This study aimed to investigate the clinical characteristics of acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) according to the 2016 World Health Organization classification and the preferred therapy for patients with AML-MRC aged 60-75 years.We retrospectively analyzed differences in clinical data among 190 patients with AML-MRC and 667 patients with AML not otherwise specified (AML-NOS). We also compared different therapeutic regimens among patients with AML-MRC aged 60-75 years.Compared with AML-NOS, patients with AML-MRC had significantly different clinical characteristics as well as worse overall survival (OS)  and complete remission rates . Multivariate analysis performed for the whole group (patients with both AML-MRC and AML-NOS) showed that AML-MRC was the independent prognostic factor (p=0.002). Additional multivariate analysis performed for 190 patients with AML-MRC indicated that age (p<0.001) and lactate dehydrogenase (p=0.031) were independent prognostic factors. Compared with the IA/DA regimen [idarubicin and cytarabine (IA) or daunorubicin and cytarabine (DA)], the DAC+CAG regimen [decitabine and half-dose CAG regimen ] was associated with better OS  in patients aged 60-75 years and categorized into the unfavorable risk group.AML-MRC cases exhibited worse clinical outcomes compared to AML-NOS. Compared to the IA/DA regimen, the DAC+CAG regimen was the optimal choice for patients with AML-MRC in the unfavorable risk group and aged 60-75 years. NPM1 or biallelic CEBPA were recategorized as having recurrent genetic abnormalities . In the supportive group, patients received hydroxyurea to inhibit the proliferation of leukemia cells or only supportive care such as transfusions of blood products when necessary and anti-infection therapy when patients had symptoms of infection.According to treatment regimens, patients with AML-MRC aged 60-75 years (n=99) were divided into three groups, including an IA/DA group (n=43), DAC+CAG group (n=49), and supportive group (n=7). Patients in the IA/DA group were treated with IA .NPM1 or biallelic mutation of CEBPA were recategorized as having recurrent genetic abnormalities , LDH , and MRC  were independent prognostic factors .In 190 patients with AML-MRC, univariate analysis suggested that LDH (p=0.031) and age (p<0.001) were prognostic factors, while WBC count, Hb, platelet count, history of MDS or MDS/MPN, MDS-related cytogenetic abnormalities, and MLD were not related to prognosis. Multivariate analysis showed that both age  and LDH  were independent prognostic factors for AML-MRC .There were 99 patients aged 60-75 years with AML-MRC, who could be categorized as belonging to the intermediate risk group (n=46) or unfavorable risk group (n=53) based on the 2017 ELN criteria. We analyzed the efficacy of different treatment regimens in each group. In the intermediate risk group, no significant difference was found between the IA/DA group and DAC+CAG group with respect to CR rate  or OS . However, in the unfavorable risk group, the OS of the DAC+CAG group was significantly better than that of the IA/DA group . The CR rate of the DAC+CAG group was higher than that of the IA/DA group, but the difference was not statistically significant .AML-MLD was first proposed in the 2001 WHO classification of myeloid neoplasms and acute leukemia and was classified as a separate category . The conWe analyzed the clinical features and prognosis of 857 AML patients including 190 patients with AML-MRC and 667 patients with AML-NOS based on the 2016 WHO classification of AML ,4,5. HowAs described above, many well-known adverse factors were observed in cases of AML-MRC, such as older age, unfavorable cytogenetics, and multidrug-resistant phenotype, which lead to unsatisfying therapeutic response and survival. Young patients in good physical condition are offered intensive chemotherapy followed by allo-HSCT. However, the optimal chemotherapy regimen for patients older than 60 years who are not eligible for allo-HCST has always been controversial. The standard 3+7 regimen, IA or DA, is the most common induction therapy, while the CAG regimen is another common choice and is often combined with decitabine. However, comparisons of the IA/DA and DAC+CAG regimens for AML-MRC patients aged 60-75 years have rarely been reported. In our study, no significant difference was found between the two regimens  in the intermediate risk group . In the tumor suppressorgene (TSG) impaired by the disorder of DNA methylation [Decitabine, a DNA-hypomethylating agent (HMA), can induce differentiation and apoptosis of leukemic blasts and activate the silenced hylation ,10,11. Rhylation ,13. Whenhylation ,15,16. Ihylation  enrolledhylation ,19,20. TDespite the efficacy of conventional chemotherapy, the survival of AML patients remains unsatisfactory. Over the decades, efforts made by researchers led to only minor improvements in the outcome of AML patients until the presence of several new therapies offered something fresh in the landscape of AML therapy. CPX-351, a liposomal formulation of cytarabine and daunorubicin, was approved by the US Food and Drug Administration in 2017 for the treatment of newly diagnosed therapy-related AML (tAML) or AML-MRC based on a phase III clinical trial named CLTR0310-301 . In thisThere are several limitations of our study. All data were collected in a retrospective manner and the scale of the cohort was small while analyzing the optimal choice for patients with AML-MRC aged 60-75 years in the unfavorable risk group.AML-MRC is associated with worse prognosis compared to AML-NOS and shows an independent prognostic effect. The DAC+CAG regimen may be preferred for patients aged 60-75 years who are classified in the unfavorable risk group.


The TC/BC interface morphology in APS TBC is one of the important factors leading to crack propagation and coating failure. Long cracks are found near the bulge on the TC/BC interface. In this study, the TBC model with the bulge on the interface is developed to explore the influence of the bulge on the coating failure. Dynamic TGO growth and crack propagation are considered in the model. The effects of the bulge on the stress state and crack propagation in the ceramic layer are examined. Moreover, the effects of the distribution and number of bulges are also investigated. The results show that the bulge on the interface results in the redistribution of local stress. The early cracking of the ceramic layer occurs near the top of the bulge. One bulge near the peak or valley of the interface leads to a coating life reduction of about 75% compared with that without a bulge. The increase in the number of bulges further decreases the coating life, which is independent of the bulge location. The results in this work indicate that a smooth TC/BC interface obtained by some possible surface treatments may be an optional scenario for improving coating life. Thermal barrier coatings (TBCs) are mainly coated on the high-temperature hot components for aero engines or gas turbines, such as combustion chambers, blades, and fuel nozzles. The thermal insulation of the ceramic material enables the substrate materials to be used at temperatures above their melting point ,2,3. TheThe TBC system has long been in service at high temperature. Since the ceramic material is almost oxygen-transparent, the Al ions in the BC layer react with the O ions from the air at high temperature to form thermally grown oxide (TGO). The TGO thickness increases with the oxidation time ,13. The The interface morphology between TC and BC severely affects the life of APS TBC. In order to obtain the optimal interface morphology, it is necessary to understand the influences of interface morphology characteristics on the stress distribution in the coating. The finite element method can be used for parametric analysis of interface morphology ,33,34,35The huge stress at the interface means that crack initiation can occur. In fact, there are a large number of two-dimensional pores between and inside the lamellae for the APS TBC, which act as cracks. The coating failure is caused by the propagation and coalescence of these cracks. In order to clarify how the TC/BC interface affects the coating failure, the influences of interface characteristic on the crack driving force and propagation behavior must be explored. Hille et al.  exploredBased on the studies of the interface morphology effects on the stress state or crack evolution, some interface optimization theories or methods have also been proposed. Yu et al.  establisThe TC/BC interface in the APS TBC is not completely smooth. Some local bulges appear on the interface, as shown in In this work, a TBC model with the bulge on the TC/BC interface was developed to study its failure mechanism under the cyclic thermal loading. The dynamic growth of TGO and the dynamic propagation of cracks are considered in the model. The effects of the bulge on the stress characteristic in the ceramic layer are first examined. Then, the dynamic propagation behavior of cracks in the coating containing the interface bulge was explored. In addition, the influences of the location and number of the bulge on the crack evolution and coating failure were also investigated.α-Al2O3 layer is obtained after the coating deposition [Here, the APS TBC is sprayed on nickel-based superalloy substrate (SUB) of 3.0 mm thickness. position ,55. The The whole model is divided into quadrilateral grids. Since the ceramic cracking near the interface is considered, the dense meshes are used near the interface and cracking path, as shown in 2 [The ceramic cracking simulation near the TC/BC interface is realized by the surface-based cohesive interaction in ABAQU software  . This me2 ,61. The 2 ,62. The 2 ,54.8YSZ is used to prepare the TC layer; NiCoCrAlTaY is used to deposit the BC layer. All layers are considered to be isotropic. Because the TC layer is a brittle material, it is treated as an elastic material. The BC layer exhibits elastic-plastic behavior. When the system temperature is higher than 750 °C, its yield strength is 200 MPa. If the temperature is lower than 300 °C, the yield strength is 1000 MPa. If the temperature increases from 300 °C to 750 °C, the yield strength changes linearly ,24. The The thickness of TGO shows a parabolic growth law with oxidation time at high temperature . The newThe TBC geometry in this study is a representative volume element of the whole sample. Therefore, the x-direction freedom of the left boundary in the model shown in N. A zero value for N represents the initial cooling process.The TBC system is often considered to be stress free at high temperature, which has been adopted in many studies ,24,63,65σ22 and shear stress σ12 contribute to the propagation of cracks parallel to the interface in the coating [Failure of APS TBC is the result of crack propagation and coalescence ,53,54. B coating ,65. TherN = 0), the tensile stress is located at the peak, and the valley is compressed, which is attributed to the larger shrinkage strain of the substrate compared with the ceramic layer. After one cycle (N = 1), the tensile stress shifts to both sides of the peak. As the thermal cycle progresses, the stress state near the interface basically no longer changes. However, the magnitude of tensile stress increases continuously, which is mainly attributed to the TGO growth at high temperature [Under the current condition, the stress distributions in the ceramic layer near the standard cosine interface without bulge are first studied, which is used to compare the results in the model with the bulge. perature ,45,62. FWhen a bulge appears in the middle of the TC/BC interface, the stress state near the interface is changed, which is shown in In order to clarify the dynamic change process of stress in the ceramic layer, the variation of normal and shear stress near the interface with the number of thermal cycles are demonstrated in The distribution of bulges on the interface in APS TBC is not regular. Moreover, the number of bulges is not fixed. Therefore, it is necessary to study the influences of the location and number of bulges on the stress state in the ceramic layer. In this study, the bulge may appear at three locations on the interface: near-valley, middle, and near-peak. The normal stress distribution characteristics under different bulge configurations are demonstrated in The change of stress distribution and magnitude in the ceramic layer can only indicate the ability of crack initiation or propagation. However, they cannot explain the influence of crack propagation on stress redistribution. Therefore, it is difficult to reveal coating failure directly by the stress state in the ceramic layer. In order to clarify the effect of the bulge on coating failure, the evolutions of the ceramic crack in the model with or without bulge on the interface are explored. It is noted that only when the damage initiation variable (CSMAXSCRT) reaches 1.0, the damage degradation (CSDMG) of the ceramic layer begins to increase from 0.0. If the CSDMG reaches 1.0, the node pairs on the crack surface begin to be released.N = 20), the stress magnitude is much lower than that in the model with a bulge (see N = 20), the maximum value is 0.88, indicating that the ceramic cracking does not occur in the model without a bulge.When there is no bulge on the interface, the stress in the ceramic layer does not increase much with the thermal cycle. After 20 cycles (ulge see . This inN = 1), the value of CSDMG near the top of the bulge is 0.28, which indicates that the damage degradation in the ceramic layer has occurred. The value of CSDMG increases with thermal cycling. When the cycle number reaches 10 (N = 10), the CSDMG has reached 0.91, which means that the ceramic cracking is about to occur. When the system undergoes more thermal cycles, the crack appears in the coating. It expands to the valley and peak. In a limited thermal cycle, the crack quickly propagates to the valley. Subsequently, the crack continues to expand to the peak. When the cycle number is equal to 20 (N = 20), the crack propagates to the near-peak. As the thermal cycle progresses, the CSDMG value in the right half of the model is always zero, indicating that the ceramic cracking in the left side does not affect the right side.When there is a bulge in the middle of the interface, the huge tensile and shear stress near the bulge leads to damage degradation in the ceramic layer, as shown in N = 20). It can be seen that no matter where the bulge is, the ceramic cracking first occurs near the top of the bulge. When the bulge is located in the middle of the interface, the crack propagation does not cross the peak. Moreover, the crack opening at the valley on the left side of the model is also significantly smaller than that in the model with a bulge near the valley or peak.Because the ceramic crack first appears near the top of the bulge, the location of the bulge may affect the initiation and total length of the crack. Besides the total length of the crack, the bulge location may also affect the cracking moment. N = 5). When the number of thermal cycles reaches 10 (N = 10), the ceramic crack has initiated from the top of the bulge at the near-valley and expanded to the near-peak region. Note that when only one bulge is located in the middle of the interface, the ceramic cracking does not occur when the cycle number is equal to 10 . However, when a bulge appears at the near-peak or near-valley location, the coating failure occurs after 16 cycles (N = 16). If two bulges appear on the interface, the number of thermal cycles at failure will be further reduced, which is independent of the bulge distribution.In order to quantitatively reveal the effect of the bulge on the coating failure, (1)The bulge results in the redistribution of local stress in the ceramic layer. Huge tensile and shear stress exists near the bulge.(2)The superposition of stress regions induced by multiple bulges causes the further increase in tensile stress region and magnitude.(3)The early ceramic cracking occurs near the top of the bulge. The bulge leads to a sharp increase in the crack length.(4)One bulge near the peak or valley of the interface results in a 75% reduction in the coating life compared with the bulge-free model.(5)The increase in the number of bulges will further decrease the coating life, which is independent of the bulge location.In this work, a TBC model with a bulge on the TC/BC interface is developed to explore the failure mechanism of the coating. A standard TBC model without bulge is also established for comparative analysis. The dynamic growth of TGO is included in the model through subroutine development. The initiation and propagation of cracks in ceramic layers are realized by the surface-based cohesive method. The effects of the bulge on the stress characteristic and crack evolution under cyclic thermal loading are implemented. The influences of bulge location and number on the coating failure are also explored. The main conclusions are as follows:


ER) and the mitochondrial UPR (UPRmt), respectively. These organellar UPRs play roles in shaping the cellular responses to proteostatic stress that occurs in aging and age-related neurodegeneration. The loss of adaptive UPRER and UPRmt signaling potency with age contributes to a feed-forward cycle of increasing protein stress and cellular dysfunction. Likewise, UPRER and UPRmt signaling is often altered in age-related neurodegenerative diseases; however, whether these changes counteract or contribute to the disease pathology appears to be context dependent. Intriguingly, altering organellar UPR signaling in animal models can reduce the pathological consequences of aging and neurodegeneration which has prompted clinical investigations of UPR signaling modulators as therapeutics. Here, we review the physiology of both the UPRER and the UPRmt, discuss how UPRER and UPRmt signaling changes in the context of aging and neurodegeneration, and highlight therapeutic strategies targeting the UPRER and UPRmt that may improve human health.Aging and age-related neurodegeneration are both associated with the accumulation of unfolded and abnormally folded proteins, highlighting the importance of protein homeostasis (termed proteostasis) in maintaining organismal health. To this end, two cellular compartments with essential protein folding functions, the endoplasmic reticulum (ER) and the mitochondria, are equipped with unique protein stress responses, known as the ER unfolded protein response (UPR ER) and the mitochondrial UPR (UPRmt), respectively. An accumulation of UPR research combining in vitro, in vivo, and human post-mortem data has revealed that these processes are intimately entwined with both aging and neurodegeneration. Here, we will review the core machinery responsible for UPRER and UPRmt function; how these components are affected with age and in age-related neurodegenerative disease (NDD); and how their genetic or pharmacological manipulation can, in turn, influence these biological processes to shape organismal health.Maintaining protein homeostasis (hereafter: proteostasis) within cells is essential for cellular health, especially in post-mitotic cells such as neurons that cannot dilute the ill-effects of misfolded, unfolded, or aggregated proteins through cell division. Several cellular compartments are more intimately in contact with proteins through their role as sites of protein synthesis and folding and are, therefore, more prone to encountering abnormal protein species over the lifetime of a cell. Two such compartments, the endoplasmic reticulum (ER) and the mitochondria, must maintain tight regulation of proteostasis on their specialized cellular functions and thus are equipped with dedicated protein stress responses. These organelles harbor molecular machinery to rapidly detect and mitigate proteotoxic stress through conserved signaling pathways that elicit defined transcriptional responses to promote proteostasis and sustain organellar and cellular health. Collectively, these elaborate networks of proteotoxic stress detection, signaling, and alleviation are termed the ER unfolded protein response , protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6 (ATF6)  see . Each prpathways .ER (hereafter: IRE1a-UPRER) (XBP1 into its active form as a potent transcription factor (XBP1s)  . Followi (XBP1s) . XBP1s t (XBP1s) . XBP1s-i (XBP1s) . Indepen (XBP1s) . ER-loca (XBP1s) .ER is governed by the protein kinase PERK (hereafter: PERK-UPRER). Like IRE1α, PERK undergoes dimerization and autophosphorylation following ER stress. Following its activation, PERK acts through its kinase domain to attenuate translation via the phosphorylation of eukaryotic initiation factor 2α (eIF2α) to prevent the influx of newly synthesized proteins into the ER . Upon ER stress, BiP dissociates from ATF6, unmasking two Golgi-localization signals that cause ATF6 to translocate to the Golgi body  neurons regulates metabolic alterations through UPRER activation in the liver  family of proteins and characterized by the permeabilization of the mitochondrial outer membrane (MOM) and release of pro-apoptotic factors including cytochrome c , can be Sustained IRE1α RNAse activity leads to the decay of a select group of precursor miRNAs that typically repress the translation of initiator protease Caspase-2 . This alER to decide cell fate, but how this balance is tipped to favor a specific fate remains an important question in the field  accumulated misfolded proteins, (2) impaired mitochondrial translation, (3) an imbalance in the ratio of mitochondria-derived to nuclear-derived proteins in the mitochondria , or (4) impaired mitochondrial protein import. Early work in the field using monkey kidney cells showed that misfolded mitochondrial proteins cause an increase in protease and chaperone transcript and protein levels , reduces protein import and activates the UPRmt  dysfunction can also initiate the UPRnd ubl-5 . Further mammals . Additiomals    . Two othha (ERα) , 2014. Amt is the ATF5-UPRmt. In both worms and mammals, the ATF5-UPRmt modulates both transcription and chromatin organization to counter mitochondrial stress  