosis of the disease. An increase in the number of NK cells is associated with a better prognosis in ALL and CLL . The pre- lymphocytes [Despite the good results of CAR-T cells, there are some expected side-effects. On the one hand, the main and most serious side-effect of CAR-T cell therapy is cytokine-release syndrome and its dangerous form, a “cytokine storm”, in which T-cells are massively activated, triggering a cascade of pro-inflammatory cytokines which cause flushing, fever and dyspnea. Although an acute cytokine storm can potentially be lethal, it has been proved that the anti-interleukin-6 receptor antibody tocilizumab is an effective treatment . Immune phocytes  and Natuphocytes ,77. NK lphocytes . Moreovephocytes . Howeverphocytes ; this cophocytes . Anotherphocytes . Althougphocytes ,84 alongphocytes . The mecphocytes . T cellsphocytes . T cellsphocytes  , NK cell-based immunotherapy as maintenance therapy for small-cell lung cancer (NCT03410368) or NK cells along with IL-2 following chemotherapy to treat advanced melanoma or kidney cancer (NCT00328861). hiPSC-derived NK cells, named FT500, are also being used in combination with Immune Checkpoint Inhibitors (ICI) in a clinical trial to treat subjects with advanced solid tumors (NCT03841110). In April 2019, Fate Therapeutics declared that the first patient treated with FT500 had successfully completed an initial safety appraisal. The patient was administered with three once-weekly doses of FT500. There were no toxicities or severe adverse events reported and the treatment cycle was well tolerated, with no dose-limiting toxicities or severe adverse events reported during the initial 28-day observation period. Not only are NK cells able to treat solid tumors, but they also play a key role in immunotherapies against hematological cancer like acute myeloid leukemia (AML), by using high doses of these cells  or infusAlthough T cells have typically been used in CAR technology-based therapy, with more than 400 clinical trials on-going and 3 commercial products, Kymriah Yescarta and TECARTUS, NK cells are also emerging as one of the new promises in this field , as showSecondly, allogenic primary NK cells from adult peripheral blood (AB) or umbilical cord blood (CB) could represent feasible, safe, off-the-shelf CAR-cell products to treat various malignancies such as hematological cancers. When studying AB CD19-CAR-NK cells, they not only successfully kill CD19-expressing target cells, but they also retain the function and expression of their native activating receptors, preserving their activity . HoweverFinally, in the last few years, hiPSCs have arisen as one of the most promising cell sources for personalized medicine. HiPSC-NK cells can be manufactured from standardized cells resulting in a homogeneous clinical scale NK cell population . These sNowadays the clinical indications of CAR-NK cell therapy in B cell hematological cancer remains an open question. Currently, it has been confirmed that various CAR-T cell therapy clinical trials are safe being an efficient therapeutic procedure for relapse or refractory B-ALL patients when used as a bridging approach before or after HSCT. Therefore, CAR-NK cells may also handle as a bridge strategy, through which patients can accomplish a low pre-infusion minimal residual disease (MRD) condition before administration of allo-HSCT. The aim of NCT02892695 clinical trial is to figure out the safety and best dose of CD19 CAR-NK cells used as a bridge therapy in patients who intend to undergo HSCT .Other potential uses of CAR-NK cellular therapy may be indicated for patients that have not enough T cell numbers for autologous CAR-T cell product manufacture. Patients with aggressive ALL or CLL will benefit from allogeneic CAR-NK permitting early treatment or later in combination with CAR-T. Additionally, for those CD19+ patients that have been relapsed after CAR-T therapy or patients who develop high toxicity (ICANS and/or cytokine-release syndrome) after CAR-T infusion.Due to the boom in immunotherapy for hematological cancers, great strides have been made in this field, especially with the arrival of CAR-T treatments. However, despite the successful results obtained with autologous CAR-T treatment, there are still some worrying aspects. Undesirable side-effects, such as cytokine storms, ICANS or GvHD caused by allogenic CAR-T cells, are driving the CAR field towards new alternatives. The lack of side-effects or GvHD in allogenic treatments has put NK cells in the spotlight.in vitro. Recently, the report from one of the few clinical trials taking place has not only proved their efficacy but also their safety when using allogenic CAR-NK cells. Hence, CAR-NK cells can serve as an off-the-shelf product to treat refractory hematological malignances, with no need for the patient-specific product request by CAR-T treatments. Moreover, the possibility of obtaining in vitro generated NK cells for this purpose could lead to massive production of universal allogenic CAR-NK cells, which could end up in a reduction of costs and more open access to this treatment. Combinations of CAR-NK products with other immunotherapies and even with other CAR-T cells are becoming a promising option.For the last few years, CAR-NK cells have proved to be an optimum product for the treatment of hematological malignances Therefore, the benefits of CAR-NK cells over CAR-T cells augur promising applications in cellular immunotherapy against hematological malignancies as an alternative or combination cell drug. Nevertheless, there are still challenges to be addressed. For instance, because of the heterogeneity of NK cells with various functional features, the selection of appropriate NK cell subsets  to specifically expand and arm CAR-NK cells has to be explored. NK cells are known for their transduction difficulty. Although several protocols with retrovirus and lentivirus have been successfully developed, there is still room for new techniques in order to improve NK cell transduction, such as mRNA electroporation or CRISPR/Cas9 technology. As CAR-NK cells are thought to be an “off-the-shelf” product, cryopreservation is also a crucial step. Focusing on not losing many NK cells at the thawing process, and re-establishing their function, it is determinant to elucidate the appropriate cryopreserving media and protocol for CAR-NK cell therapy success. Due to their short persistence in vivo, the NK cell cytolytic effect could also be restricted, but they are probably unable to trigger cytokine storms or on-target/off-tumor effects; continuous cytokine support or several infusions of CAR-NK cells may