Healthcare workers (HCWs) in long-term care facilities (LTCFs) are disproportionately affected by severe acute respiratory coronavirus virus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19). To characterize factors associated with SARS-CoV-2 positivity among LTCF HCWs, we performed a retrospective cohort study among HCWs in 32 LTCFs in the Minneapolis–St Paul region.We analyzed the outcome of SARS-CoV-2 polymerase chain reaction (PCR) positivity among LTCF HCWs during weeks 34–52 of 2020. LTCF and HCW-level characteristics, including facility size, facility risk score for resident-HCW contact, and resident-facing job role, were modeled in univariable and multivariable generalized linear regressions to determine their association with SARS-CoV-2 positivity.Between weeks 34 and 52, 440 (20.7%) of 2,130 unique HCWs tested positive for SARS-CoV-2 at least once. In the univariable model, non–resident-facing HCWs had lower odds of infection . In the multivariable model, the odds remained lower for non–resident-facing HCW , and those in medium- versus low-risk facilities experienced higher odds of testing positive for SARS-CoV-2 .Our findings suggest that COVID-19 cases are related to contact between HCW and residents in LTCFs. This association should be considered when formulating infection prevention and control policies to mitigate the spread of SARS-CoV-2 in LTCFs. Surveillance testing for early detection of severe acute respiratory coronavirus virus 2 (SARS-CoV-2), the etiologic agent of COVID-19, is important in mitigating the spread of infection in LTCFs. Various factors have a documented association with COVID-19 in healthcare settings, including geographical setting, facility size, and community incidence. In this study, we assessed individual and facility-level characteristics associated with SARS-CoV-2 positivity among LTCF healthcare workers (HCWs) in a surveillance testing program.Residents of long-term care facilities (LTCFs) have experienced disproportionately high morbidity and mortality during the coronavirus disease 2019 (COVID-19) pandemic.This retrospective cohort study included HCWs from 32 LTCFs within a single Minneapolis–St. Paul (MSP) area healthcare system who underwent surveillance testing for SARS-CoV-2. Across the 32 LTCF sites, a range of care is provided: skilled nursing care, memory care, transitional care and/or short-term rehabilitation for persons transitioning to home after a hospital stay, and assistance for independently living older adults. The study period was March 1, 2020, to December 21, 2020. A positivity rate <5% required that LTCF HCWs test once monthly; a rate of 5%–10% required once weekly testing; and a rate >10% required twice weekly testing. The source of data for the 14-day county positivity rate was the Centers for Medicare and Medicaid Services (CMS) website, which publicized county-specific SARS-CoV-2 test-positivity rates. Detection of SARS-CoV-2 in an LTCF prompted a facility-wide PPS. Facility PPSs were repeated every 3–7 days until 14 days passed since the last potential exposure to a SARS-CoV-2–positive individual. Individuals who tested positive were not retested for 90 days based on public health recommendations. We collected individual-level data within the Occupational Health and Safety Management  electronic record, into which data on SARS-CoV-2 testing was manually entered.Passive surveillance SARS-CoV-2 testing, defined as testing that occurred in individuals with symptoms compatible with COVID-19, was performed for HCWs and LTCF residents throughout the study period. Active surveillance testing, defined as testing irrespective of COVID-19 symptoms, was introduced in June 2020. All tests were reverse-transcriptase polymerase chain reaction (RT-PCR) assays of nasopharyngeal specimens for SARS-CoV-2 RNA. An initial point-prevalence survey (PPS) of all HCWs and residents at study sites was conducted in June 2020 (weeks 23–26). From June to August 2020 , the active surveillance testing goal for LTCFs was set at capturing 20% of HCWs weekly. In September 2020 (week 37 onward), active surveillance was dictated by the 14-day test-positivity rate in the geographic county of each LTCF.The outcome was positive RT-PCR for SARS-CoV-2 RNA reported at the individual level.HCW covariables included job role, work site, and testing results. Facility covariables included facility type, facility size, and facility risk score. Facility type was dichotomized into assisted living–independent living and skilled nursing facility. Resident-facing status was defined as “yes” for HCWs whose job roles entailed direct resident contact and “no” for those whose job roles did not entail direct resident contact . A COVID-19–dedicated unit was opened during week 48; activities on this unit are not captured in our covariables. LTCFs were categorized into quartiles based on the combined number of residents and HCWs: (1) smallest (100–150 residents), small (151–183 residents), moderate (184–223 residents), and large (224–415 residents). LTCF personnel categorized the facility risk score for resident-to-HCW contact as highest risk, medium risk, and lowest risk. Facility risk was proportionate to the skilled needs of residents; the highest-risk facilities housed the largest number of residents occupying memory care beds or who depended on HCWs for activities of daily living. The multivariable regression analysis was also adjusted for weekly county-level SARS-CoV-2 positivity data, which were retrieved from the CMS website.Descriptive information concerning numbers tested, numbers diagnosed, and proportion positive were assessed over the study period: March 1, 2020, to December 21, 2020. Our multivariable analysis comprised a shorter study period, from August 17, 2020, to December 21, 2020, because SARS-CoV-2 community prevalence data from CMS were only available beginning August 17, 2020. Two analyses were conducted. The first analysis assessed infection risk factors at the individual level with emphasis on individual level risk factors. Logistic regression models were utilized in SAS proc genmod software. Participants were conditioned as random effects to account for repeated measures or clustering by facility. A second analysis was performed at the facility level and assessed facility-level factors predicting overall cumulative incidence within facilities. County SARS-CoV-2 prevalence for each facility was time varying and was treated as a continuous variable. These analyses were conducted using generalized linear regression models. Data were cleaned and analyzed with SAS version 9.4 software .Ethical approval was obtained from the University of Minnesota Institutional Review Board.SARS-CoV-2 test positivity peaked in week 18 at 25% and subsequently ranged from 0.31% to 6.3% between weeks 20 and 52 , 14,526 tests were obtained from 2,130 HCWs, 445 of which were positive (Table P < .0001). The association between facility risk score and SARS-CoV-2 positivity was not statistically significant in the univariable regression (P = .25).Our univariable analysis shows that resident-facing status was associated with higher odds of SARS-CoV-2 positivity Table . HCWs whP < .0001), and county positivity rate was associated with higher odds of a positive tests result . Although facility risk factors did not have any overall effect on risk of infection, working in a facility with medium risk compared to those with lower risk was associated with increased risk of positivity   may be unique to LTC settings and distinct from the patient-to-HCW interactions in acute-care settings. An additional explanation for the association between LTCF job role and COVID-19 infection is that personal protective equipment (PPE) use, which has been demonstrated as effective in mitigating the risk of COVID-19 transmission in HCWs, is challenged in LTC settings. A recent investigation in Minnesota found that significantly fewer HCWs used PPE during exposure to residents with COVID-19. Appropriate use of PPE requires both an adequate supply and knowledge of proper use. Due to inadequate PPE supplies and high HCW turnover, HCWs in LTCFs often did not have PPE available, and even when PPE was available, a lack of proficiency with PPE diminished its potential effectiveness.Published data on the association between SARS-CoV-2 positivity and HCW role are somewhat mixed. Although several investigations have identified SARS-CoV-2 seropositivity risk among patient-facing versus non–patient-facing HCW roles in LTCFs, and Hispanic ethnicity have been associated with SARS-CoV-2 seropositivity in HCWs. In larger samples of county-level data, COVID-19 risk has been associated with not only race and ethnicity affiliation but also income inequality, education