an upregulation of the expression of mitochondrial chaperones, proteases, protein importers, and ETC components; (2) a reduction in global protein translation in the cytosol; (3) a decrease in protein translation within mitochondria; and (4) a reprogramming of mitochondrial metabolism  responds to proteotoxic stress within the mitochondrial inner membrane space (IMS)  . Proteotce (IMS) . ERα incrial IMS . Interesctivated . This sumt signals through SIRT3 and the transcription factor FOXO3 (hereafter: SIRT3-UPRmt)  . SIRT3-U changes . The SIR changes . As an e changes .mt and SIRT3-UPRmt, there is significant evidence that the SIRT3-UPRmt is a bona fide unique and parallel arm of the UPRmt that is separable from the ATF5-UPRmt  pathway, whereby xbp-1 coordinates with daf-16, the C. elegans ortholog of FOXO3, to upregulate genes involved in longevity and ER stress resistance  mutants under high nutrient conditions  regeneration to maintain proliferative homeostasis , a non-specific pore in the MIM that allows for increased movement of contents between the mitochondrial matrix and IMS , including Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) . Two comAlzheimer’s disease is a NDD that primarily affects cognitive function, and represents the most common cause of dementia worldwide . HistoloER activity in AD. In the affected brain regions of AD patients, the molecular chaperone BiP is elevated in neurons at an early stage of AD pathology, suggesting that the UPRER is activated early as a protective response in AD  rescues structural abnormalities and memory deficits  restores cognitive function and reduces Aβ deposition , a generator of ROS that is acutely toxic to dopaminergic neurons  and chromosome 9 open reading frame 72 (c9ORF72), can cause ALS, FTD, or both (c9ORF72, the highest frequency familial ALS/FTD causing lesion (Amyotrophic lateral sclerosis is a NDD characterized cellularly by motor neuron death and clinically by muscle weakness and atrophy leading to paralysis and death typically within 2–5 years of disease onset . Additio or both . The maj or both . Howeverg lesion .ER chaperones appears to be a common feature in ALS patient samples. Both BiP and PDI have been observed in ALS spinal cord tissue (ER transcription factors XBP1 (c9ORF72 ALS patient tissue identified widespread alterations to genes involved in the UPRER in both cerebellum and cortex samples (ER activation is also associated with FTD; p-IRE1α, p-PERK, and p-eIF2α are elevated in c9ORF72 FTD patient samples, observed in close association with DPR inclusions (ER activation appear across the ALS/FTD continuum, suggesting that UPRER activity may represent a common pathological feature amongst the range of lesions known to cause these diseases.The activation of UPRd tissue , as haveors XBP1  and CHOPors XBP1 . Furtherors XBP1 . Finally samples . UPRER aclusions . TherefoER components to disease pathology. Knockdown of IRE1α or Xbp1 reduces mutant SOD1 aggregation in motor neurons both in vitro and in mutant SOD1 mice, subsequently extending lifespan and reducing apoptotic cell death (Xbp1 deficiency impairs ERAD function but upregulates autophagy, suggesting that compensatory degradative pathways are activated in this paradigm (ER acts protectively in ALS pathogenesis (Atf4 in mutant SOD1 mice reduces developmental viability but ameliorates disease phenotypes, possibly via the reduction of apoptotic effector CHOP (ER to mutant SOD1 forms of ALS/FTD remains up for debate, whilst its participation in other forms of ALS/FTD pathology is largely unexplored.Experimental models of ALS/FTD have been used to investigate the contribution of upregulated UPRll death . Intriguparadigm . Initialogenesis , 2014a, ogenesis . Howevertor CHOP . Thus, tmt is intimately involved in AD pathobiology. Genes involved in the UPRmt, such as YMEL1L, which encodes a mitochondrial AAA + protease and HSP60, which encodes a chaperone, are upregulated in post-mortem brain tissue obtained from patients with AD (mt-related genes is also recapitulated in the nervous systems of animal models of AD (mt. Additionally, the finding that resveratrol, an activator of sirtuins, reduces Aβ toxicity in a ubl-5-dependent fashion suggests that Aβ may activate both the ATF5-UPRmt and SIRT3-UPRmt (mt is activated in AD, it is clear from multiple independent studies that the activation is largely beneficial. Brief exposure of human neuroblastoma-derived SH-SY5Y cells to Aβ fragments causes the accumulation of the mitochondrial proteases LONP1, HTRA2, and CLPP and as well as the mitochondrial chaperone HSP60, indicating that the UPRmt has been activated (mt prevents Aβ-induced cellular toxicity which suggests that acute UPRmt activation may represent a beneficial compensatory cellular response (atfs-1 in a C. elegans model of AD further induces the UPRmt and improves healthspan suggesting that the induction of the UPRmt offsets some of the pathological effects of Aβ aggregation (mt worsens cellular features commonly associated with AD such as an increased Aβ42:Aβ40 ratio and increased phosphorylated tau in an AD-like human cerebral organoid model (Data from both animal models and humans show that the UPR with AD . Upregulls of AD . Based oT3-UPRmt . Regardlctivated . This inresponse . Additioregation . Lastly,id model .mt (mt activation. However, transcript and protein levels of UPRmt-related proteases and chaperones are increased in human cell lines exposed to the PD-causing toxin MPP+ suggesting that the UPRmt may indeed be activated in PD (mt plays a role in PD in humans is the finding that some PD patients have mutations in HTRA2, a key player in the ERα-UPRmt (mt has been shown to exert both protective and damaging effects. In MPP+-treated SH-SY5Y cells, further activation of the UPRmt reduces cell death (mt decreases lifespan and worsens dopaminergic neuron loss (mt component CLPP, which encodes a mitochondrial matrix protease, in induced pluripotent stem cell-derived neurons from PD patients reduces pathogenic α-syn phosphorylation, reduces mitochondrial oxidative stress, and restores neuron morphology (mt is detrimental for lifespan and healthspan in C. elegans overexpressing a mutant form of α-syn. It is likely that this chronic UPRmt hyperactivation exerts detrimental cellular effects in concert with aggregates of α-syn. Mechanistically, it appears that aggregation-prone α-syn accumulates within mitochondria to activate the UPRmt directly (mt is beneficial under many circumstances, save those where the UPRmt is chronically over-activated.Mitochondrial dysfunction is a key player in the pathophysiology of PD, and many genetic and toxic causes of PD are also known to activate the UPRmt . To our ed in PD . BolsterRα-UPRmt . In animll death . Additioron loss . Furtherrphology . In contdirectly . Overallmt is activated in the context of ALS/FTD-related mutant TDP-43, SOD1, CHCHD10, and FUS; however, it remains unclear what role this UPRmt activation plays in the ALS/FTD pathobiology (mt, likely through inhibition of ETC complex I leading to reduced mitochondrial ATP synthesis, in both human cell lines and transgenic Drosophila models of ALS/FTD (mt acts to counter the detrimental effects of TDP-43 (SOD1, the ATF5-UPRmt appears to be activated in the early stages of ALS disease in mouse spinal cord (mt effectors such as the IMS protease HTRA2 (CHCHD10 cause mitochondrial dysfunction, impaired mitochondrial proteostasis, and the activation of the UPRmt (mt and SIRT3-UPRmt as evidenced by enhanced activation of the transcription factors ATF4, ATF5, and FOXO3 (FUS leads to disruption of the ATP synthase complex and activation of the UPRmt, both of which contribute to the FUS-induced neurodegeneration (mt, although it cannot be ruled out that differences in disease models account for the contradictory effects of UPRmt activation on cellular survival.Mounting evidence suggests that the UPRobiology . Mutant  ALS/FTD . In thesf TDP-43 . In trannal cord . Additiose HTRA2 . Mutatiohe UPRmt . The losnd FOXO3 . Lastly,neration . These dER and UPRmt activation is a common feature in patient post-mortem nervous system tissue. Whether this activation represents protective or detrimental signaling is complex. Activation of the UPRER by virally delivered XBP1 or XBP1/ATF6 fusion protein appears to be protective across multiple NDD contexts (ER signaling can drive pathogenesis under certain disease states, and so future work aimed at better defining both protective and detrimental UPRER signatures in disease models will be critical moving forward to allow targeting of therapies to the appropriate disease populations. Disease stage and context also appear to define the contribution of the UPRmt to disease onset and progression. The currently available data paint a picture where the UPRmt is acutely activated early in the course of age-related NDD to preserve cellular health. At this stage, it is likely that moderate levels of proteostasis impairment trigger the UPRmt to preserve mitochondrial and cellular integrity and health. This view is bolstered by data suggesting that inducing the accumulation of proteins within the mitochondria does not lead to cell death but does activate the UPRmt (mt, likely in response to chronic or severe proteotoxic stress that occurs with the progression of age-related NDD, can elicit detrimental effects (The inability of cells and tissues to maintain proteostasis, including mitochondrial proteostasis, appears to be central to the pathophysiology of many age-related NDDs . Thus, icontexts . Howeverhe UPRmt . However effects . Thus, tER and UPRmt signaling has been shown to affect aging and age-related NDDs. Pharmacological agents targeting the UPRER and UPRmt may, therefore, hold promise as therapeutics (see ER and UPRmt signaling to elicit beneficial effects; we also discuss the next steps needed to translate these pre-clinical findings into clinical therapeutics. For a more comprehensive review of therapeutic strategies targeting UPR function, including chemical chaperones, we refer readers to a recent review (Modification of UPRtics see . Here, wt review .ER has received little attention in the context of aging and NDD. However, a recent study screening for compounds that activate XBP1 splicing through IRE1α identified three promising options. IXA1, IXA4, and IXA6 selectively activate XBP1 signaling without affecting other functions of IRE1α, and IXA4 and IXA6 reduce levels of Aβ by improving proteostasis (ER signaling and further testing of these strategies in NDD models is warranted.Pharmacological targeting of the IRE1α-UPReostasis . Thus, sER, pharmacological modulation of the PERK-UPRER signaling pathway has been investigated extensively. Enhancing PERK-eIF2α signaling through the inhibition of eIF2α dephosphorylation has provided mixed results, with evidence that it can either protect (In contrast to the IRE1α-UPR protect  or exace protect  NDDs pat protect , but als protect , highlig protect , and has protect , suggestER, pharmacological strategies targeting ATF6 in aging and neurodegeneration have not yet been