will be needed. Additionally, various NK cell sources have been studied , and therefore in the future the foremost appropriate one could be used for refractory malignancies. Moreover, the best configuration of CARs to boost the activation, proliferation, cytolytic activity and cytokine secretion of NK cells has not yet been found. Perhaps the future of these CAR constructs for NK cells lies on exploring NK cell activating domains such as NKG2D, DAP10, DAP12 and 2B4 in order to improve their performance. Moreover, future directions of CAR-NK cell therapy could be administrated in combination with other therapies, being lymphodepletion, radiation, immune checkpoint blockers or even CAR-T cells. Notwithstanding, as a result of the recent advances, quick developments and future challenges for improvement, CAR-NK cell-based immunotherapy constitutes an encouraging scenario for cancer treatment. Tumor-associated antigens do not require the recognition of HLA molecules from patients, which makes it possible to manufacture off the shelf NK cell banks rather than manufacturing individualized CAR-NK cells. In conclusion, as more evidence from clinical trials is procured within the coming years, CAR-NK cell therapies could provide meaningful progress in tumor immunotherapy. Moreover, CAR-NK therapies in combination with other immunotherapies or even other CAR-T cells may pave a new way for CAR-NK cell-based immunotherapy in the future.CAR-NK cells have excellent potential as an innovative and “off-the-shelf” cellular immunotherapy against hematological cancer that could be quick, accessible and safe for clinical practice. With the growing safety and encouraging work reported in preclinical studies and clinical trials, together with progressive achievements addressing the remaining challenges, it is envisioned that CAR-NK cell therapy will progress to emerge and contribute to significant improvement in the survival of relapsed or refractory hematological cancer patients.


To assess the relationship of serum brain-derived neurotrophic factor (BDNF) levels with the subsequent short-term decline in cognitive functioning in community-dwelling older adults.Two-year prospective, observational study.The study included 405 adults aged 65–84 years, initially free of a dementia diagnosis who were living in Tokyo, Japan.Participants underwent health assessments at baseline (2011) and follow-up (2013). Serum BDNF levels and scores from the Montreal Cognitive Assessment-Japanese version (MoCA-J) were systematically measured. Logistic regression was used to estimate the odds of cognitive decline between baseline and follow-up assessments in the full MoCA-J scale , as well as in MoCA-J subscales , as a function of serum BDNF level, adjusting for baseline demographics, prevalent chronic diseases, and baseline cognitive scores.p = 0.013). Regarding MoCA-J subscales, adjusted odds of decline in the executive function subscale, but not in the other five subscales, were substantially low among those with highest baseline serum BDNF levels (top quartile), as compared to those with the lowest (bottom quartile), i.e., OR: 0.27 .Among individuals who performed worse on the full MoCA-J at baseline , but not among those who performed better (top 3 quartiles), those with highest baseline serum BDNF levels (top quartile) had lower odds of subsequent decline in the full MoCA-J scale than those with lowest (bottom quartile); i.e., odds ratio (OR): 0.10 (95% confidence interval [CI]: 0.02–0.62; Higher serum BDNF levels were associated with a lower risk of decline in cognitive function in a sample of community-dwelling older Japanese adults. Risk varied across cognitive subdomains and according to baseline cognition. This warrants further research to evaluate the added-value of serum BDNF in health promotion initiatives directed toward cognitive decline prevention in community-dwelling older adults. As people age, their cognitive function declines more rapidly in areas of fluid intelligence, including executive function, processing speed, working memory, and reasoning . The preBrain deterioration in both structure and function underlie age-related cognitive decline. For example, the prefrontal cortex starts shrinking in the second decade of life, and the hippocampus begins shrinking in the sixth decade. Both of these areas exert key control over fluid intelligence . ImproveBrain-derived neurotrophic factor (BDNF) is a neurotrophin that is widely distributed in the brain. BDNF and its tropomyosin-related receptor kinase B (TrkB) are highly expressed in cortical structures, especially in the hippocampus, and play an important role in learning and memory . BDNF isReflecting the growing interest in the assessment of BDNF as a clinically useful biomarker for the prevention of cognitive decline, there has been an increasing number of observational studies reporting data on the association of circulating BDNF levels with cognitive functioning. Most studies to date, however, have been performed on patients with specific neuropsychiatric disorders and chronic diseases , and datThis report uses data from a subset of participants in the Otassha Study, a large observational study, of older adults living in areas surrounding the Tokyo Metropolitan Institute of Gerontology (TMIG), Tokyo, Japan. Study invitations were sent by postal mail to 6,699 community-dwelling older adults living in nine regions across the Itabashi Ward, in the Northwestern part of Tokyo. Nursing home residents were not eligible. A total of 876 study volunteers aged 65–84 years old were recruited for the study’s comprehensive baseline health assessment, which took place at a research TMIG research examination room in October 2011. Of those, a follow-up assessment was conducted for 410 participants in 2013. Included in this study were those who participated in both baseline and follow-up assessments, were free of a dementia diagnosis, and had data on serum BDNF samples and relevant cognitive tests (n = 405). All subjects provided written informed consent to participate in the study, which was approved by the Institutional Review Board and Ethics Committee of the TMIG .Blood was drawn from participants within minutes, and samples were centrifuged at 3,000 rpm at 4°C for 15 min. Sera were then transferred to a new set of polyethylene tubes. The serum samples were stored at −80°C immediately after separate serum and serum BDNF levels were measured within 3 months using the BDNF Emax immunoassay system , according to the supplier’s protocol . The senTrained research assistants administered the Montreal Cognitive Assessment-Japanese version (MoCA-J) . For thiDetails of the MoCA-J items have been described elsewhere . RegardiThe outcome of interest was a meaningful short-term decline in MoCA-J performance. However, there are currently no well-established criteria to define what constitutes a meaningful decline in the full MoCA-J scale or its subscales. In this context, we arbitrarily used the following operational definition approach. First, for each participant, we calculated the difference in score between the participant’s baseline and follow-up assessments. Second, we examined the distribution of the within-person difference in scores and, for each cognitive outcome, identified the cut-off point that identified 25% of participants who declined the most in each cognitive outcome. For example, the subset of 25% of study participants who experienced the greatest decline in the full MoCA-J scale had a score decline of two or more points; thus, cognitive decline in the full MoCA-J scale was operationally defined as a decline in a score of two or more points in that scale. Analogously, for all MoCA-J subscales, cognitive decline was defined as a decline of one or more points from baseline to the follow-up visit 2 years later.a priori from available data collected as part of available health assessment data as potential confounders: (a) socio-demographic variables, including age (continuous), sex (dichotomous), years of formal education (≥ vs. <12 years); (b) health habits, including smoking and drinking status ; and (c) clinical indicators, including a previous history of being told by a physician they had a diagnosis of hypertension, cardiac disease, diabetes, and/or stroke (≥ vs. < 2 diseases), and depressive symptoms. The latter was assessed using the Self-Rating Depression Scale (SDS) (The following parameters collected at baseline were selected le (SDS) . Traditile (SDS) . The selUnivariate distributions of study variables were reported as means ± standard deviations (SD), medians (interquartile range [IQR]), and proportions with absolute cell numbers. A bivariate analysis of baseline variables involved comparisons of the distribution of mean age, median cognitive scores, and categorical variables across the 3-level serum BDNF categorical variable using one-way analysis of variance, Kruskal-Wallis, and chi-square tests, respectively.p = 0.007, as per the likelihood ratio test). Thus, logistic regression models assessing the odds of cognitive decline the full MoCA-J scale was stratified by MoCA-J baseline performance.We also compared the observed proportion of study participants who met criteria for cognitive decline in each cognitive test score across categories of serum BDNF levels at baseline using chi-square tests for homogeneity of proportions. Multivariate logistic regression estimated the odds of meeting criterion for subsequent cognitive decline in the full MoCA-J scale as well as in its different subscales between baseline and follow-up visits as a function of serum BDNF levels at baseline. For the full MoCA-J scale, but not for the subscales, there was evidence that the association of baseline BDNF with subsequent cognitive decline differed between the subset of participants with better  vs. worse  at baseline. Specifically, the addition of the interaction term representing the multiplication of baseline full MoCA-J performance category (better vs. worse) times the 3-level serum BDNF levels at baseline resulted in a statistically significant improvement in model fit . However, considering the small study sample size, and the lack of an association of those additional covariates considered for adjustment with baseline serum BDNF, they ended up not being included in final logistic regression models. As a post-hoc sensitivity analysis, we performed a re-analysis, excluding study participants who had a history of prevalent stroke at baseline. Stata version 14.2 software was used to this end .Adjustments were conducted in a hierarchical fashion and similarly for all cognitive outcomes. For each MoCA-J-related outcome, three models were constructed: model 1 was unadjusted; model included adjustments for age (continuous), sex (dichotomous), and education (≥ vs. < 12 years); and model 3 included adjustments for all variables in model 2 plus a number of chronic diseases (2–4 vs. 0–1), depression symptom burden , and baseline cognitive score. All above-listed covariates considered for adjustment were selected p = 0.035). Dose-response relationships characterized by incrementally higher baseline BDNF levels with an incrementally lower proportion of participants experiencing cognitive decline were observed in the executive function subscale, as well as in the attention and working memory subscale, though the dose-response relationship was stronger and only statistically significant for the executive function subscale (p = 0.003).p-value = 0.013). There was no association between serum BDNF levels at baseline and subsequent decline among those who performed better (scores in the top three quartiles) in the full MoCA-J scale at baseline. Regarding the MoCA-J executive function subscale, the odds of cognitive decline from baseline to the follow-up visit was substantially lower among those with highest than lowest baseline BDNF levels; i.e., OR: 0.27 . No other meaningful, statistically significant association of serum BDNF levels at baseline with other MoCA-J subscales. Post-hoc analyses similar to those presented in Logistic regression models estimated adjusted odds of cognitive decline between baseline and follow-up visit 2 years later as a function of baseline levels of BDNF (bottom vs. intermediate vs. top quartiles), adjusted for age, sex, education, number of chronic diseases, depressive symptom burden, and cognitive score at baseline. Among participants with baseline full MoCA-J scale scores ≤ 21 (worst quartile), those with the highest baseline BDNF levels were associated with substantially lower odds of cognitive decline in the full MoCA-J scale from baseline to the follow-up visit, as compared those with lowest BDNF levels; i.e., OR: 0.10  in the full MoCA-J scale, consistent with a mild cognitive impairment status .We studied a community-dwelling population that had a high education level and did not include subjects with a known clinical diagnosis of dementia. The average full MoCA-J scale score was slightly above that observed in a population-based sample used for the derivation of normative data on the distribution of MoCA-J scores in a community-dwelling older Japanese population. Our findings are thus consistent with the hypothesis that higher serum levels of BDNF at earlier