deprivation, and underinvestment in infrastructure. In addition, although we controlled for community positivity, we were unable to assess known occupational or household COVID-19 in our sample, and these variables are known to influence SARS-CoV-2 positivity in HCWs. Furthermore, although the COVID-19–dedicated unit in our sample was not opened until week 48 of 52, we did not assess its impact on SARS-CoV-2 positivity among HCWs, which is a limitation given prior work demonstrating higher risk in HCWs on COVID-19–dedicated units in LTCFs.This study had several limitations. Our study sample lacked HCW demographic data. Variables related to social determinants of health were unmeasured in our sample and may be a source of confounding. Importantly, black raceOur study was not designed to ascertain the directionality of SARS-CoV-2 transmission in LTCFs. We hypothesized that SARS-CoV-2 entry into LTCFs occurred principally via infected HCWs, based on our presumption that visitor restrictions in place at the time of our investigation served as a substantial barrier to transmission from non-HCWs. Following the introduction of SARS-CoV-2 into a facility, both residents and HCWs become reservoirs for transmission. we found that facility size was not associated with the risk of COVID-19 infection. Other investigations have identified facility staffing levels, facility profit status, and staff-to-resident ratio as being associated with SARS-CoV-2 positivity. These variables were not available for inclusion in our analysis and may be other sources of confounding in our analysis.Although recent studies have reported that larger facility size was consistently associated with increased risk of COVID-19 infections and outbreaks, Facility size characterization was based on capacity and not actual census, so it may not accurately represent the numbers of residents and HCWs. SARS-CoV-2 testing by PCR of nasopharyngeal swab samples was not compulsory or universal; thus, we may have missed convalescent infection. Possibly, testing was biased toward a subset of HCWs. Our study period occurred prior to vaccine availability for COVID-19; thus, we did not consider the vaccination status of HCW and residents.The strengths of our study include the large size of our sample and the large number of tests performed. In addition to those previously mentioned, this study had several limitations. We calculated odds ratios for our outcomes of interest, and we acknowledge that these estimates may differ substantially from risk ratios in our sample given the high number of observed outcomes. Our results cannot be generalized to other congregate settings because our investigation was limited to the subpopulations of HCWs in LTCFs in the Minneapolis–St. Paul area. The findings from our urban sample may not be generalizable to LTCFs in rural areas. we hypothesize that universal testing of HCWs in LTCFs may be an important component of infection control policies to mitigate SARS-CoV-2 transmission. This hypothesis warrants further study.We did not assess the impact of active surveillance SARS-CoV-2 testing on the positivity trends in LTCFs. Based on the sizeable proportion of persons who transmit SARS-CoV-2 asymptomatically,In conclusion, we observed that HCWs with job roles involving direct resident contact in LTCFs are at higher risk for COVID-19 than HCWs whose job roles at LTCF do not involve resident contact. This finding should inform COVID-19 mitigation strategies at LTCFs, including policies related to PPE.


Gangliosides are glycosphingolipids with prevalence in nervous tissue and their involvement in certain neuronal diseases have been widely known. Interestingly, many recent studies highlighted their importance in the development and progression of various cancers through orchestration of multiple attributes of tumorigenesis, i.e., promoting migration, invasion, escaping the host immune system, and influencing other cancer hallmarks. Therefore, the multidimensional role of gangliosides in different cancers has established them as potential cancer targets. However, the tremendous structural complexity and functional heterogeneity are the major challenges in ganglioside research. Moreover, despite numerous immunotherapeutic attempts to target different gangliosides, it has failed to yield consistent results in clinical trials owing to their poor immunogenicity, a broad range of cross-reactivity, severe side effects, lack of uniform expression as well as heterogeneity. The recent identification of selective O-acetylated ganglioside expression in cancer tissues, but not in normal tissues, has strengthened their potential as a better and specific target for treating cancer patients. It was further supported by reduced cross-reactivity and side effects in clinical trials, although poor immunogenicity remains a major concern. Therefore, in addition to characterization and identification of the biological importance of O-acetylated gangliosides, their specific and efficient targeting in cancer through engineered antibodies is an emerging area of glycobiology research. This review highlights the modulatory effect of select gangliosides on different hallmarks of cancer and presents the overall development of ganglioside targeted immunotherapies along with recent progress. Here, we have also discussed its potential for future modifications aimed towards improvement in ganglioside-based cancer therapies. Gangliosides, sialic acid containing glycosphingolipids, localized in the external leaflet of the plasma membrane, have gained much importance in the recent years for their involvement in manipulating the established and widely recognized Hallmarks of Cancer, apart from their well-known function of maintaining membrane integrity, regulating cellular communication, and transducing cellular signals. Accumulating evidence from recent progress in the study of gangliosides revealed their crucial involvement in various cancers. High structural variation and discrete expression in the different developmental stages as well as in various pathophysiological conditions make them one of the most interesting candidates for further functional exploration.The structural complexity of gangliosides primarily arises out of a high degree of structural diversity owing to the variations in the arrangement of oligosaccharide moieties, as well as sialic acid residues, which makes them an intriguing yet extremely complex subject to study their biological role and function. Structural analysis of different gangliosides reveals their enormous variation, e.g., ganglioside GM3 and GD3 are structurally separated by only one sialic acid residue whereas, GM1 and GM2 are differentiated by only one galactose moiety. A close structural similarity is noted in the case of ganglioside GM1a and GM1b. Not only the subtle structural changes and positional differences of sialic acids make gangliosides highly heterogeneous but differential acetylation in the sialic acid residues makes them more diverse. O-acetylation of gangliosides is one of the major modifications in the sialic acids and most prevalent in cancer tissues compared to normal tissues . Gangliode novo biosynthesis of gangliosides is initiated with the formation of the common precursor, ceramide (Cer) within the endoplasmic reticulum (ER) by ceramide synthases , GD3-synthase was overexpressed to assess the role of b-series gangliosides. GD3-synthase overexpressed cells exhibited an increased proliferation compared to the control cells. It was further observed that overexpression of GD3-synthase was associated with an increased phosphorylation of ERK1, suggesting the activation of proliferative signaling. This increased proliferation was further restricted by incubating the culture medium with anti-GD2 antibody, which indicates the positive role of GD2 in the proliferation of SCLC in vitro. In breast cancer cell line MDA-MB-231 stable overexpression of GD3-synthase was associated with increased proliferative activity owing to the activation of MEK/ERK and PI3K/Akt signaling pathway. Further analysis identified the constitutive activation of an upstream element c-Met leading to GD3-synthase dependent proliferative activity in MDA-MB-231 cells  mediated proliferation of mouse embryonic fibroblast cell line Swiss 3T3 was significantly blocked by chemical inhibition of ganglioside synthesis by d-l-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol-HCl (PPPP) , indicat31 cells . Further of BMCs . In norm of BMCs . Thus, s of BMCs , murine  of BMCs  and huma of BMCs . A parad of BMCs . Althoug of BMCs . Interes of BMCs . Figure ex-vivo . Reduced activity of transcription factor NFAT1 explains the molecular events behind the reduced tumorigenesis in GM3 