thoroughly explored. A recent screen to identify small regulators of ER proteostasis identified a number of regulators of ATF6-dependent transcriptional pathways (Similar to the IRE1α-UPRpathways . One compathways . Moving mt to avert the deleterious effects of aging and neurodegeneration. First, antibiotics that inhibit mitochondrial protein synthesis, namely doxycycline and chloramphenicol, have been shown to upregulate the expression of ATF5-dependent UPRmt genes and extend lifespan in C. elegans (mt-linked genes (+, or mimic this effect, appear to activate the ATF5-UPRmt and SIRT3-UPRmt to extend lifespan and protect against NDD-related cellular and organismal decline in various model systems (Two therapeutic strategies appear capable of activating the ATF5-UPR elegans . Furthered genes . Second, systems .mt that have been specifically investigated in the context of aging or neurodegeneration. As current data suggest that the ERα-UPRmt does not play a role in modulating aging nor neurodegeneration, more research is needed in this area to assess whether pharmacological targeting of this pathway is a worthwhile endeavor.Currently, there exist no drugs targeting the ERα-UPR+ modulators not only activate the ATF5-UPRmt but also the SIRT3-UPRmt. As drugs that increase the cellular NAD+ concentration appear to extend lifespan and protect against neurodegeneration in model systems, future research investigating whether activation of the SIRT3-UPRmt independently of the ATF5-UPRmt achieves these same effects could aid in the design of more targeted therapeutics with the potential to increase resistance to the ill-effects of aging and NDD.As discussed previously, many NADER or UPRmt may hold promise as a therapeutic strategy to combat the ill-effects of aging and NDDs. However, many issues remain to be solved before therapeutic strategies targeting the UPRER and UPRmt can be translated to humans. First, it remains a key question how best to harness beneficial UPR signaling without inducing the pathological effects of chronic hyperactivity, though recent studies of the UPRER support the idea that fine-tuning UPR activity may provide a better template than directly targeting the master regulators. Second, it remains to be seen whether different neuronal subtypes respond similarly to modulators of the UPRs; activation of either of the organellar UPRs may be beneficial in some neurons but harmful in others. Third, as both the UPRER and UPRmt are composed of multiple, parallel signaling pathways, more research is needed to assess how individual pathways may compensate for drug-induced changes in another. Fourth, activation of the UPRER or UPRmt appears to be beneficial in some contexts and detrimental in others. Therefore, UPR-modifying therapies will rely upon the identification of biomarkers that can differentiate beneficial from detrimental circumstances. Lastly, numerous reports have suggested that there are non-cell autonomous effects of activating the UPR in one tissue; understanding how the activation of the UPR in multiple tissues simultaneously affects organismal health will be important when considering if UPR modulators represent an avenue to stave off the detrimental effects of aging and NDD.Taken together, current data suggest that targeting either the UPRER and UPRmt both have complex relationships with aging and age-related NDDs. These relationships share common features as both UPRs experience a functional decline with age, while evidence of their activation is frequently observed in post-mortem NDD patient tissue. Additionally, genetic studies in model organisms demonstrate that manipulations of either UPR can influence aging and neurodegeneration, though these changes remain complex and context dependent. Activating specific components of either mechanism can prove beneficial in ameliorating disease phenotypes and extending lifespan under some circumstances, though sustained activation results in dysregulated signaling and can instead exacerbate pathology. Thus, how to best harness beneficial UPR functionality, while avoiding the consequences of sustained activity, remains a critical question when considering the therapeutic potential of UPR modulation for aging and age-related NDDs.The decline of proteostasis and mitochondrial dysfunction are both closely tied to the deterioration of organismal health, and it is clear from the data collected thus far that the UPRAPKW and AWS conducted the literature search and wrote the first draft of the manuscript. APKW designed the figures. All authors contributed to conceptualizing, reviewing, and editing the manuscript, approved the submission 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.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.


Drosophila melanogaster) causes the membrane potential to become more negative. The mechanism for this change was investigated in this study. It appears this may be due to a potassium ion leaving the cell, and this response is independent of calcium ions flowing into the cell. The direct action of a toxin, lipopolysaccharide, which is released from bacteria, on tissues is still not well understood. Skeletal muscle exposed to lipopolysaccharide in the larvae of the fruit fly , or the opening of Cl− channels. The purpose of this study was to further investigate the mechanism of the hyperpolarization of the larval Drosophila muscle due to exposure of LPS using several different experimental paradigms. It appears this response is unlikely related to activation of the Na-K pump or Ca2+ influx. The unknown activation of a K+ efflux could be responsible. This will be an important factor to consider in treatments of bacterial septicemia and cellular energy demands.The endotoxin lipopolysaccharide (LPS) from Gram-negative bacteria exerts a direct and rapid effect on tissues. While most attention is given to the downstream actions of the immune system in response to LPS, this study focuses on the direct actions of LPS on skeletal muscle in  Escherichia coli, Pseudomonas aeruginosa, and Serratia marcescens o. Each preparation showed a depolarization over time during incubation with ouabain  and the preparations in which LPS was used in combination with ouabain hyperpolarized . The 1 mM and 10 mM exposure to ouabain depolarized the membrane to the same extent, suggesting 1 mM is sufficient to block the pump. With either concentration of ouabain, the membrane still rapidly hyperpolarized with exposure to LPS in saline containing the same concentration of ouabain during the 10 min pre-exposure.  current to account for the hyperpolarization, a Ca2+-free saline was used. Preparations were bathed in the saline for 2 min prior to exposure to LPS. The initial saline was flushed three times with the saline not containing any added Ca2+. This was followed by the saline without Ca2+ but with LPS. In every preparation, there was depolarization as a result of switching the saline to one without Ca2+ added. However, the addition of LPS with saline without Ca2+ still produced a pronounced hyperpolarization in the preparations . Upon switching the saline back to one containing Ca2+ without LPS, there was still a slight hyperpolarization. This response could be related to compensation for the saline without Ca2+ or an effect of LPS still present on the membrane and responding once Ca2+ was added. To examine the potential of the influx of CaDrosophila conducts electrical depolarization by voltage-gated Na+ and Ca2+ channels on the plasma membrane. Since Ca2+ entry can potentially activate K(Ca) channels, Cd2+ was substituted for Ca2+ in the saline prior to LPS exposure. The incubation in Ca2+-free saline containing Cd2+ would block all voltage-gated Ca2+ channels on the plasma membrane. The Ca2+-free saline still produced depolarization of the membrane, indicating Cd2+ does not replace the effect of Ca2+ in this regard. This effect was present in all preparations examined . The additional exposure to LPS in saline without Ca2+ but containing Cd2+ still produced a rapid hyperpolarization . Initially, the effect of Cd2+ was examined in standard saline containing Ca2+, but since the effect of exposure to LPS was still prominent in a hyperpolarization  a saline without Ca2+ added was examined, assuming that no potential Ca2+ would enter the muscle. An additional approach to potentially block Ca2+ influx and not activate K(Ca) channels was examined using Ba2+ and gadolinium chloride (Gd3+). The same trends were observed for [Ca2+]o that was substituted for the same [Ba2+]o or [Gd3+]o. In each preparation, the membrane was depolarized . Additionally, LPS exposure in saline with the same [Ca2+]o substituted for [Ba2+]o or [Gd3+]o still showed rapid hyperpolarization .The skeletal muscle of larval 2+ influx was induced by LPS. In addition, some preparations were not utilized due to the muscle contracting when exposed to LPS. As noted in earlier studies, sometimes there would be a burst of vesicular fusion events with LPS exposure, which could induce the muscle to contract o is reduced [2+ added. It has been suggested that the depolarization of the membrane potential due to reduced [Ca2+]o and the hyperpolarization due to raising [Ca2+]o is likely induced by calcium-activated potassium channels K(Ca) o for [Ba2+]o, [Cd2+]o, and [Gd3+]o would indicate that there is likely a LPS-activated type of K+ channel or a K(Ca) current not blocked by these ionic substitutions responsible for the hyperpolarization.Given that the larval  reduced . It was ls K(Ca) . So, to Drosophila. However, the membrane potential did gradually depolarize with exposure to ouabain over 10 min, which was also shown to occur in prior studies o was not a high enough concentration to reduce the effect of Ca2+ on K(Ca) or induction of muscle contraction. In saline substituting [Ca2+]o for [Ba2+]o, depolarization of the membrane potential still occurred. So, Ba2+ does not appear to block the effect of the membrane depolarization by reduced [Ca2+]o. The addition of LPS in saline with Ba2+ substituted for Ca2+ greatly reduced the hyperpolarization. Thus, it is feasible that the depolarization of muscle with lowered [Ca2+]o is due to an effect on screening of the membrane and proteins by Ca2+ o was due to a depressed K(Ca) current or a screening of membrane charge, [Ca2+]o was substituted for [Cd2+]o. Cd2+ normally blocks voltage-gated Ca2+ channels. Interestingly, the membrane still depolarized with [Ca2+]o (1 mM) substituted for [Cd2+]o (1 mM); furthermore, the membrane was still hyperpolarized when exposed to LPS. Thus, this would suggest that if K(Ca) current is responsible for the LPS-induced hyperpolarization, Cd2+ is activating a K(Ca) current. This is feasible given that cadmium or strontium can activate K(Ca) channels in neurons of mollusks o as a substitute for [Ca2+]o still resulted in depolarization of the membrane, and the addition LPS still produced hyperpolarization. This would indicate that the depolarization is induced by a Ca2+ mechanism that cannot be substituted by Gd3+. The LPS hyperpolarization may be connected to a similar mechanism