stages of the cognitive decline process might reflect an effective compensatory response to pathophysiologic insults . This stOur observational data suggest that the relationship between serum BDNF levels and cognition in community-dwelling older populations might vary by cognitive subdomain. We found a relationship between higher serum BDNF and executive function in the whole population, but not in other MoCA-J subscales reflective of other cognitive subdomains. The reason underlying these differential associations by cognitive subdomains remains unclear. Considering previous evidence that decline in executive function, which involves higher-order cognitive abilities, often precedes a decline in other cognitive subdomains , it mighOur results should be interpreted in light of important study limitations. The follow-up period was short (2 years), and the sample size relatively small. There is no validated, widely accepted standard for defining what constitutes clinically meaningful score declines in the full MoCA-J or its subscales; thus, we used an arbitrary operational definition for cognitive decline in study analyses. The cohort solely consisted of Japanese adults living in an urban area of Tokyo; therefore, the influence of race and lifestyle was limited. This was an observational study, which restricts inferences on causality, or the potential impact of intervention-resulting changes in serum BDNF levels on cognition. Cognitive decline was defined using a statistical distribution-based operational definition, given the lack of an established standard for what constitutes clinically meaningful score declines in the full MoCA-J and its subscales. This study’s strengths include its population-based setting, data collection done in a standardized fashion, and the contribution of novel data on the prospective relationship of serum BDNF levels as a biomarker for declines in different cognitive subdomains in community-dwelling older adults.Our results show that higher serum BDNF levels were associated with a lower risk of decline in cognitive function in community-dwelling older adults. They also demonstrate that the type of cognitive decline and the subsets of individuals at risk may vary according to cognitive subdomains and baseline cognitive performance level. This study contributes information relevant for public health researchers seeking to establish the value of serum BDNF levels as part of novel interventions and approaches to prevent age-related cognitive decline.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Institutional Review Board and Ethic Committee of the TMIG. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.YF, MHc, and KI: conceptualization. HK: data curation. YF and MHc: formal analysis. YF, MHc, HS, MHs, HK, HH, SO, MK, and KI: investigation. HK, HH, HS, YF, SO, and KI: project administration. SO and KI: resources. YF: writing–original draft. YF, MHc, PC, and KI: writing–review and editing. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Exclusive breastmilk feeding during the delivery hospitalization, a Joint Commission indicator of perinatal care quality, is associated with longer-term breastfeeding success. Marked racial and ethnic disparities in breastfeeding exclusivity and duration existed prior to COVID-19. The pandemic, accompanied by uncertainty regarding intrapartum and postpartum safety practices, may have influenced disparities in infant feeding practices. Our objective was to examine whether the first wave of the COVID-19 pandemic in New York City was associated with a change in racial and ethnic disparities in exclusive breastmilk feeding during the delivery stay.n=3122 deliveries) to disparities in a pre-pandemic cohort . We defined exclusive breastmilk feeding as receipt of only breastmilk during delivery hospitalization, regardless of route of administration. We ascertained severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection status from reverse transcription-polymerase chain reaction tests from nasopharyngeal swab at admission. For each DID model (e.g. Black-white disparity), we used covariate-adjusted log binomial regression models to estimate racial and ethnic risk differences, pandemic versus pre-pandemic cohort risk differences, and an interaction term representing the DID estimator.We conducted a cross-sectional study of electronic medical records from 14,964 births in two New York City hospitals. We conducted a difference-in-differences (DID) analysis to compare Black-white, Latina-white, and Asian-white disparities in exclusive breastmilk feeding in a pandemic cohort  and Asian  women, but not Black  or Latina  women overall. There was an increase in the Latina-white exclusive breastmilk feeding disparity associated with the pandemic ). We found decreased breastmilk feeding specifically among SARS-CoV-2 positive Latina women (20.1% pre-pandemic vs. 9.1% pandemic p=0.013), and no change in Black-white or Asian-white disparities.Exclusive breastmilk feeding increased from pre-pandemic to pandemic among white (40.8% to 46.6%, We observed a pandemic-related increase in the Latina-white disparity in exclusive breastmilk feeding, urging hospital policies and programs to increase equity in breastmilk feeding and perinatal care quality during and beyond this health emergency.The online version contains supplementary material available at 10.1186/s12884-022-04570-w. Breastfeeding is associated with myriad health benefits, including improved maternal-infant attachment, reductions in infectious and chronic disease risk for the dyad, decreased postpartum depression, and improved childhood cognitive development , 2. The Women who breastfeed exclusively during their delivery hospitalization are more likely to continue breastfeeding exclusively through the first month of life , and EBFOur objective was to evaluate whether the COVID-19 pandemic was associated with a change in racial and ethnic disparities in EBF at two hospitals in a major New York City (NYC) hospital system. At our institution, all women, including SARS-CoV-2 positive women, have been encouraged to room-in with infants since the late March 2020 onset of the first wave of the pandemic. We conducted a difference-in-differences (DID) analysis of EBF rates among singleton, term births, comparing the first wave of the COVID-19 pandemic to births in the previous year.n=15,779). Gestational age was recorded in the EMR based on best obstetric estimate.Electronic medical records (EMR) on 18,904 births from January 1, 2019 through July 31, 2020 were available for analysis. We followed criteria for the Joint Commission EBF quality metric , full