synthase knocked down cells  suggested an association of estrogen receptor in GD2 mediated tumorigenesis. Promoter study of ICAM1 gene in GD3 synthase overexpressed condition showed a supressed transcriptional activity in ER-breast cancer cell lines  with oncogenic V12-H-Ras mutations (HMLER cells), a subset of cells showed a striking overexpression of ganglioside GD2. Upon further analysis, these GD2 overexpressing subsets showed greater migratory activity compared to the GD2-populations along with high expression of MMPs  and differential expression of EMT associated genes. GD2+ cells showed an increased expression of N-cadherin, Vimentin and a reduction in E-cadherin expression levels compared to the GD2-sub-population suggesting the direct involvement of GD2 in promoting pro-metastatic ability of the cancer cells . In a siex-vivo . In mousex-vivo . The rolwn cells . The rolwn cells . Initiale of GD3 . In malill lines . Functiopatients . Though ll lines . Furtherll lines . Overexpll lines . In a coll lines . In anotll lines . This su+ progenitor cells with ganglioside secreting neuroblastoma cells, resulted in a substantial inhibition of dendritic cell generation (Dendropoiesis) in vitro by up to 90%. However, pretreatment of neuroblastoma cells with a glucosylceramide synthase inhibitor, DL-threo-1-phenyl-2-decanolylamine-3-morpholino-1-propanol HCl (D-PDMP), at a concentration of 10 µM hampers ganglioside synthesis, and represses the ability of neuroblastoma cells to inhibit dendritic cell generation. Further studies identified the enrichment of GD2 and GM3 within the neuroblastoma derived tumor shed glycolipid components. Interestingly, tumor shed GD2 and GM3 together inhibited the generation of dendritic cells from bone marrow progenitor cells but only GM3 was shown to hamper the antigen presenting property of these dendritic cells in neuroblastoma. Reduction in the antigen presentation by dendritic cells led to poor T-cell mediated immune response against tumor derived antigens, which further assisted in the progression of tumorigenesis (in vitro study demonstrated that tumor shed gangliosides augment the infiltration of myeloid derived suppressor cells (MDSCs) at the tumor sites and impedes host anti-tumor immune responses. However, the intricate mechanism behind ganglioside mediated MDSC infiltration is yet to be clearly understood , which resulted in successful development of antibodies against GM3 in breast cancer patients in a phase I clinical study. There were few side-effects reported during the study. Short-term pain at the injection site and local skin responses, such as induration, erythema, soreness, and oedema, were the most common side effects among the patients. Additionally, a few patients experienced fever, chills, myalgias, arthralgias which were disappeared by usual antipyretic treatment (in vivo by exerting antibody-dependent cell-mediated cytotoxicity (ADCC)  . Conjugay (ADCC) . MoreoveTherapeutic approaches in cancer by targeting gangliosides generally include using either specific chemotherapeutic agents targeted towards specific gangliosides or selective activation of an immune network against gangliosides using active or passive immunization. Minnelide, an analog of Triptolide , an epoxSpecificity and availability of the antigen are two most crucial bottlenecks for a successful immunotherapeutic approach. To overcome that, CAR-T cell-based therapies targeting specific antigen is an interesting and developing field in cancer immunotherapy. A CAR-T cell-based therapy employs T cells from cancer patients which were genetically modified to express chimeric antigen-specific receptors that target overexpressed antigens thereby targeting cancer cells while leaving the normal cells unharmed. Targeting specific ganglioside using CAR-T cells is a growing field towards glyco-immunotherapy. Initial clinical studies with GD2. CAR-T in neuroblastoma patients failed to achieve significant clinical response, but further modification of the CARs and inclusion of an IL-15 cassette (GD2. CAR.15) exhibited superior antitumor activity toward neuroblastoma by promoting enhanced tumor cell apoptosis and elevated CAR-T cell survival in the peripheral blood in a preclinical murine model . An ongoin vitro. But unfortunately, residual Retinoblastoma cells became resistant to these CAR-GD2 T cells by selective attenuated expression of GD2, and increased PD-L1 expression on tumor cells, suggesting a combinatorial approach along with the genetically modified T cells to achieve a more pronounced antitumor effect in Retinoblastoma. However, the prevalence of gangliosides on the normal tissues and nerve fibers, limited the comprehensive success of targeted therapy. An extensive study on tumor glycobiology leading to the identification of the O-acetyl derivatives of ganglioside GD2 and their specific localization on tumor tissues (The cytotoxic efficacy of GD2 targeting CAR-T cells in Retinoblastoma was also demonstrated in a preclinical model. The fourth generation GD2 targeting CAR-T cells have shown improved T cell generation and elevated cancer cell specific killing effect  tissues establishThis article delved into the diverse functions and intricate involvement that gangliosides play in the different aspects of tumorigenesis. Further, this review also focussed on the recent developments in the field of ganglioside biology and their direct implication in cancer immunotherapies either by directly targeting gangliosides or by modulating its downstream effectors in different cancers. Numerous studies highlighted the significance of gangliosides and raised the prospect of utilizing these glycolipids both as biomarkers, as well as potential therapeutic targets against various cancers. Among most gangliosides, the involvement of GD3 and GD2 has been investigated rigorously in the context of tumour initiation, development, progression, as well as an immunotherapeutic target against cancer. The role of GD3 and GD2 in the regulation of cellular proliferation, differentiation , and evaThe importance of gangliosides as an impactful regulator of cellular function has increased significantly over the past few years following several studies indicating its capability to regulate the hallmarks of cancer directly or indirectly. Following initial studies that suggested such an involvement, further studies have solidified this link. Gangliosides are not only reported to be engaged in modulating several hallmarks such as migration and invasion, promoting proliferative signalling, evading host immune response, angiogenesis or avoiding cell death signals, but also their role in these regulations were reported to be multi-dimensional, and often context-dependent. Such contrasting dynamics in ganglioside behaviour have invited more and more studies which included attempts of targeting select gangliosides for immunotherapy, providing us with a scope of venturing effective and targeted therapeutic strategy against cancer. Despite promising results, several challenges remain in ganglioside-based anti-cancer therapeutic approaches. The necessity of anti-ganglioside conjugates, owing to poor immunogenicity of gangliosides, in general have been presented with moderate to severe side effects in several clinical trials, despite positive therapeutic achievements. Hence, the need for a targeted approach which will provide better specificity towards cancer cells. Interestingly, the absence of O-acetyl GD2 in normal tissues and peripheral nervous system reduces tumor off-target effects resulting in significantly reduced toxicity with O-acetyl-GD2 mAb 8B6 . Constan


Electrochromic optical recording (ECORE) is a label-free method that utilizeselectrochromism to optically detect electrical signals in biological cells witha high signal-to-noise ratio and is suitable for long-term recording. However,ECORE usually requires a large and intricate optical setup, making itrelatively difficult to transport and to study specimens on a large scale.Here, we present a compact ECORE apparatus that drastically reduces the spatialfootprint and complexity of the ECORE setup whilst maintaining highsensitivity. An autobalancing differential photodetector automates common-modenoise rejection, removing the need for manually adjustable optics, and acompact laser module conserves space compared to a typical laser mount. Theresult is a simple, easy-to-use, and relatively low cost system that achieves asensitivity of 16.7 {\mu}V (within a factor of 5 of the shot noise limit), andreliably detects action potentials from Human-induced pluripotent stem cell(HiPSC) derived cardiomyocytes. This setup can be further improved to within1.5 dB of the shot noise limit by filtering out power-line interference.