for all ionic substitutions. The LPS-induced hyperpolarization was not blocked in any of the ionic substitution experiments. Both phenomena of membrane potential changes indicate that K+ channels or K(Ca) channels could account for the effects. Some studies have used a substitution of Ca2+ for Gd3+ to block K(Ca) effects in smooth muscle [3+ is toxic at high concentrations, as it is known to block presynaptic Ca2+ channels in nerves and Ca2+ channels in cardiac tissue [The use of [Gdh muscle , but thic tissue ,46,47,482+ substitutes for Ca2+, an internal release of Ca2+ from a sarcoplasmic reticulum (SER) is suggested. However, larval Drosophila skeletal muscle does not have a robust SER. It would appear that Cd2+ can also substitute for Ca2+ in binding to troponin to induce skeletal muscle contraction, as is reported in other studies [2+ was able to move into the cytoplasm of the muscle to come into contact with troponin.Since the muscle can still contract when Cd studies ,50. So, − channel. Considering that the chloride equilibrium potential (−40 to −20 mV in Cl− loaded fibers) is above resting membrane potential in the body wall muscle of Drosophila larvae, this possibility was not an option [Drosophila muscle did not block the LPS-induced hyperpolarization [Other possible scenarios in the mechanism behind the LPS-induced hyperpolarization were considered, such as activating a Cln option ,30. Muscn option . TEA incrization ; howeverrization .Drosophila muscle and moth muscle indicated that the muscle potential for K+ may exceed −90 mV [+ reversal potential of around −50 to −60 mV was rationalized by pointing out the more active metabolic processes, as compared to those taken into consideration in the experimental paradigms to measure K+ reversal potential [+ current and the resting potential [+ current measures, and not all insects will show similar responses to LPS exposure to the body wall muscles. In addition, the larval and adult forms of body wall muscles may likely differ in equilibrium potential for K+. How the membrane can become so rapidly hyperpolarized to values such as −80 mV by LPS exposure needs to be addressed by ionic driving gradients to account for the change. Early studies in adult d −90 mV ,53. Thisotential ,51,53,54otential . This il+ equilibrium potential and measured K+ reversal potential. The equilibrium potential for K+ is very negative. The presence of a Na+ leak, a constituently active K(Ca) current, or even a Cl− leak in an aggerate of fluxes could maintain the membrane potential at rest more depolarized than the equilibrium potential for K+. It is likely these other currents were not blocked or experimentally considered when determining the K+ reversal potential as a measure of the K+ equilibrium potential in earlier studies [Since the dissections and time taken to measure membrane potentials are relatively rapid, it makes it unlikely that active metabolic processes can explain the differences in measured K studies .+ current either directly or through K(Ca) channels to result in transient hyperpolarization. It is feasible that the hyperpolarization slowly decreases as the membrane repolarizes due to the activity of the Na-K ATPase pump. Since the pump is electrogenic and not as active with hyperpolarization as depolarization, this homeostatic drive is slower with hyperpolarization. However, the prolonged exposure to LPS will result in a depolarized state, which is possibly due to plasma membrane break downing as the resting membrane potential is lost. This cell degradation might be induced by an excess of Ca2+ or Na+ loading over time.It is most likely that LPS exposure activates a KDrosophila, as used in mammalian tissues, can be tested to target K+ channels and pumps. However, the agents need to be confirmed to work on Drosophila isoforms of the proteins [Additional pharmacological approaches for examination in larval proteins . It woulproteins ,23,58,59proteins ,61.


The crystallinity of the synthesized NFNPs was confirmed using X-ray diffraction (XRD) technique. The saturation magnetization (46.7 emug−1) was measured using vibrating sample magnetometer (VSM) and 0.35-tesla magnetron by magnetic resonance imaging (MRI). The adverse effects of NFNPs on blood biochemistry and histoarchitecture of the liver, kidneys, spleen, brain, and heart of the rabbits were determined. A total of sixteen adult rabbits, healthy and free from any apparent infection, were blindly placed in two groups. The rabbits in group A served as control, while the rabbits in group B received a single dose (via ear vein) of NFNPs for ten days. The blood and visceral tissues were collected from each rabbit at days 5 and 10 of posttreatment. The results on blood and serum biochemistry profile indicated significant variation in hematological and serum biomarkers in NFNP-treated rabbits. The results showed an increased quantity of oxidative stress and depletion of antioxidant enzymes in treated rabbits. Various serum biochemical tests exhibited significantly higher concentrations of different liver function tests, kidney function tests, and cardiac biomarkers. Histopathologically, the liver showed congestion, edema, atrophy, and degeneration of hepatocytes. The kidneys exhibited hemorrhages, atrophy of renal tubule, degeneration, and necrosis of renal tubules, whereas coagulative necrosis, neutrophilic infiltration, and severe myocarditis were seen in different sections of the heart. The brain of the treated rabbits revealed necrosis of neurons, neuron atrophy, and microgliosis. In conclusion, the current study results indicated that the highest concentration of NPs induced adverse effects on multiple tissues of the rabbits.From the past few decades, attention towards the biological evaluation of nanoparticles (NPs) has increased due to the persistent and extensive application of NPs in various fields, including biomedical science, modern industry, magnetic resonance imaging, and the construction of sensors. Therefore, in the current study, magnetic nickel ferrite (NiFe During the last few decades, application of nanotechnology in different fields like treatment therapy, biotechnology, nutrition, aerospace engineering, and electronics has rapidly grown across the globe. Nanoparticles (NPs) are extensively and persistently investigated for their toxic impacts on the cells, immune tissues, and DNA damages , 2. The All the chemicals were of analytical grade and obtained from Sigma-Aldrich (USA) and Merck (Germany). Commercial chemicals were procured from Sigma and utilized without any further purification. NFNPs were produced using the coprecipitation technique , 34.α1 radiation, λ = 1.54 Å). The Scherrer equation (Dhkl = 0.9λ/β cos θ) was employed to investigate the average crystallite size of the produced samples [The X-ray diffraction (XRD) technique was carried out to confirm the crystalline structure of the synthesized nanoparticles. The crystallinity, average particle size, and phase identification of nanoparticles were determined using a Bruker-D8 Advance Laboratory Diffractometer  was used to examine the microstructure morphology of the synthesized NPs. Also, it confirmed the size of the synthesized nanoparticles. A suitable statistical histogram plot was used to determine the mean particle size , 38.The VSM analysis was used to confirm the magnetic behavior of the synthesized material. According to the previous literature , 40, theMRI was used to investigate the contrasting impact of our synthesized samples in rabbit liver and spleen. MRI was also used to examine the intensity of the region of interest in rabbit organs. Relaxivity of the liver and spleen was determined using MRI data , 42.Oryctolagus cuniculus) of the same age and body mass were obtained from a local private market of district Bahawalpur, Punjab Province, Pakistan. Study rabbits were kept under similar housing conditions. All the animals were free from any clinical signs of disease and had free access to normal food and clean water during the trial. The animal house was cleaned and disinfected before the start of the trial. After a few days of adaptation to laboratory conditions, the rabbits were randomly picked, divided, and placed in two groups equally. After that, the rabbits in group B were administered with NFNPs via ear vein, while the rabbits in group A were considered normal control animals. All the experimental rabbits were monitored for clinical and behavioral changes throughout the trial.For biological evaluation, a total of 16 rabbits  of each rabbits at days 5 and 10 of postexposure. All the blood samples were immediately processed for hematological and serum biochemistry changes. The hematological profile of each rabbit was determined using an automated hematology analyzer  according to a previous protocol , 44. TheThe quantity of different serum antioxidant enzymes like catalase (CAT), supper oxide dismutase (SOD), reduced glutathione, and oxidative stress parameters such as malondialdehyde concentrations (MDA) were determined with the help of spectrophotometer according to the previous procedure .For gross and microscopic changes, four rabbit from each group were euthanized after blood collection on days 5 and 10 of the experiment. The heart, brain, liver, spleen, and kidneys were carefully examined, and a small piece of each tissue was fixed in neutral buffered formalin (10%). After a few days of preservation, all the tissues were processed for microscopic observation using standard histological procedures of commonly used hematoxylin and eosin staining .The data on different blood and serum profile was subjected to suitable statistical software for a significant difference. For the pictorial presentation, MRI (RadiAnt DICOM Viewer), XRD , VSM (Origin pro), and TEM (ImageJ) software were used.D = 0.9λ/β cos θ was 30 nm. The structure of synthesized NFNPs by TEM analyses is indicated in −1.The results on XRD patterns of NFNPs with prominent peaks having hkl values (220), (311), (222), (400), (422), (511), and (440) planes at 2theta angles of 30.4, 35.9, 37.3, 43.7, 53.9, 57.7, and 63.1 fully matched with JCPDS (File no. 10-0325) are presented in I = 94.2, SD = 28) and spleen . T1 and T2 relaxivity  were significantly higher in treated rabbits on day 10 of the trial .At the necropsy level, no gross ailments were observed in the visceral organs of control and treated rabbits. Microscopic observation of kidneys of nanoparticle-treated rabbits did not exhibit histological alterations at day 5 of posttreatment. However, different microscopic abnormalities in the kidneys of rabbits at day 10 of postexposure tissue like severe necrosis, hemorrhages, congestion, widening of urinary space, atrophy of renal tubule, degeneration of glomeruli, and necrosis of renal tubules were observed. Different histological alterations like necrosis of hepatocyte, congestion, edema, atrophy, and degeneration of hepatocyte and congestion were observed in liver sections of treated rabbits at day 10 of exposure. At the microscopic level, various sections of the heart of treated rabbits exhibited congestion, coagulative