term [390/7-406/7 completed weeks], late term [410/7-416/7], and post-term [420/7+ weeks]), and month of delivery to account for potential seasonal trends.We evaluated covariates including maternal age , insurance type , pre-pregnancy body mass index , parity , gestational age , non-Latina Asian (Asian), non-Latina white (white), and Latina births. Comparisons used chi-square tests with a two-sided We estimated a DID equation using log binomial regression. We specified main effects for the Black versus white risk difference, pandemic versus pre-pandemic cohort risk difference, and an interaction term representing the DID estimator. The DID estimator provides the additional disparity resulting from the pandemic, beyond pre-existing disparities. We repeated the model for Latina versus white and for Asian versus white births. To parse the potential direct influence on breastmilk feeding of SARS-CoV-2 infection from that of delivering during the pandemic era, we carried out the same DID analyses restricting the pandemic cohort to SARS-CoV-2 positive or negative status. Multivariable models adjusted DID estimates for maternal age, insurance type, pre-pregnancy BMI, parity, and month of delivery. The DID approach is generally robust to confounding if the balance of covariates between treatment and control groups is constant over time . We exclWe evaluated the robustness of our results through multiple sensitivity analyses. First, we specified an alternate control group using a pre-pandemic time frame  with months identical to the pandemic period. Second, we used a spurious treatment group of August through December 2019. Third, we conducted an interrupted time series analysis to account for secular trends in EBF by examining whether the slope of EBF changed in the pandemic compared to the pre-pandemic period . Finallyp=0.023, Table We did not find notable changes in study characteristics over time Table , nor witp<0.001, data not shown in table). Rates increased among white  and Asian  women, and not among Black  or Latina  women  cohort  of infants born to SARS-CoV-2 positive women were breastfed exclusively or mostly [At a NYC hospital with similar policies during the pandemic onset, 40.6% , while Black and Latina women may be more likely to mix both formula and breastmilk feeding from the outset. Since accounting for early discharge did not entirely explain the EBF increase among white women, we explored whether our findings reflected a decrease in cesarean delivery during the pandemic in a post-hoc analysis. However, adjusting for vaginal versus cesarean delivery did not attenuate the EBF increase comparing births to white women in the pre-pandemic and pandemic cohorts.Our finding of a significant decrease in EBF among births to SARS-CoV-2 positive Latina women is potentially concerning. While estimates were based on a small number of cases, they suggest an influence of factors such as the disruption in breastfeeding education during the pandemic, when hospital group classes were discontinued. There may have been specific misperceptions about viral transmission that inhibited breastmilk feeding among Latina women, suggesting a need for more targeted patient consultation and education on safe breastmilk feeding and infant care practices in the hospital.It is unclear why the unexpected benefit of hospital COVID-19 precautions on EBF was not observed among the Latina and Black women included in this study. We can hypothesize several mechanisms, including direct effects of maternal SARS-CoV-2 infection, the psychosocial corollaries of giving birth during the pandemic, and disparate experiences of delivery care. Rates of SARS-CoV-2 infection at delivery were higher among Black and Latina than among white women, and SARS-CoV-2 positive women may have refrained from breastmilk feeding out of fear of infecting the neonate. Latina women may also encounter language barriers, preventing access to up-to-date information about breastmilk feeding safety during the pandemic . The decA growing literature has documented obstetric and neonatal risks of SARS-CoV-2 infection that may impact breastfeeding. Studies suggest increased risks of maternal complications –37, cesaOur study has several limitations. Our EBF measure was subject to error or variation between clinicians or hospitals in recording infant feedings in the EMR. However, this variation would likely be consistent over time, so while absolute EBF percentages may be an under or over-estimate we expect that the change in disparity would be accurately measured. Second, although we probed potential mechanisms, we were not able to quantify the extent to which observed differences were due to changes in rooming-in, early discharge, fear of viral transmission, or another pathway. We did not have information on galactosemia or parenteral infusion, which are exclusion criteria for the Joint Commission measure, but replicated the measure as closely as possible through study restrictions. We did not analyze women of other or unknown race/ethnicity for interpretability of results. NYC was a racially diverse epicenter of the pandemic, and our results may not generalize to other settings or to hospitals with different COVID-era policies.Strengths of our study include use of a strict EBF definition from nursing notes on all infant feedings, and ascertainment of SARS-CoV-2 PCR status from EMR lab data with universal screening of obstetric patients. We employed a robust DID design that allowed us to isolate changes in disparities associated with the pandemic. We conducted multiple sensitivity analyses, including an ITSA to test for the influence of secular trends and use of an alternate EBF measure to account for the early discharge policy.We provide a novel analysis of the implications of the COVID-19 pandemic on EBF during the delivery hospitalization, a perinatal metric with important short- and long-term health effects. We observed improvements in EBF associated with the pandemic among white and Asian but not Black or Latina women, who already face a substantial baseline disparity, and evidence of a decrease among SARS-CoV-2 positive Latina women. Changes resulted in a widening of the EBF gap between white and Latina women. Health care providers should advocate for hospital policies and programs to promote equity in breastmilk feeding, which may be particularly needed during the pandemic. Further, our findings emphasize the importance of reporting perinatal quality measures disaggregated by sociodemographic characteristics to monitor the influence of the pandemic on perinatal health care and disparities.Additional file 1.