In several diseases, however, the presence of Tregs interferes with a desirable effect to combat disease. This is specifically important in malignancy where Tregs have been shown to reduce the anti-tumor effects of immune checkpoint inhibitor (ICI) such as anti-PD-(L)1 (programmed cell death 1 (ligand) protein 1 or anti-CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) and thereby promote secondary resistance. On the other hand, a lack of functional Tregs may enhance autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, severe allergy, and allogeneic immune responses such as allograft rejection or graft-versus-host disease (GvHD).Regulatory T cells (Tregs) represent a group of specialized CD4In this editorial, the authors cover recent advances on Tregs including the role of glycoprotein A repetitions predominant (GARP), ectonucleotidases CD39 and CD73, as well as nanodrugs targeting T cells.via multiple mechanisms including suppression of antigen-presenting cells (APCs), secretion of anti-inflammatory cytokines, reprogramming of metabolic functions, and direct cytotoxic effect.Tregs exert immunosuppression + Tregs in food allergy results in increased gastrointestinal inflammation - activating transmembrane GARP. They demonstrated that depletion of GARPammation . Interes has shown to be a promising approach, thereby maximizing on-target while minimizing any off-target effects. NCs targeting Teff and Tregs are an especially useful tool as they can be used for depletion/reprogramming of immunosuppressive cells, and APCs mediated T- cell activation or enhancement of immunogenic cell death. One such approach is the use of protein-based nanodrug (Nab-paclitaxel) that exhibited rapid tumor response for patients with metastatic breast cancer . Other gIn summary, this editorial provides an overview over several novel interventions employing a variety of therapeutic moieties that result in therapeutic up- or down-modulation of Treg function. Future studies will reveal whether some of these modalities are effective and safe enough to become part of our treatment armamentarium to combat autoimmunity and cancer.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Microglia, the innate immune cells of the CNS, are critical for the regulation of the neuronal network, from the early neuronal developmental stages to adulthood . They acGan et al. used oxymatrine (OMT), a natural quinoxaline alkaloid extracted from the root of Sophora flavescens, and provided evidence for its ability to reduce neuroinflammation in the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) mouse model of Parkinson’s disease (PD). They also used 1-methyl-4-phenylpyridinium (MPP+)-induced mice primary microglia, primary murine neuron-microglia co-cultures, and primary microglia infected with cathepsin D (CathD)-overexpressed lentivirus. OMT dose-dependently inhibited MPTP-induced motor deficits and provided dopamine neuroprotection. OMT also inhibited microglia activation following exposure to the MPTP/MPP+-induced release of pro-inflammatory cytokines, downregulated the expression of CathD, and inhibited the activation of the HMGB1/TLR4 signaling pathway and the nuclear translocation of NF-κB. Their study supports a potential role for OMT in ameliorating PD and proposes that OMT may be useful in the treatment of PD.In this Research Topic, novel and well-characterized compounds are being discussed as microglia-selective neuro-therapies that can alter the functional state of microglia and modulate neuroinflammation . Gan et Deng et al. used isorhynchophylline (IRN), a tetracyclic oxindole alkaloid extracted from Uncaria rhynchophylla with anti-inflammatory effects in microglial cells, and provided evidence for its ability to provide protection against ischemia/reperfusion injury induced by middle cerebral artery occlusion (MCAO) by employing a rat model of stroke. IRN attenuated the infarct volume, improved the neurological function, and reduced the neuronal death rate, brain water content, and aquaporin-4 expression in the brains. IRN also inhibited IκB-α degradation, NF-κB p65 activation, CX3CR1 expression, microglial activation, and the inflammatory response.Zou et al. provided evidence for the ability of ethyl ferulate (EF) to improve ischemic stroke outcomes. EF suppressed the lipopolysaccharide (LPS)-induced pro-inflammatory response in the primary microglia and immortalized cell lines and post-stroke neuroinflammation in the transient middle cerebral artery occlusion (tMCAO) stroke model in mice. EF was shown to bind and inhibit the activity of monoamine oxidase B to reduce the pro-inflammatory response.Wagner et al. used the hallucinogen and mushroom Amanita muscaria and provided evidence of the ability of A. muscaria extract (AME-1) to modify the inflammatory responses in a human microglia cell line (HMC3) by using flow cytometry. AME-1 upregulated expression of CD86, CXCR4, CD125, and TLR4 receptors. Although AME-1 at higher concentrations increased IL-8 production of HMC3, AME-1 potentiated HMC3 production of IL-8 in response to poly(I:C). Metabolomics analysis revealed that AME-1 contains the autophagy inducer trehalose, which also potentiated the HMC3 poly(I:C)-mediated production of IL-8.Ortiz-Romero et al. used chronic treatment with the natural phenol compound curcumin, the non-selective voltage-dependent calcium channel verapamil, and a combination of both in a mouse model of the rare neurodevelopmental disorder Williams–Beuren syndrome (WBS) (WBS complete deletion (CD) mice characterized by a 1.3 Mb heterozygous deletion). CD mice have a distinctive cognitive phenotype compared to wild-type, marked by hypersociability and lower performance in the marble burying test. CD mice showed more activated microglia in the motor cortex and CA1 hippocampal region, which were prevented by co-treatment of curcumin and verapamil. Behavioral improvement with the combination of curcumin and verapamil on hypersociability correlated with the molecular recovery of several affected pathways involving MAPK signaling and was important in the control of synaptic transmission and inflammation. The whole study highlighted a possible pharmacological targeting of microglia for a symptomatic treatment of this complex disease that is, currently devoid of treatment.McKee et al. contributed a mini-review about microglia as a target in age-related cognitive decline (ACD) and Alzheimer’s disease (AD), discussing microglial pathways of interest for the prevention and treatment of ACD and AD. In addition, they explored the heterogeneity of microglia in these conditions and how pharmacological agents could target specific microglial states. The infiltration of immune cells into the brain might play a role in these detrimental effects of activated microglia. As recently shown by Finally, These timely studies and this mini-review support the potential of microglial and infiltrating immune cells as therapeutic targets in neurodegenerative conditions. Future efforts are warranted to further develop and test these interventions in pre-clinical models and subsequently in patients.