necrosis, edema, neutrophilic infiltration, severe myocarditis, and degeneration of cardiac myocytes . Microsc−1. Further doping the metals such as nickel and others can be used to increase the further high magnetic moment of ferrites [Due to the widespread use of nanoparticles in biomedical and biological sciences, the studies have been focused on the monitoring of deleterious impacts of nanoparticles both on the environment and public health , 49. Dueferrites .As a consequence, the proposed NFNPs could be used as a negative contrast in diagnostic testing to identify organ pathology even in MRI equipment with specialized fields . NFNPs wIn our experimental study, various blood parameters, including lymphocytes, monocytes, hemoglobin, and red blood cells, significantly reduced, while white blood cells and neutrophils significantly increased. The lower hematological parameters in NFNP-treated rabbits could be due to the deleterious effects of NFNPs on blood-forming tissues. The lower hematological profile could also be related to the induction of oxidative stress on bone marrow. The increased total white blood cells and neutrophils can be due to injuries in association with the increased and rapid generation of free radicals in treated rabbits . The sigIn the present study, significantly increased concentrations of different serum biochemical biomarkers, including serum enzymes like aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase along with serum bilirubin, were significantly increased in treated rabbits. The increased concentration of liver function test, kidney function test, and different other serum parameters in treated rabbits could be due to the toxic effects of NFNPs on the liver, kidney, and different other tissues of the body. The significantly increased concentration of serum biochemistry parameters might be due to oxidative stress in the liver and kidneys in association with induction of tumor necrosis factor in NFNPs . PreviouIn the published literature, scanty information is available about the microscopic changes in rabbits due to nickel ferrite nanoparticles. The microscopic changes in our study in rabbits might be due to the rapid depletion of antioxidant enzymes (as evident in this study) and increased amount of reactive oxygen species (ROS), leading to an increased process of lipid peroxidation (oxidative stress) induced by NFNPs. However, different earlier studies have observed various histopathological alterations in the liver  in rats treated with oxidic nickel nanoparticles , 77. The−1 of synthesized material, it is suggested that these particles can be used in MRI as a contrast agent. Different characteristics of NFNPs such as drug delivery, MRI, and relaxivity are important tools for in vivo biological applications. Furthermore, the results on the biological evaluation of nanoparticles indicated that nickel-coated iron oxide nanoparticles induce adverse effects on hematology, serum biochemistry, oxidative responses, and microscopic alterations in multiple visceral tissues of rabbits.NiFe2O4 nanoparticles with a size of about 30 nm were synthesized by the coprecipitation method. XRD characterization showed exact confirmation of crystallinity and its phase identification. VSM evaluated magnetic behavior. Due to high saturation magnetization value of 46.7 emug


Basigin  is a multifunctional protein involved in cancer cell survival, mostly by controlling lactate transport through its interaction with monocarboxylate transporters (MCTs) such as MCT1. Previous studies have found that single nucleotide polymorphisms (SNPs) in the gene coding for BSG and MCT1, as well as levels of the soluble form of BSG (sBSG), are potential biomarkers in various diseases. The goal of this study was to confirm BSG and MCT1 RNA overexpression in AML cell lines, as well as to analyse soluble BSG levels and selected BSG/MCT1 genetic variants as potential biomarkers in AML patients. We found that BSG and MCT1 were overexpressed in most AML cell lines. Soluble BSG was increased in AML patients compared to healthy controls, and correlated with various clinical parameters. High soluble BSG was associated with worse overall survival, higher bone marrow blast percentage, and higher white blood cell count. BSG SNPs rs4919859 and rs4682, as well as MCT1 SNP rs1049434, were also associated with overall survival of AML patients. In conclusion, this study confirms the importance of BSG/MCT1 in AML, and suggests that soluble BSG and BSG/MCT1 genetic variants may act as potential AML biomarkers. Acute myeloid leukaemia (AML) is a haematologic malignancy associated with uncontrolled proliferation of immature leukemic blasts that can lead to bone marrow failure . It is tBasigin (BSG), also known as cluster of differentiation 147 (CD147) and extracellular matrix metalloproteinase inducer (EMMPRIN), is a glycoprotein that belongs to the immunoglobulin superfamily. It is a transmembrane protein commonly expressed on many human cell types and first identified as a stimulator of matrix metalloproteinase (MMP) production, although it is now known to also be involved in many other cellular pathways . It was While many studies exist on BSG in solid tumours ,21,22,23Apart from its membrane-bound form, BSG can also be secreted in soluble form. This can be done either by cleavage of its extracellular domain by MMPs/ADAM12, or by release of microvesicles ,29,30. SIn the present study, we aimed to confirm the expression status of BSG and associated proteins (MCT1 and VEGF) in AML. Furthermore, we wanted to determine whether soluble BSG, as well as BSG and MCT1 genetic variants, can be used as potential markers of AML susceptibility, survival, and progression.2 at 37 °C. Cells and cell culture media were collected for further analyses.Six established human AML cell lines, as well as normal human primary bone marrow CD34+ cells, were cultured in vitro for the analysis of BSG, MCT1, and VEGF expression (as previously described ). The non = 27) from the smaller serum group were also included in the larger genetic group. Blood samples for all patients were obtained at diagnosis. Approval from the Wroclaw Medical University Bioethical Committee  was obtained for the study. Clinical data analysed in the study are included in The study involved a group of 37 newly diagnosed AML patients and 25 healthy blood donors for the analysis of serum soluble BSG, as well as a larger group of 92 newly diagnosed AML patients and 135 healthy blood donors for the analysis of BSG and MCT1 genetic variants. The healthy blood donors served as a control group. Patients in the larger group were aged 20–93, and the median age was 61. The group included 54 male and 38 female patients. The smaller group used for serum analyses was aged 25–93, and the median age was 62; there were 18 males and 19 females. Some of the patients  according to the manufacturer’s instructions. RNA purity and integrity were verified on the DeNovix DS-11 spectrophotometer  and by gel electrophoresis. Using the High Capacity cDNA Reverse Transcriptase kit , 2000 ng of isolated RNA was then reverse transcribed into cDNA and an RNase Inhibitor  was added into the reaction mix. Reverse transcription was performed in a SimpliAmp Thermal Cycler  according to the manufacturer’s instructions. cDNA was then stored at −70 °C until further use.−ΔΔCt method.Quantitative real-time PCR was used to evaluate BSG, MCT1, and VEGF gene expression, and the expression data were normalized to β-actin (ACTB), acting as an internal control. TaqMan Gene Expression assays and TaqMan Gene Expression Master Mix  were used for the PCR. The probes used for the reaction were: Hs00936295_m1 (BSG), Hs01560299_m1 (MCT1), Hs00900055_m1 (VEGF), and Hs01060665_g1 (ACTB). Samples were run in triplicate, and three independent experiments were performed. PCR was performed on a LightCycler 480 II  according to the manufacturer’s instructions. Relative expression was calculated using the 2g. Serum was collected and stored at −70 °C until further use. Likewise, cell culture supernatants were collected  was used for cell lysate preparation. The total protein concentration of the lysates was measured using the modified Lowry method  according to the manufacturer’s instructions. Subsequently, 10–25 μg of protein samples were separated on polyacrylamide gel, and three repeats were performed, for a total of three polyacrylamide gel electrophoreses. The separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes with a pore size of 0.45 μm . The membranes were then incubated for 1 h with 5% non-fat dry milk in 0.1% Tris-buffered saline/Tween-20 (TBST) , washed four times with TBST, and incubated overnight at 4 °C with an anti-BSG primary mouse antibody . Subsequently, the membranes were washed four times with TBST, incubated for 1 h with a horseradish peroxidase (HRP)-conjugated secondary anti-mouse antibody, and then washed again four times with TBST. The Clarity Western ECL Substrate  was used for chemiluminescence, and bands corresponding to BSG expression were visualized on a ChemiDoc Imaging System . For β-actin detection, the membranes were first incubated for 40 min in 100% methanol to remove the remaining ECL signal, then washed four times with TBST, blocked for 1 h with 5% non-fat dry milk, and washed again four times with TBST. The membranes were then incubated with a mouse anti-β-actin-HRP monoclonal antibody  for 1 h and washed four times with TBST. Detection was performed as described above. Densitometry was performed using ImageJ 1.48v software  and the results were normalized to β-actin.Genomic DNA was extracted from peripheral blood taken on EDTA from 92 AML patients and 135 healthy individuals using a Qiagen DNA Isolation Kit . DNA was also isolated from AML cell lines. DNA purity and concentration were verified on the DeNovix DS-11 spectrophotometer . Isolated DNA was subsequently stored at −20 °C until further use.BSG and MCT1 SNPs were selected based on three criteria: (1) minor allele frequency (MAF) in European populations higher than 0.15, (2) a functional effect on expression/protein structure predicted by the National Institute of Environmental Health Sciences SNP Function Prediction tool , and (3)p-value was used to compare differences between BSG/MCT1/VEGF expression/sBSG/total BSG level in AML cell lines vs. normal CD34+ cells. Survival was analysed using the Kaplan–Meier method, while Spearman’s coefficient was used to assess the correlation between sBSG and clinical parameters and VEGF. The Mann–Whitney U test, Kaplan–Meier survival analysis, and Spearman’s correlation analysis were performed in a Real Statistics Resource Pack for Microsoft Excel 2013 , logistical regression was performed in RStudio , and the ANOVA with Dunnett’s test was performed in GraphPad Prism . Linkage disequilibrium and Hardy–Weinberg equilibrium analyses were performed with the Haploview 4.2 software. Fisher’s exact test was used to test if allele/genotype frequencies differed between patients and controls, as well as between subgroups of patients. This was calculated using the