Due to the fast global spreading of the Severe Acute Respiratory Syndrome Coronavirus – 2 (SARS-CoV-2), prevention and treatment options are direly needed in order to control infection-related morbidity, mortality, and economic losses. Although drug and inactivated and attenuated virus vaccine development can require significant amounts of time and resources, DNA and RNA vaccines offer a quick, simple, and cheap treatment alternative, even when produced on a large scale. The spike protein, which has been shown as the most antigenic SARS-CoV-2 protein, has been widely selected as the target of choice for DNA/RNA vaccines. Vaccination campaigns have reported high vaccination rates and protection, but numerous unintended effects, ranging from muscle pain to death, have led to concerns about the safety of RNA/DNA vaccines. In parallel to these studies, several open reading frames (ORFs) have been found to be overlapping SARS-CoV-2 accessory genes, two of which, ORF2b and ORF-Sh, overlap the spike protein sequence. Thus, the presence of these, and potentially other ORFs on SARS-CoV-2 DNA/RNA vaccines, could lead to the translation of undesired proteins during vaccination. Herein, we discuss the translation of overlapping genes in connection with DNA/RNA vaccines. Two mRNA vaccine spike protein sequences, which have been made publicly-available, were compared to the wild-type sequence in order to uncover possible differences in putative overlapping ORFs. Notably, the Moderna mRNA-1273 vaccine sequence is predicted to contain no frameshifted ORFs on the positive sense strand, which highlights the utility of codon optimization in DNA/RNA vaccine design to remove undesired overlapping ORFs. Since little information is available on ORF2b or ORF-Sh, we use structural bioinformatics techniques to investigate the structure-function relationship of these proteins. The presence of putative ORFs on DNA/RNA vaccine candidates implies that overlapping genes may contribute to the translation of smaller peptides, potentially leading to unintended clinical outcomes, and that the protein-coding potential of DNA/RNA vaccines should be rigorously examined prior to administration. The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA virus that was first described in late 2019 . The heaThe SARS-CoV-2 genome codes for at least 30 proteins, three of which are exposed on the virion surface and can be recognized by the immune cell system –10. The Numerous companies and academic institutions across the globe have developed or are currently developing DNA/RNA vaccines for the SARS-CoV-2 spike protein . GeneralAlthough SARS-CoV-2 DNA/RNA vaccines have been subjected to health and safety testing prior to bulk dissemination, a diverse assortment of both systemic and local (near injection site) side effects, ranging from mild to severe, following vaccination have been described , 27. SymThe understanding that one mRNA codes for one gene in eukaryotes, not considering alternative splicing, has been challenged after several studies have revealed the presence and translation of multiple open reading frames (ORFs) within one expressed mRNA , 37. AddSeveral ORFs have been found to overlap previously annotated genes in the SARS-CoV-2 proteome . For insSince little has been reported on the functionality of ORF2b or ORF-Sh in infected cells  and no homologous domains were found using sequence analysis tools, we used structural bioinformatics methodologies, as previously performed to elucidate the three-dimensional features of the SARS-CoV-2 proteome, to model the structural characteristics both ORFs –61. As shttps://github.com/NAalytics; https://berthub.eu/articles/posts/reverse-engineering-source-code-of-the-biontech-pfizer-vaccine/). The posting of these data allows direct comparative analyses between the vaccine-formulated and wild-type spike protein sequences.Although the presence of overlapping genes on the wild-type nucleotide sequence of the spike protein challenges the effectiveness of DNA/RNA vaccines, precautionary steps can be taken to prevent the translation of these smaller, internal ORFs. For example, vaccine nucleotide sequences can be selectively codon optimized, as is normally performed to enhance translation efficiency in host tissues, to remove alternative start codons and internal ribosome entry sites, thus preventing non-specific recognition by ribosomal complexes . Codon oComparing the nucleotide sequences of the wild-type and vaccine mRNA spike proteins may reveal the extent to which the sequences have been changed during codon optimization, thus potentially altering translation efficiency of the spike protein and overlapping ORFs. Of note, prior to codon optimization, both companies have reported including proline mutations to stabilize and preserve the spike protein structure, thus implying small changes in spike amino acid content as well. Using the EMBOSS Needle pairwise sequence alignment tool, the wild-type spike sequence (NCBI accession: NC_045512) is found to be 68.7% and 45.3% identical to the mRNA-1273 and BNT162b2 vaccine spike sequences (as opposed to the entirety of the mRNA sequence), respectively, and the mRNA-1273 and BNT162b2 spike sequences are 48.6% identical to one another , which has been noted to be an accurate reflector of gene translation, was calculated for all three spike sequences using the COUSIN and CAIcal web servers –71. As ahttps://www.ncbi.nlm.nih.gov/orffinder/). Although ORF identification using this tool does not imply translation, an overview of the available reading frames may provide insights into coding potential differences between the wild-type and vaccine candidate sequences. Minimum ORF length was set to the default 75 nucleotides, no alternative initiation codons were allowed, and only “ATG” start sites were considered.Considering the extensive codon optimization performed on the vaccine spike sequences, the comparison of putative ORFs in the wild-type and selected vaccine mRNA sequences may shed light on the protein-coding potential of DNA/RNA sequences used in SARS-CoV-2 vaccines. Thus, in order to examine the differences between the available ORFs on the wild-type, Moderna mRNA-1273, and Pfizer BNT162b2 sequences, putative ORFs of all three nucleotide sequences were detected using the NCBI ORFfinder web server  of the protein sequence that code for the most antigenic regions, as exemplified by the Pfizer BNT162b1 mRNA vaccine that codes for a trimeric construct of the receptor-binding domain of the SARS-CoV-2 spike protein . A shortMultimeric vaccine DNA/RNA sequences that include antigenic regions of different viral proteins could also be used to increase immunogenicity while shortening the length of the construct and, thus, controlling for the presence of overlapping ORFs –77. For The use of experimental techniques, such as ribosomal profiling or mass spectrometry, on vaccinated patient or laboratory animal samples or pseudovirus-infected tissue cultures may help determine whether the overlapping ORFs are translated and to what degree they are translated compared to the intended protein. Thus, several potential checkpoints can be utilized to control for the translation of small ORFs within DNA/RNA vaccine candidate sequences. Otherwise, unintended proteins could be translated by the host cell, which may lead to side effects resembling that of viral infection symptoms.DNA/RNA vaccines have proven to be an effective way to develop vaccines quickly for emerging pathogens. However, with a new set of solutions, comes a new set of problems . AlthougThe original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.CB, MB, and AV contributed to conception and design of the study. CB, MB, and AV contributed to sequence and structural analyses. All authors contributed to manuscript writing and revision. All authors contributed to the article and approved the submitted version.TB thanks the Wellcome Trust for support through an Investigator Award . CB was supported by Antibiotic Research UK (PHZJ/687).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.