Extracellular vesicles (EVs) derived from neural stem cells (NSC-EVs), astrocytes (ADEVs), and microglia (MDEVs) have neuroregenerative properties. This review discusses the therapeutic efficacy of NSC-EVs, ADEVs, and MDEVs in traumatic brain injury (TBI) models. The translational value and future directions for such EV therapy are also deliberated. Studies have demonstrated that NSC-EV or ADEV therapy can mediate neuroprotective effects and improve motor and cognitive function after TBI. Furthermore, NSC-EVs or ADEVs generated after priming parental cells with growth factors or brain-injury extracts can mediate improved therapeutic benefits. However, the therapeutic effects of naïve MDEVs are yet to be tested rigorously in TBI models. Studies using activated MDEVs have reported both adverse and beneficial effects. NSC-EV, ADEV, or MDEV therapy for TBI is not ready for clinical translation. Rigorous testing of their efficacy for preventing chronic neuroinflammatory cascades and enduring motor and cognitive impairments after treatment in the acute phase of TBI, an exhaustive evaluation of their miRNA or protein cargo, and the effects of delayed EV administration post-TBI for reversing chronic neuroinflammation and enduring brain impairments, are needed. Moreover, the most beneficial route of administration for targeting EVs into different neural cells in the brain after TBI and the efficacy of well-characterized EVs from NSCs, astrocytes, or microglia derived from human pluripotent stem cells need to be evaluated. EV isolation methods for generating clinical-grade EVs must also be developed. Overall, NSC-EVs and ADEVs promise to mitigate TBI-induced brain dysfunction, but additional preclinical studies are needed before their clinical translation.  Neural stem cell-derived extracellular vesicles (NSC-EVs) or astrocyte-derived EVs (ADEVs) can mediate neuroprotective effects and improve motor and cognitive function in models of traumatic brain injury (TBI). Furthermore, such EVs generated after priming parental cells with growth factors or brain-injury extracts can mediate improved therapeutic benefits via a specific miRNA or protein in their cargo. However, NSC-EV or ADEV therapy for TBI is not ready for clinical translation. Additional studies are needed, including testing their efficacy for preventing neuroinflammatory cascades and enduring cognitive dysfunction and examining the beneficial effects of delayed EV treatment after TBI.1-6 TBI is classified as either primary or secondary. The primary injury involves brain tissue loss from the initial insult, whereas the secondary injury is a response to the initial penetrating or closed head injury, which includes complex pathophysiological processes such as oxidative stress, neuroinflammation, altered autophagy, and neuronal death.7-9 The severity of TBI varies greatly depending on the secondary molecular injury cascades that could persist for years after the initial TBI, resulting in long-term cognitive and behavioral impairments, including the development of mental disorders in surviving patients.10-12 Treatment of TBI in the acute phase of injury is crucial for reducing tissue loss and improving outcomes.13 Currently, TBI treatment is limited to preserving nervous tissue function through surgery and pharmaceuticals to reduce oxidative stress.15 However, due to the intricate pathophysiology of secondary TBI, participating molecular targets are often ambiguous, lacking target identification, and development of effective therapies.5 Understanding the fundamental pathological mechanism of TBI is needed first to effectively treat and restore function after injury.3Traumatic brain injury (TBI), neurological damage resulting from trauma, is a leading cause of disability and death worldwide, with approximately 50 million TBI cases occurring annually, creating a global financial burden of $70 billion and making TBI a major health problem.17 Mitochondria produce ROS as a byproduct of oxidative phosphorylation, but mitochondrial dysfunction following TBI causes excessive ROS production, leading to oxidative damage, and apoptosis.18-20 Another critical pathophysiological process of secondary TBI is neuroinflammation, initiated by microglia that become activated by damage-associated molecular patterns (DAMPs) such as ROS, cellular debris, and heme released from microhemorrhages.22 Upon activation, microglia become mainly polarized to proinflammatory phenotypes, inducing adverse changes in astrocytes and exacerbating secondary injury.21-25 Therefore, regulating microglia activation and downstream inflammatory signaling cascades has attracted attention as a potential avenue for treating secondary TBI and preventing long-term cognitive and mood impairments.One mechanism of neuronal injury following TBI is oxidative stress, resulting from an imbalance between reactive oxygen species (ROS) and antioxidants.27 EVs are two-layered, membrane-bound, nanosized vesicles secreted by virtually all cell types.28-32 EVs contain nucleic acids, proteins, and lipids and function as critical mediators of intercellular communication by transporting DNA, mRNAs, miRNAs, and proteins between cells and across the blood-brain barrier (BBB) in the nervous system.33 Broadly, there are two types of EVs: exosomes and microvesicles.34 Exosomes, ranging in size from 30 to 150 nm, originate from the endosomal pathway, while microvesicles, ranging in size from 100 to 1000 nm, bud off directly from the plasma membrane.34 Cargo loading into EVs is not random but a highly controlled process.36 EVs can be derived from various sources, including neural stem cells (NSCs), astrocytes, and microglia, and studies suggest that EVs containing large amounts of miRNAs in their cargo have potential therapeutic effects.35 Other studies have demonstrated that EVs carrying small molecules and drugs can be targeted to the brain to deliver antioxidants to neurons to reduce ROS levels.37-40 Such targeted delivery of small molecules via EVs could potentially treat neurological conditions and diseases, like Alzheimer’s disease and Parkinson’s disease.42Recently, the idea of employing neural cell-derived extracellular vesicles (EVs) to combat oxidative stress and acute neuroinflammation after TBI has emerged because of their antioxidant and anti-inflammatory cargo.43 NSCs were once considered a means to replace neurons lost in the central nervous system (CNS).44 NSCs can be obtained in unlimited quantities from human embryonic or induced pluripotent stem cells, and such cells have shown promise for brain repair following injury or disease. However, widespread clinical translation of NSC therapy is yet to be realized due to potential complications linked to their use.44 These include immune rejection without significant immune suppression, low cell engraftment, the risk of developing teratomas, and the invasive approach of intracerebral transplantation.44 Furthermore, there is growing evidence that NSCs do not mediate their restorative effects via direct cell replacement but through “bystander” effects from secreted factors, including EVs.45-48 Increasingly, NSC-derived EVs (NSC-EVs) are considered an excellent alternative to NSCs as they carry NSC secretome but do not have the risks associated with NSCs, and can readily cross the BBB following intravenous or intranasal delivery.49 Thus, NSC-EVs offer a potential “cell-free” alternative to using NSCs that will convey therapeutic benefits without adverse side effects of NSCs.4EVs from NSCs, astrocytes, and microglia are attractive for treating TBI due to the multiple therapeutic miRNAs and proteins they carry in their cargo.50-52 Even in physiological conditions, the EVs secreted by astrocytes (ADEVs) display neurotrophic and neuroprotective properties. However, astrocytes shed EVs carrying both neuroprotective and neuroreparative proteins/molecules in ischemic and other stressful conditions.53-55 ADEVs are also considered potent mediators of neural plasticity.57 Microglia are resident immune cells in the brain. Hence, naïve microglia-derived EVs (nMDEVs) likely have anti-inflammatory, neuroprotective, or neuroregenerative properties.58-61 On the other hand, activated MDEVs (aMDEVs) might have therapeutic or adverse effects depending on their activation state.58-61 Overall, due to the lack of immune rejection and their roles in neural recovery and immune