web-based tool http://vassarstats.net/tab2x2.html (accessed on 29 September 2021). p-values < 0.050 were considered statistically significant, while those between 0.050 and 0.100 were considered to be indicative of a trend.A non-parametric Mann–Whitney U test was used for comparison of VEGF levels and clinical parameters  among AML patients with high/low sBSG. The Mann–Whitney U test and logistical regression were used for comparison of sBSG levels between patients and healthy individuals. A one-way ANOVA with Dunnett’s multiple comparison test and multiplicity adjusted p < 0.001; To assess the general BSG expression level in AML, we analysed BSG mRNA levels in six AML cell lines , as well as in normal primary bone marrow CD34+ cells. Relative BSG expression was significantly higher in all AML lines compared to the normal CD34+ cells (p < 0.010), which had slightly lower MCT1 expression compared to normal CD34+ cells  and healthy individuals (n = 25). The median and interquartile range were 4186.45 pg/mL (IQR = 3658.70–5710.65) for the former and 3894.45 pg/mL (IQR = 2903.50–4544.15) for the latter group. Comparing sBSG levels in the two groups, we found that sBSG was higher in the serum of AML patients compared to healthy individuals . This indicates that serum sBSG may be a potential marker of AML.Having proven the increased expression of BSG in AML, we attempted to analyse the expression of soluble BSG (sBSG) in the serum of AML patients  and those with low sBSG . Using the Kaplan–Meier method, we found that patients in the high sBSG group had significantly worse overall survival (OS) than patients in the low sBSG group , and negatively with haemoglobin , and a trend for a positive correlation with white blood cell (WBC) count was observed . In terms of groups with different  sBSG expression, patients with high sBSG were characterized by a higher percentage of bone marrow blasts .In the next step, we compared sBSG with various clinical parameters of AML. Soluble BSG was positively correlated with the percentage of blasts in the bone marrow .Soluble BSG was also measured in supernatants of AML cell lines and normal CD34+ cells. All AML lines except for NB4 (2928.93 pg/mL) secreted lower sBSG levels than normal CD34+ cells  and healthy individuals (n = 135) for four BSG  and two MCT1 (rs1049434 and rs9429505) SNPs. The frequencies for all the SNPs were in accordance with the Hardy–Weinberg equilibrium in both groups. No statistically significant differences between patients and healthy individuals were detected, suggesting no association of selected SNPs with the predisposition to AML. The distribution of genotypes is presented in We genotyped a group of AML patients . No association between the SNPs and soluble BSG levels was found in AML patients.The Kaplan–Meier method was used to assess the effects of BSG and MCT1 SNPs on overall survival. The analysis showed that BSG alleles rs4919859 C and rs4682 C were associated with significantly worse OS . Additionally, MCT1 allele rs9429505 G was associated with higher CRP (p = 0.049).We also analysed BSG and MCT1 SNPs in relation to various clinical parameters of AML. We observed that BSG alleles rs4682 C and rs8259 A were more common in AML subtypes M0–M2 (myeloblastic leukaemia with/without maturation) than in other subtypes (BSG CD147) is a multifunctional glycoprotein involved in several regulatory pathways associated with cell adhesion, migration, proliferation, angiogenesis, ion transport, or drug efflux. Recent findings on its potential involvement in SARS Coronavirus 2 (SARS-CoV-2) infection increased the need to better describe BSG expression patterns in various tissues ,7. BSG w is a mulIn the present study, we aimed to clarify the expression status of BSG and MCT1 RNA in AML cells and to study soluble serum BSG as well as BSG and MCT1 genetic variants as potential biomarkers of AML risk, survival, and progression. We found BSG and MCT1 mRNA expression to be significantly increased in almost all AML cell lines compared to normal primary bone marrow CD34+ cells. This confirms earlier reports regarding potential BSG and MCT1 overexpression in AML ,27,41. TIt is established that angiogenesis plays a role in AML . Since BA major goal of our study was to determine whether BSG is a potential biomarker in AML. While it works primarily as a membrane-bound protein, various studies have found soluble circulating BSG to be a marker of disease ,32,33. IAnother aspect of this study was to analyse SNPs in the gene coding for BSG and MCT1 in the context of AML. We previously showed that SNPs can act as biomarkers in AML ,47, and We also analysed two MCT1 SNPs, one of which, rs1049434 , was associated with better overall survival. This SNP represents a missense Asp (A) to Glu (T) mutation, and the Glu (T) isoform is thought to be better at transporting lactate ,50,51,52In conclusion, our study shows that BSG and MCT1 are overexpressed in most AML cells compared to normal primary bone marrow CD34+ cells. Soluble serum BSG is increased in AML patients compared to healthy individuals and its high level is associated with lower overall survival and worse clinical parameters. BSG and MCT1 genetic variants are associated with overall survival and other clinical parameters. This study confirms the role of BSG and MCT1 in AML and shows that soluble serum BSG and BSG/MCT1 genetic variants may act as markers of AML survival.


Glenoid component offset in SI and humeral head centering in AP remained unchanged throughout the follow-up. Radiolucency (Lazarus classification) was recorded in 21 cases (17.3%) with a Lazarus score of 1 (15 cases) and 2 (6 cases). The EQ-5D health slider, WOOS and ASES, and ROM confirmed continuous improvements from the preoperative scores to the two-year follow-up (p < 0.05). Regression models showed no correlation between glenoid component radiolucency at two years and the postoperative week six glenoid component position; however, female gender was a significant variable. Conclusion: Glenoid component changes from its original native glenoid were observed following TSA. Glenoid inclination was improved more than version from baseline, and the humeral head remained well-centered in AP and SI at two years. Radiolucency of the glenoid at two years is not negatively associated with PROMs or component position; however, female gender was identified as a significant predictor and warrants further investigation. Complications are not associated with glenoid position or radiolucency, but longer-term follow-up is required.Background: Malpositioning of the glenoid component in total shoulder arthroplasty (TSA) remains the primary source of loosening. The purpose of this study is firstly, to quantify postoperative glenoid component position in patients having a TSA and secondly, to explore whether glenoid component radiolucency is associated with glenoid position, clinical outcomes and patient-reported measures in the short-term (two year) follow-up period. Methods: This study was a sub-study of a larger clinical trial that included patients who underwent a TSA and who were randomized into two different glenoid types with a minimum two-year follow-up period. Post-operative radiographic assessments (six weeks and two years) were used to measure glenoid component position  and humeral head centering anterior–posterior (AP) and superior–inferior (SI), and to assess glenoid component radiolucent scoring (modified Lazarus). Pre-operative X-rays were used to measure glenoid version, inclination and Walch classification. Patient-reported measures (PROMs) included the EQ-5D health slider and the Western Ontario Osteoarthritis (WOOS) and American Shoulder and Elbow Surgeons (ASES) score and were captured at baseline and two years postoperative. Clinical outcomes including range of motion and complications were also documented. Statistical analysis included t-tests and regression modeling. Results: Ninety-one patients with an average age of 69.9 ± 6.2 years were included in this study. Glenoid component position improved significantly in version (−19.4 ± 8.6° to −17.7 ± 8.5°;  Glenoid version, inclination and wear patterns are important factors to consider when performing an anatomic total shoulder arthroplasty (TSA) ,2,3. TheGlenoid component malposition is a contributing factor to loosening and poorer clinical outcomes ,6. GregoThe purpose of this study is firstly, to quantify glenoid component position in patients having a TSA and secondly, to explore whether glenoid component radiolucency is associated with glenoid position, clinical outcomes or patient-reported measures in the short-term (two year) follow-up period. We hypothesize that changes will be observed in glenoid component position and that glenoid component radiolucency is associated with glenoid position or PROMs at two years postoperative.Patients included in the current study were part of a prospective, randomized controlled multi-center study comparing two glenoid components . This stPatients presenting to one of five shoulder surgeons between June 2012 and December 2016 were screened for eligibility . Inclusi® (TM) glenoid  and a cemented all-polyethylene (PE) glenoid   . RandomiSurgical techniques and postoperative details are described in Chin et al. . In the Shoulder X-rays were taken in true–anterior posterior (AP), lateral and axial views at baseline, six weeks and two years postoperative. Postoperative day one images were not used in this study because they were only available in AP and not in axial views as patients’ pain levels did not allow for second plane imaging.