Background: Myocardial blush grading is considered to be a novel tool for assessment of coronary microvasculature and myocardial perfusion in patients undergoing coronary angiography and angioplasty, and its reduction identifies patients at high risk. Our study aimed to evaluate the association between acute insulin resistance and myocardial blush in non-diabetic patients with ST-segment elevation myocardial infarction (STEMI).Methods: Two hundred forty non-diabetic patients with STEMI who underwent primary percutaneous coronary intervention were consecutively recruited. The relationship of homeostasis model assessment—estimated insulin resistance (HOMA-IR) to myocardial blush and in-hospital outcome was investigated.Results: Higher HOMA-IR tertile was observed in obese patients, with hyperinsulinemia, had Killip class >1, with higher CPK-MB level and was correlated to impaired myocardial blush after adjusting for the other confounding risk factors. It was also concluded that higher HOMA-IR was independently associated with no/minimal myocardial blush after STEMI. Moreover, it was founded to be an independent predictor of pulmonary edema and impaired left ventricular systolic function.Conclusion: This study revealed that acute insulin resistance was prevalent in non-diabetic patients with STEMI and was an independent predictor for post-infarction myocardial and microvascular injury and poor in-hospital outcome.Trial Registration: The trial was registered at the registry of Clinicaltrials.gov, ClinicalTrials.gov Identifier: NCT04651842, Date of registration: 2nd December 2020 Registry URL, https://clinicaltrials.gov/ct2/show/NCT04385589?cond=Dapagliflozin+in+diabetic+patients&cntry=EG&draw=2&rank=1. Complete myocardial reperfusion and restoration of coronary microcirculatory function (CMF) is a therapeutic goal in ST-segment elevation myocardial infarction (STEMI) . HoweverIt was found that insulin resistance (IR) plays substantial role in the development of cardiac-vascular infirmities and carries bad prognostic outcome for acute myocardial infarction (AMI) .IR was found to be related to cardiac injuries on both muscular and microvascular levels after STEMI. Likewise, after adjusting for other integrals of metabolic syndrome, IR was associated with myocardial injury after elective PCI .Now, IR was assessed by the homeostatic model assessment (HOMA) index in the early phase of acute coronary syndrome in non-diabetic patients. This “acute IR,” considered a part of the acute glycol-metabolic response to stress, may be transient and also can occur even in patients without chronic glycol-metabolic derangements .Acute IR includes acute hyperglycemia and/or acute hyperinsulinemia. Hyperglycemia has the importance of prognostic relevance of hyperinsulinemia in STEMI patients, but its relationship with coronary flow is still unclear , 8. HoweMyocardial blush is a qualitative visual assessment of the amount of contrast medium filling a territory supplied by a pericardial coronary artery  was fir. DecreasThe current study hypothesis was that acute IR can occur in the early post pPCI period even in non-diabetic patients as a dynamic phenomenon, and it could be related to the development of microvascular injury. Myocardial blush is defined as a marker of coronary microvascular function; accordingly, IR was evaluated in relation to myocardial blush in nondiabetic STEMI patients treated by pPCI as a primary endpoint. The residual ST-segment elevation, post-TFC% and major adverse cardiovascular events (MACE) were secondary endpoints. The HOMA index is a simple and inexpensive marker of IR, primary used in chronic states. It was recently validated in STEMI patients as feasible for assessing IR during myocardial infarction and therefore used in the current study .*Power 3 software. With a power of 95% and type I error of 5% (α = 0.05 and β = 95%), the minimum required sample was 210 patients  for an effect size of 10% in rate of myocardial blush grade.This cross-sectional comparative study included 240 non-diabetic patients with acute STEMI selected from the Cardiology Department, Assiut University Hospital, who underwent primary percutaneous coronary intervention (PCI) between May 1, 2018 and May 1, 2019. Sample size was calculated using the GExclusion criteria were diabetic patients, renal insufficiency, advanced hepatic dysfunction, had malignancy, chronic heart failure/cardiomyopathy, prior myocardial infarction, and diseases requiring steroid therapy; patients on antioxidant supplement/therapy within 4 weeks before enrolment in the study, pregnant females, alcoholic, patients allergic to radiographic contrast, and uncooperative/refusal.Ethical approval was obtained from the Medical Faculty, Assiut University (IRB No. 17300510) and was adherent to the guidelines of the declaration of Helsinki.Proper history taking including history for the presence of cardiovascular risk factors: hypertension, diabetes mellitus, dyslipidemia; smoking and family history of premature coronary artery disease, history of ischemia, previous CCU admission, and previous PCI.2 (kilograms per square meter).Complete physical examination including body mass index (BMI) was calculated using the formula of weight/heightBlood pressure was measured in a seated position after a 10-min rest, using an electronic blood pressure monitor .Diagnosis of diabetes was made according to the criteria of the American Diabetes Association, and prediabetes was defined by fasting blood glucose of 100 mg to <126 mg/dl, 2-h plasma glucose of 140 to <200 mg/dl, or HbA1C of 5.7 to <6.5% , and hyperlipidemia was diagnosed according to the guidelines of the National Cholesterol Education Program (ATP III) , 14.Blood samples were collected in an air-conditioned and quiet room. Serum glucose, blood urea, creatinine, total cholesterol, low-density lipoprotein—cholesterol (LDL-C), high-density lipoprotein—cholesterol (HDLC), and triglycerides were assessed using a HITACHI 912 Analyzer . Insulin and creatine kinase MB isoform level were assessed by AIA 900, fluorescence enzyme immunoassay (FEIA) method (Japanese).After obtaining informed consent, the included patients were subjected to:15. Normal reference levels for HOMA-IR range between 0.7 and 2.0. HOMA-IR is a preferred estimate for insulin resistance as glucose clamp methods, the current gold standard, are resource intensive and time consuming. Several studies