regulation in the CNS, NSC-EVs, ADEVs, and nMDEVs are potentially beneficial for treating TBI and other neurodegenerative conditions involving oxidative stress and chronic neuroinflammation.49 Both NSC-EVs and ADEVs also promise to provide neuroprotection, increase hippocampal neurogenesis, and improve cognitive and mood function in disease conditions.62-64 This concise review aims to discuss highlights from studies that have tested the efficacy of NSC-EVs, ADEVs, nMDEVs, and aMDEVs in prototypes of TBI and to deliberate the translational value, limitations, and future directions for NSC-EV, ADEV or MDEV therapy for TBI.Astrocytes, a type of glia in the CNS, play an essential role in regulating CNS development, homeostasis, defense, and function.The NSC-EV studies in brain injury models discussed in this section are listed in 4 In this study, NSC-EVs isolated from human embryonic H9 cell culture media through ultrafiltration were administered intravenously to rats at 4-6 h, 24-26 h, and 48-50 h post-CCI .post-CCI . Assessmpost-CCI .4 However, the study did not examine the reasons underlying sex-specific differences in the therapeutic properties of NSC-EVs. It could be due to increased brain damage and accelerated neuropathological changes after CCI in females due to sex hormones. Since the study did not perform the dose-response study, it remains to be addressed whether higher doses of NSC-EVs would be proficient in inducing functional recovery in females. Also, while the study noted the migration of endogenous NSCs to the lesion site and upregulation of VEGF activity, the exact mechanism by which NSC-EVs promoted neuroprotection and motor functional recovery remains elusive.4 Additional limitations of this study include not investigating the incorporation of intravenously administered NSC-EVs into neurons and glia and not studying the effects of NSC-EV therapy on CCI-induced chronic neuroinflammation and long-term cognitive and mood impairments.The overall therapeutic effect was gender-specific, with female rats showing reduced beneficial effects of NSC-EVs than male rats after TBI.35 The NSCs from the embryonic day 15 rat cerebral cortex were expanded as neurospheres in a standard NSC medium or an IGF1-containing NSC medium  on neuroinflammation, apoptosis, and regeneration after a spinal cord injury (SCI).C medium . EVs she35 The animals received tail vein injections of standard or IGF1-EVs immediately after SCI, and hindlimb motor function was assessed at 1, 3-, 7-, 14-, and 28-days post-SCI using the Basso Beattie Bresnahan (BBB) locomotor rating scale and slanting board test  mice. While ADEV treatment reduced ROS and hydrogen peroxide levels with increased SOD and CAT levels in Nrf2+/+ mice, such effects were not seen in Nrf2-KO mice. The authors also demonstrated that ADEVs decrease apoptosis through the activation of Nrf2  and western Blot analysis of CD9 and CD63. Through genome-wide microarray analysis, the authors identified miR-873a-5p as one of the top 5 miRNAs among ~132 miRNAs within these ADEVs. Based on the bioinformatics database, the authors identified miR-873a-5p as the likely candidate affecting nuclear factor kappa B (NF-kB) signaling and focused all further studies on this miRNA. They confirmed the upregulation of miR-873a-5p in human traumatic brain tissue samples from necrotic and edema areas. Next, they demonstrated that transfection of miR-873a-5p into primary microglia stimulated with lipopolysaccharide (LPS) can result in a decreased secretion of inducible nitric oxide synthase (iNOS), high mobility group box protein 1 (HMGB1), and proinflammatory cytokines interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α) and IL-6, compared to microglia challenged with LPS alone. Additional characterization also demonstrated that intervention with miR-873a-5p can inhibit the activation of NF-kB signaling in LPS-treated primary microglia.21Long et al. investigated ADEVs collected from C57BL/6 mouse primary astrocytes following treatment with human traumatic brain tissue extracts.21 Additional characterization revealed that miR-873a-5p promoted M2 polarization by inhibiting M1 signature genes iNOS, CD32, IL-1β and promoting M2 genes CD206, IL-4, and arginase-1  and human mitochondrial transcription factor A (mtTFA), the activators of mitochondrial biosynthesis, Tom20, important in mitochondrial internal transporter function, cytochrome c oxidase subunit 2 (mTCO2), ATP content, and lactate/pyruvate ratio. GJA1-20k expressing ADEVs also promoted recovery of mitochondrial ultrastructure and neuronal morphology. However, such effects were not seen when damaged neurons were treated with GJA1-20k KO EVs, implying that GJA1-20k is a significant neuroprotectant in ADEVs.3To determine if ADEVs carrying gap junction alpha 1-20k (GJA1-20k), the mRNA of a chaperone protein maintaining mitochondrial stability, are neuroprotective after TBI, Chen et al. established a transwell culture of rat astrocytes and neurons.GJA1-20k . Next, G3 The results showed that TBI rats treated with GJA1-20k expressing ADEVs displayed better preservation of brain tissues than TBI rats receiving GJA1-20k KO ADEVs. The study identified GJA1-20k as a vital component of ADEVs mediating neuroprotection. However, this study has limitations, including no testing of cognitive, behavioral, or motor function after administering GJA1-20k containing ADEVs or GJA1-20k KO ADEVs in TBI rats to assess the functional effects of GJA1-20k. Also, the mechanisms underlying neuroprotection mediated by standard ADEVs were not evaluated.To verify the results of the cell culture assay, the authors tested the efficacy of GJA1-20k expressing ADEVs and GJA1-20k KO ADEVs in a rat model of TBI utilizing hydraulic injury . The EVs52 NKILA has a binding site for miR-195, and miR-195 bound to nucleotide-binding leucine-rich repeat-containing family member X1 (NLRX1) stimulates ROS production to amplify NF-κB signaling while unbound NLRX1 inhibits it. Therefore, the authors hypothesized that ADEVs carrying NKILA promotes recovery after TBI, as binding of NKILA to miR-195 facilitates NLRX1 to inhibit NF-κB signaling. The authors first tested these EVs in an in vitro model of TBI comprising mechanically injured human neurons.52 Then, they tested the efficacy of these EVs for neuroprotection in a mouse model of CCI. In the cell culture model, authors found that NKILA was downregulated in injured neurons but coculturing with astrocytes increased NKILA expression in injured neurons with corresponding increases in cell viability. Further analysis revealed that NKILA from astrocytes was transferred into injured neurons via EVs, which resulted in the reduction of miR-195 and upregulation of NLRX1. These changes facilitated increased proliferation and decreased apoptosis of neurons, which was evident from the reduced expression of proapoptotic genes Bax and caspase-3. The authors also demonstrated the ability of NKILA to upregulate NLRX1 by competitively binding to miR195 and the overexpression of NKILA suppressing the apoptosis of injured neurons through upregulation of NLRX1.52He and associates investigated the effects of ADEVs overexpressing the nuclear transcription factor NF-κB interacting long noncoding RNA (NKILA) on TBI.52 TBI mice receiving NKILA enriched EVs displayed higher levels of NKILA and NLRX1, reduced levels of miR-195, and better brain tissue preservation  with primary neurons isolated from embryonic day 18 mice undergoing stretch injury. aMDEVs were next isolated through ultracentrifugation and labeled with PKH26. Labeled aMDEVs (2.5 × 1010) were administered intravenously through tail vein injections into Thy1-green fluorescent protein (GFP) knock-in mice subjected to lateral fluid percussion injury (FPI). The study employed a moderate TBI model with the peak impact pressure controlled at 1.9-2.1 atmospheres (atm), equivalent to ~30 pounds per square inch (PSI). aMDEVs incorporated into pyramidal neurons in cortical layers V and VI, and decreased growth-associated protein-43 (GAP-43) expression in neurons in the peri-contusion region. Such changes were associated with increased foot slips in FPI mice receiving aMDEVs compared to FPI mice receiving vehicle treatment in the beam walking test.58 Moreover, spine density in apical dendrites of pyramidal neurons was decreased significantly in the peri-contusion region of