® (TM) glenoid component loosening was, by necessity, modified from the Lazarus score to include osteolysis around the (TM) component  independent from the main trial completed all radiographic measurements. The independent raters were blinded to the patient clinical history and scores. Radiographic measures were standardized and reviewed by both raters prior to evaluation. One rater was an orthopaedic fellow and the other was an orthopaedic resident. Each observer/rater was blinded to the other’s measurements.Demographic variables included age, gender, body mass index (BMI), dominant and operated arm. Information related to the implants and surgical procedures were documented on the day of surgery. Clinical assessment and patient-reported outcome measures (PROMs) were performed pre-operatively (baseline) and postoperatively at six weeks and then at two years . The assDescriptive statistics were calculated for demographic, radiological and clinical variables, as well as change from pre-operation to two-year post-operation for all clinical and patient-reported outcomes. Changes in glenoid position variables between pre-operative and six weeks postoperative, and between six weeks and two years postoperative were analyzed via t-test to assess the significance of change at each time point. Within-group comparisons for ROM and PROMS between baseline and two years postoperative were made via paired t-tests. Inter-rater reliability of radiographic evaluations was assessed via intraclass correlation coefficient (ICC).Glenoid component radiolucency (binary) was regressed on 6-week glenoid version, glenoid inclination, and humeral head centering (AP and SI views in one model), using logistic regression controlling for age, sex, BMI and dominant hand operated (yes/no) and was also adjusted for deltas (pre-to-postop) and pre-operative Walch score. The continuous PROMS measures such as WOOS were regressed on two-year glenoid component radiolucency in linear regression models adjusted for age, sex, BMI and dominant hand operated (yes/no). Logistic model fit was assessed via the Hosmer and Lemeshow goodness of fit test . Linear Analyses were performed on the entire study population, as well as stratified by glenoid type: PE vs. TM. Analyses were performed using SAS version 9.4 .Ninety-one patients were included in this study, 44 with a TM-glenoid and 47 with a PE-glenoid . Two casPreoperative imaging demonstrated an average glenoid version of −19.4 ± 8.6° and glenoid inclination of 11.5 ± 7.1° . Glenoidp < 0.045 and p < 0.00001, respectively) (Postoperative week six measures demonstrated that the glenoid component was positioned at −17.72 ± 8.53° version and an inclination of 5.87 ± 6.31° which was significantly different than the pre-operative measures (ctively) . Overallctively) . One casctively) . There wp < 0.0001) (n = 15) and Lazarus 2 (n = 6) at the two-year mark (n = 13) versus TM glenoids (n = 8), but no differences between glenoid component type and component position were detected.Postoperative year two measurements showed no significant change from six weeks postoperative in the glenoid component version (−18.24 ± 9.00°); however, inclination (6.38 ± 6.33°) was significantly different ( 0.0001) . The hum 0.0001) . Howeverear mark . There wExcept for the six-week postoperative AP humeral head centering (0.277), all measurements demonstrated moderate (0.5–0.75) or good (>0.75–0.9) inter-rater reliability .p < 0.0001 and p < 0.0001). Significant improvements were also observed with the EQ-5D health slider score from baseline to two-year follow-up . There were no statistically or clinically relevant differences in PROMs between the two glenoid types at two-year follow-up [PROMs demonstrated a significant improvement from pre-operative status overall and in subgroup analyses. Combined ASES and WOOS scores significantly improved from 35.47 ± 18.04 and 29.07 ± 18.37 preoperatively to 89.07 ± 14.07 and 92.40 ± 11.95 at the two-year follow-up, respectively (ollow-up .p < 0.0001) in the two-year follow-up. External rotation (ER) also significantly increased from 16.56 ± 18.64° preoperatively to 51.06 ± 18.52° at two years postoperative (p < 0.0001). There were no statistically or clinically relevant differences in PROMs between the two glenoid types at two-year follow-up [Forward elevation (FE) improved significantly from 102.0 ± 27.17° to 139.00 ± 18.83°  . Six casLogistic regression models showed no correlation between glenoid component radiolucency at two years and post-operative week six glenoid component position when controlled for preoperative glenoid wear, age, BMI and whether the operated arm was the dominant arm . InteresLinear regression models for the two-year PROMs showed no correlation between glenoid component radiolucency at two years when controlled for gender, age, BMI and whether the operated arm was the dominant arm .p = 0.045) and inclination (p < 0.00001) were changed significantly with the latter demonstrating greater post-operative correction at six weeks. The humeral head remained well centered in AP and SI at two years. There was no association between glenoid radiolucency and component position or PROMs; however, female gender was identified as a significant predictor. ROM and PROMs improved over the study period and were unaffected by glenoid component position or type. Complications were not related to glenoid position.The current study quantified changes in glenoid component position compared with native glenoid following TSA. Glenoid component version  and second-generation TM glenoid components (n = 8). Second generation TM glenoids, with the addition of a porous tantalum keel, has improved the component design of the first generation where early loosening and high rates of revision surgery were reported [Radiolucency around the posterior glenoid component is important to detect and quantify as it can identify early problems with loosening. The original radiolucency classification described by Lazarus et al. , was usereported ,30. HoweOne interesting finding with respect to glenoid component radiolucency, is that female gender had a statistically higher odds ratio in the six-week regression models for component position . Reasons to explain this finding are unclear and warrant further investigation, notably longer-term follow-up.The current study has limitations. The radiographic images were obtained in multiple facilities as it was a multi-center study and strict standardization of shoulder X-ray protocol was not feasible. Furthermore, non-standardized pre- and post-operative radiographs might have a bearing on the marginal improvement between pre-operative and postoperative changes in the glenoid version. CT scans are the gold standard to identify changes in bone and particularly radiolucent lines around glenoid components . HoweverThe strengths of the presented study include the collection of prospective data within a level I study. Results indicate good ICC for radiographic measures. Patients were closely followed with multiple PROMs and ROM, and there was minimal patient drop out.Changes in glenoid component position compared with the preoperative native glenoid were observed following TSA. Glenoid inclination was improved more than version, and the humeral head remained well-centered in AP and SI at two years. Radiolucency of the glenoid at two years is not negatively associated with PROMs or component position; however, female gender was identified as a significant predictor and warrants further investigation. Complications were not associated with glenoid position or radiolucency, but longer-term follow-up is required.


Taken together, these facts allow us to consider the model we have created to be a useful and successful development of this technology.The study of human neurons and their interaction with neurochemicals is difficult due to the inability to collect primary biomaterial. However, recent advances in the cultivation of human stem cells, methods for their neuronal differentiation and chimeric fluorescent calcium indicators have allowed the creation of model systems in vitro. In this paper we report on the development of a method to obtain human neurons with the GCaMP6s calcium indicator, based on a human iPSC line with the TetON–NGN2 transgene complex. The protocol we developed allows us quickly, conveniently and efficiently obtain significant amounts of human neurons suitable for the study of various neurochemicals and their effects on specific neurophysiological activity, which can be easily registered using fluorescence microscopy. In the neurons we obtained, glutamate (Glu) induces rises in [CaThe online version contains supplementary material available at 10.1007/s11064-021-03497-6. The study of human neurons is difficult due to tight restrictions on obtaining primary biomaterial. However, the discovery of induced pluripotent stem cells (iPSCs) has made it possible to obtain human neurons in vitro. Such models have formed a powerful platform for studying the molecular, morphological and physiological properties of human neurons. In addition, the use of iPSCs derived from readily available human skin cells has enabled us to obtain autologous neurons from different patient-carriers of various genetic neurodegenerative diseases. The approach can also be useful for the development of individual patient-specific therapeutic strategies for various pathologies.Neurogenin-2 (NGN2) in the iPSCs. NGN2 is a pro-neural transcription factor that is responsible for the initiation of their differentiation into neurons in vertebrates i measurements were carried out using the “endogenous” Ca2+ sensor, GCaMP6s, or using the fluorescent Ca2+ indicator Fura-2 . A fluorescent indicator SBFI (ThermoFisher) was used to measure [Na+]i. The indicators were introduced into the cells in the form of acetoxymethyl esters  for 50–60 min at 37 °C. Stock solutions of the AM esters in DMSO were pre-mixed with the non-ionic detergent, Pluronic F-127 , to facilitate penetration of the esters into the cells and then added to the incubation medium at the concentrations indicated above.i and [Na+]i were performed using an image analysis system based on a Nikon Ti2 microscope (Japan), an LED-based illumination system , a triple-band beam-splitter mirror DM468/526/596, a set of excitation filters 340 ± 13 and 387 ± 6 nm for Fura-2 and SBFI, and 442 ± 21 nm for GCaMP6s. The emissions from the Fura-2, SBFI, and GCaMP6s were recorded at 544 ± 12 nm. The light filters and cube were manufactured by Semrock . Exposure times were 200–500 ms, with the LED radiation intensity in the range of 5–20% of the maximum value.Measurements of changes in i . Measurements of the GCaMP6s signals . These data indicate the presence of AMPA and/or kainate receptors in the studied culture. The differences in Glu-induced [Ca2+]i elevations as well as the differences in the relative decreases of [Ca2+]i in the presence of iGluRs inhibitors  in the cells. Adding Glu to cells in a nominally calcium-free buffer, in which the Ca2+ concentration was reduced by replacing Ca2+ with EGTA (100 μM), abolished the effect of Glu on [Ca2+]i . Inhibition was significant using both the synthetic Ca2+ indicator Fura-2    Fig. . The alm neurons –28.2+]i and [Na+]i by recording fluorescence signals from GCaMP6s and the synthetic Na+ indicator SBFI. The addition of Glu led to a rapid increase in both [Ca2+]i and [Na+]i   caused a decrease of both i in all cells stained with Fura-2   Fig. B. ATP, iKCl Fig. .NGN2 overexpression meant that slightly different cultures were obtained from different protocols. The type and morphology of neurons depends on the specific cultivation conditions, the pluripotent stem cell line used, the presence of growth factors in the culture medium, and the type of substrate i and [Na+]i in NGN2-induced neurons , the main excitatory agonist of neurons is glutamate (Glu), which is able to activate ionotropic receptors (iGluRs), as well as metabotropic receptors (mGluRs). The iGluRs provide an influx of Catructure , 43, 44,lum, ER) , 46. We ons Fig. . The dyn+]i Fig. . A simill cortex . Differeolic ATP , 47.2+]i rises induced by Glu do not exceed half of the maximum increase observed under the action of ionomycin  induces [Ca2+]i rises by units of micromoles/liter, and during prolonged exposure, by tens of micromoles/liter [2+]i rise in calcium-free buffer, and the suppression of [Ca2+]i rise by ~ 74% upon addition of a mixture of MK-801 and CNQX indicates a dominant role of ionotropic glutamate receptors in Ca2+ signaling Below is the link to the electronic supplementary material.