use 2.0 as cutoff for increased insulin resistance /405After the angioplasty procedure immediately, epicardial and myocardial reperfusion was assessed and graded on the angiograms. In each patient, the best projection was chosen to assess the myocardial region of the infarct-related coronary artery, preferably without super positioning of non-infarcted myocardium. Angiographic runs must be long enough to allow some filling of the venous coronary system, and backflow of the contrast agent into the aorta have to be present to be sure of adequate contrast filling of the epicardial coronary artery.All angiograms will be made after 400 μg of nitroglycerin intracoronary has been given immediately after the primary angioplasty procedures, and this procedure allows quantitative coronary artery analysis : First, Myocardial reperfusion had been also determined by measuring ST-segment resolution (STR) analysis so ECGs had been done on admission (first ECG) and 90 min after the primary PCI in the coronary care unit (second ECG). The second ECGs had been classified with regard to the ST segment resolution into the following grades: Normalized, defined as no residual ST segment elevation; Improved, defined as a residual ST segment elevation <70% of that on the first ECG; Unchanged, defined as a residual ST segment elevation > 70% of that on the first ECG .For all patients, postprocedural transthoracic echocardiography left ventricular ejection fraction was measured by biplane Simpson method using the end diastolic and end systolic apical four- and two-chamber views for estimation of left ventricular volume and calculation of ejection fraction. In hospital primary angioplasty procedures, major adverse cardiovascular events (MACEs) were determined and defined as the composite of death, stent thrombosis, re-infarction, cardiogenic shock, and stroke .2 and Monte Carlo exact test was used to compare the difference in the distribution of frequencies among different groups. One-way ANOVA was used to determine the mean difference between groups. Binary logistic forward regression analysis was used to define the independent predictors of MBG variables that showed significant differences among the studied groups. A value of p < 0.05 was considered statistically significant.Data entry, verification, and validation were carried out by the researcher, and analyses were performed via IBM-SPSS software . Numerical data were expressed as mean ± SD, and frequency tables with percentages were used for categorical variables. χThis study aimed to evaluate the impact of insulin resistance in non-diabetic (IR) on myocardial blush and in hospital MACE in the setting of STEMI patients treated with PCI. Non-diabetic patients (274), with anterior and non-anterior STEMI, who were admitted to the ICU during the study period, were recruited. Thirty-four patients were excluded for recently discovered diabetes mellitus, left main thrombosis, aortic valve prostheses with embolization, stent thrombosis, and defect of data counting. Hence, 240 patients  were available for analysis .The majority of the studied patients were males aged >50 years with insignificant difference between groups in relation to HOMA-IR. Moreover, acute insulin resistance as estimated by HOMA-IR tertile was significantly higher in obese, those with heart failure (Killip class >1), and higher plasma insulin level, CPK-MB, and fasting blood glucose level .Although, patients with anterior wall infarction (AWI) had insignificantly higher HOMA-IR levels, those with higher HOMA-IR experienced significantly low-grade baseline TIMI as higher tertile was founded on grade 0 baseline TIMI . Also, pIt was found that patients with MBG 0, 1, 2 had higher HOMA-IR level than those with grade 3, and those who had unchanged ST segment in their ECG after reperfusion had higher HOMA-IR level. Furthermore, postprocedural TIMI frame count (TFC) was longer in those with higher HOMA-IR level. Patients with low, intermediate HOMA-IR levels showed higher postprocedural TIMI flow grade than Grade 3 .A higher level of HOMA-IR was detected in those with post MI pulmonary edema; however, prolonged hospital stays, arrhythmias, LVF, cardiogenic shock, re-infarction, and early stent thrombosis complicated higher HOMA-IR level but with insignificant difference .p = 0.001) and by 10% in the multivariate analysis  . Importantly, HOMA-IR level is considered an independent risk for decreasing myocardial blush, i.e., with one unit increase in HOMA-IR level, there was decreasing myocardial blush by 20% in univariate analysis  .The present study analyzed IR in the acute phase of STEMI in non-diabetic patients treated by pPCI. First, it confirmed that acute IR in the early post pPCI period is common even in non-diabetic patients; second, IR, as estimated by HOMA-IR, affects myocardial perfusion especially MBG; and third, IR was related to the in-hospital MACE.IR in the early phase of STEMI is considered part of the acute glycol-metabolic response to stress  GenerallMore cardiac necrosis was reported in those with higher IR. The relationship between acute IR in non-diabetic STEMI patients and myocardial damage in terms of peak enzymes was previously reported , 29. In per se predicted impaired epicardial flow before pPCI  were considered important predictors of markedly reduced myocardial blush. This may be explained by IR in the setting of STIMI being associated with poor myocardial reperfusion, impaired coronary microcirculation  and collThe current study revealed that acute IR was prevalent in non-diabetic patients with STEMI and was identified as an independent predictor of post infarction myocardial and microvascular injury. Likewise, it was found to be associated with poor in-hospital outcome.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by prof/Mahmoud abdel-Aleem- Medical ethics committee IRB no.17300510. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.SK and MA conceptualized and designed the study, conducted a literature search, conducted the clinical studies, prepared the experimental study manuscript, and edited and reviewed the article. AH was in charge of the definition of intellectual content, literature search and manuscript review, clinical studies, experimental studies, and data acquisition. GS conducted the clinical studies, experimental studies, and acquired the data. All 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.