mice receiving aMDEVs. In contrast, intravenous administration of nMDEVs isolated from naïve BV2 primary microglial cultures significantly increased apical dendrite spine density.Zhao et al. tested the inhibitory effects of aMDEVs on neurite outgrowth and synapse recovery after TBI.58 Overall, the study demonstrated that the administration of nMDEVs promotes spine formation after TBI. In contrast, aMDEV treatment inhibits spine density in cortical pyramidal neurons and exacerbates neurological dysfunction after TBI due to reduced expression of miR-2151 in aMDEVs. The study also uncovered the beneficial effects of aMDEVs overexpressing miR-2151 in promoting neurite outgrowth and synapse recovery after a moderate TBI by inhibiting the RhoA-Rho kinase pathway. The limitations of this study include no testing of nMDEVs on functional recovery after TBI and no examination of the effects of a MDEVs overexpressing miR-5121 on cognitive function.Cell culture studies revealed that adverse effects of aMDEVs were due to reduced expression of miR-5121, as overexpression of miR-5121 in these EVs increased the expression of GAP-43 and other synaptic proteins and enhanced dendritic complexity in cultured neurons. Besides, administration of aMDEVs overexpressing miR-5121 into FPI mice enhanced GAP-43 expression, apical dendrite spine density in the peri-contusion region, and improved motor coordination. Additional investigation revealed that MDEVs overexpressing miR-5121 also decreased the upstream activator of the RhoA-Rho kinase pathway and its downstream effector RhoA-GTP in the peri-contusion region.61 miR-124 MDEVs were isolated from cultures of BV2 microglia treated with repetitive TBI mouse brain extracts. In an in vitro scratch neuronal injury model, administration of miR-124 MDEVs promoted neuroprotective effects by regulating the exaggerated upregulation of autophagy and apoptotic markers typically seen in injured neurons by targeting 3ʹ UTR of family-interacting protein of 200 kDa (FIP200) mRNA. While autophagy is beneficial in physiological conditions because of its ability to maintain cellular homeostasis by eliminating damaged cellular components, overly upregulated autophagy after TBI can contribute to increased neurodegeneration.61 Indeed, studies have shown that inhibition of excessive autophagy after TBI can improve neurological outcomes.68 Therefore, in this study, the authors tested the effects of intravenous administration of miR-124 MDEVs in rCCI mice. Following treatment with miR-124 MDEVs, rCCI mice displayed reduced mNSS scores, improved motor performance in the rotarod test, and enhanced spatial learning and spatial memory retrieval ability compared to the rCCI alone group.61 The results demonstrated the efficacy of miR-124 MDEVs for mediating functional recovery after rCCI. However, the authors did not test the effects of miR-124 MDEVs on autophagy in the brain, which is a limitation of the study. Therefore, mechanisms underlying miR-124 MDEV-treatment mediated improved functional recovery in rCCI mice remain to be determined, though suppression of autophagy is one of the likely mechanisms.Li et al. tested the efficacy of MDEVs enriched with miR-124-3p (miR-124 MDEVs) in a mouse model of repetitive CCI (rCCI) that employed 4 impacts at 24-h intervals, at a velocity of 3.6 m/s and a deformation depth of 1.2 mm.65 The RES-MDEVs were isolated through ultracentrifugation and were administered 14 days post-SCI. The authors demonstrated that resveratrol in RES-MDEVs was more stable than free resveratrol, with increased concentrations detected in the lung, liver, brain, and spinal cord in vivo. RES-MDEV treatment after SCI improved muscle tension in hind limbs and functional movements in the foot, compared to the SCI alone group or SCI group receiving free resveratrol. The SCI rats receiving RES-MDEV treatment also displayed better BBB scores. The beneficial effects of RES-MDEV treatment were associated with the upregulation of autophagy-related proteins microtubule-associated protein light chain 3 beta (LC3B) and beclin-1, and inhibition of apoptosis-related protein cleaved caspase-3. The results suggested that RES-MDEVs can improve motor recovery in rats after SCI through increased autophagy and decreased apoptosis. However, the study has several limitations. Apart from resveratrol analysis, the study did not characterize the protein or miRNA cargo of MDEVs. Also, the study did not employ a naïve MDEV treatment group. Hence, it is unclear whether the effects were due to the delivery of resveratrol by MDEVs or the therapeutic cargo of MDEVs.Fan et al. investigated the effects of the administration of MDEVs generated from microglia cultured from fetal spinal cords and treated with the antioxidant and anti-inflammatory compound resveratrol (RES-MDEVs) in a rat model of SCI that employed Allen’s method.58 Whereas, another study showed that aMDEVs isolated from cultures of BV2 microglia treated with repetitive TBI mouse brain extracts could improve functional recovery in a model of rCCI.61 Thus, therapeutic or adverse effects of aMDEVs likely depend on the microglial activation state. Considering these, it may be necessary to utilize MDEVs shed from microglia displaying either non-inflammatory or anti-inflammatory properties.NSC-EVs and ADEVs have the potential to treat TBI. Several preclinical studies show that NSC-EV administration after a CCI or SCI can mediate neuroprotective effects and promote motor recovery. Also, EVs from NSCs primed with IGF1 have better antiapoptotic effects because of specific miRNAs in their cargo. Furthermore, early intervention with ADEVs after TBI appears promising for promoting motor and cognitive recovery by antioxidant effects via enhanced Nrf2 signaling. The ADEVs generated from primary astrocytes treated with traumatic brain tissue extracts appear to be loaded with a specific miRNA that can transform activated microglia into non-inflammatory or anti-inflammatory M2 phenotypes by inhibiting NF-κB signaling. Additionally, ADEVs generated from astrocytes cultured in the presence of injured neuron factors carry high levels of GJA1-20k, having robust neuroprotective activity. Another study has demonstrated that ADEVs overexpressing NKILA could further enhance the antioxidant, NF-kB signaling inhibition, and antiapoptotic effects of ADEVs. Concerning MDEVs, the therapeutic effects of nMDEVs are yet to be tested rigorously in TBI models. On the other hand, studies using aMDEVs have reported both adverse and beneficial effects. One study demonstrated that aMDEV treatment inhibits spine density in cortical pyramidal neurons and exacerbates neurological dysfunction after TBI due to reduced expression of miR-2151 in aMDEVs.69-71 the effect of early NSC-EV, ADEV, or MDEV treatment after TBI needs to be examined on chronic neuroinflammatory cascades associated with long-term cognitive and mood impairments. Third, the efficacy of delayed interventions using NSC-EVs, ADEVs, or MDEVs after TBI has not been ascertained. Such studies are vital to determine whether neuroinflammatory cascades and enduring cognitive and mood impairments could be reversed through single or intermittent administration of NSC-EVs, ADEVs, or MDEVs commencing days or weeks after experiencing TBI. Fourth, the best route of EV administration for treating TBI has not been evaluated. Most studies employed tail vein injections in rodent models but have not quantified the fraction of EVs that permeate the brain or get incorporated into neurons and glia. Since intravenous administration leads to the entry of EVs into the systemic circulation, the amount of EVs entering the brain is likely minimal. Therefore, testing the efficacy of intranasal administration in TBI models will be necessary as such administration in other models has shown quick entry of EVs into virtually all regions of the brain as well as their incorporation into neurons, microglia, and astrocytes.72Nonetheless, several issues remain regarding the use of NSC-EVs, ADEVs, or MDEVs as “biologic” for treating TBI in the future. First, rigorous and long-term testing of motor, cognitive, and mood function after NSC-EV, ADEV, or MDEV administration after TBI has not been performed. Several studies have employed only mNSS to gauge therapeutic effects. Second, most studies have mainly tested the neuroprotective, anti-apoptotic, and anti-inflammatory effects of such EVs in the acute phase of TBI. Since secondary changes after TBI evolve over weeks and months after TBI,26 in penetrating TBI and closed head injury models will be needed in the future to translate EV therapy for both mild and moderate TBIs in the clinic. Also, EV isolation methods compatible with generating clinical grade EVs as per FDA guidelines must be developed before clinical translation of NSC-EVs, ADEVs, or MDEVs is considered for treating TBIs in the clinic. In conclusion, NSC-EVs and ADEVs have promise for mitigating TBI-induced brain dysfunction. However, significant additional preclinical studies in various models of TBI using NSC-EVs and ADEVs that have undergone thorough characterization of their cargo as well as therapeutic and adverse properties are needed prior to clinical translation.Furthermore, while some studies have identified one or more miRNA or protein cargo having neuroprotective, antioxidant, or anti-inflammatory effects, an exhaustive evaluation of miRNA or protein cargo of NSC-EVs, ADEVs or MDEVs has not been conducted. Such characterization is vital as NSCs, astrocytes, and microglia grown in different culture conditions could carry some adverse miRNAs and proteins in addition to beneficial molecules. Therefore, evaluating the function of highly enriched miRNAs and proteins within NSC-EVs, ADEVs, and MDEVs through gain of function and loss of function studies in cell culture assays will be crucial. Since secondary changes contribute to illness at extended time points after TBI, a thorough analysis of NSC-EV, ADEV, or MDEV cargo will be vital to understand the potential mechanisms by which EVs mediate recovery of function and further improve their therapeutic efficacy. Moreover, most studies have employed NSC-EVs, ADEVs, or MDEVs from rat or mouse NSCs, astrocytes, and microglia. Hence, testing the efficacy of well-characterized EVs from NSCs, astrocytes, or microglia derived from human embryonic or induced pluripotent stem cells


Six healthy volunteers underwent 129Xe DW-MRI at baseline only. Moreover, 3He DW-MRI was indirectly co-registered to CT via spatially aligned 3He ventilation and structural 1H MRI. A voxel-wise comparison of the overlapping 3He apparent diffusion coefficient (ADC) and mean acinar dimension (LmD) maps with CALIPER CT patterns was performed at baseline and after 1 year. The abnormal lung percentage classified with the LmD value, based on a healthy volunteer 129Xe LmD, and CALIPER was compared with a Bland–Altman analysis. The largest DW-MRI metrics were found in the regions classified as honeycombing, and longitudinal DW-MRI changes were observed in the baseline-classified reticular changes and ground-glass opacities regions. A mean bias of −15.3%  towards CALIPER was observed for the abnormal lung percentage. This suggests DW-MRI may detect microstructural changes in areas of the lung that are determined visibly and quantitatively normal by CT.The patterns of idiopathic pulmonary fibrosis (IPF) lung disease that directly correspond to elevated hyperpolarised gas diffusion-weighted (DW) MRI metrics are currently unknown. This study aims to develop a spatial co-registration framework for a voxel-wise comparison of hyperpolarised gas DW-MRI and CALIPER quantitative CT patterns. Sixteen IPF patients underwent  Idiopathic pulmonary fibrosis (IPF) is a group of lung diseases that are defined by the lack of an underlying cause and are characterised by the presence of usual interstitial pneumonia (UIP) and pathological fibroblastic activity . UIP is Several texture-based or machine learning algorithms ,14,15,163He) or xenon-129 (129Xe) is an imaging technique that is sensitive to changes in acinar microstructure . Ho. Ho36]. piration , and conD values were observed in the regions classified as honeycombing. In addition, longitudinal DW-MRI changes were predominantly observed in reticular change and ground-glass opacity regions. Furthermore, the LmD values in voxels with a CALIPER normal pattern were larger than those from age-matched healthy volunteers, thus suggesting DW-MRI may detect microstructural changes even in areas of the lung that are determined as structurally normal by CT scans. The quantitative biomarkers from hyperpolarised gas DW-MRI and CT could play a role in future clinical trials, whereby IPF disease progression and response to new treatments is assessed. With the transition from hyperpolarised 3He to 129Xe for clinical lung imaging studies [129Xe DW-MRI. Furthermore, it can be used to explore quantitative CT patterns in different ILD subtypes and pulmonary diseases.The spatial co-registration of hyperpolarised gas DW-MRI and CALIPER quantitative CT maps in IPF patients demonstrated that the largest ADC and Lm studies , this sp


Growing evidence suggests that infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may trigger idiopathic inflammatory myopathies (IIM). Few studies have described individual juvenile IIM (JIIM) cases following SARS-CoV-2 infection, and none explored its potential effects on JIIM clinical presentation. We aim to investigate the impact of SARS-CoV-2 on JIIM in patients diagnosed before and after the onset of the Coronavirus Disease 2019 (COVID-19) pandemic.Patients diagnosed with JIIM before age 19 at The Children’s Hospital at Montefiore were included. Demographics, clinical and laboratory data, and evidence of SARS-CoV-2 exposure were collected retrospectively. Patients were grouped by pre-COVID-19  and post-COVID-19 . Descriptive statistics were used to summarize each variable. Non-parametric testing was performed using Fischer’s exact test and Mann-Whitney U test.Fifty-one patients were included, 13 (25%) diagnosed in the post-COVID-19 era. Of these, 10 (77%) had onset of JIIM symptoms after January 1, 2020; 6 (60%) with known or suspected SARS-CoV-2 exposure. Though not statistically significant, post-pandemic patients tended to be older, female, and have non-specific cutaneous manifestations. Despite reported delays in care for other pediatric diagnoses during the pandemic, fewer post-pandemic patients had delays in JIIM diagnosis.This is the first study to explore the effects of SARS-CoV-2 on JIIM clinical presentation. While our exploratory single-center study did not find significant differences in JIIM diagnosed pre- and post-pandemic, larger prospective multicenter studies are warranted to evaluate this association and to explore clinical variances over time. The link between infectious organisms and autoimmunity has been well-described –3. GivenSince the onset of the COVID-19 pandemic, there have been observations worldwide of new diagnosis or flare of existing IIM after SARS-CoV-2 infection and reports of increased IIM incidence . In the Although these observational reports identify possible differences in IIM since the COVID-19 pandemic, much remains unknown. While a few reports identify a clear temporal link with evidence of SARS-CoV-2 infection preceding development or flare of IIM , 8, the A search of The Children’s Hospital at Montefiore (CHAM) electronic medical record, Epic, was performed using ATLAS, a web-based open-source software application that supports clinical analyses. The search identified JIIM patients seen by a pediatric rheumatologist at CHAM between January 1, 2008 and July 15, 2022. The CHAM catchment and referral area is broad and includes New York City and its suburbs (including Westchester County), New York State, and, less commonly, surrounding states.JIIM was defined as systemic autoimmune disease characterized by weakness and objective evidence of chronic inflammation of skeletal muscles with onset of symptoms before 19 years of age, based on Rider et al’s definition . ObjectiIn this retrospective cross-sectional study, manual chart review was performed to assess baseline clinical data. Sociodemographic variables, date of diagnosis and symptom onset, laboratory data, and clinical manifestations were recorded. Muscle weakness was determined using the Childhood Myositis Assessment Scale (CMAS) when available, otherwise physical exam was used. Weakness was categorized using CMAS score based on cutoffs determined by CMAS validation studies  or by suPatients were grouped by date of diagnosis as pre-pandemic  or post-pandemic ; January 1, 2020 was considered the cutoff date since cases of COVID-19 were first reported in multiple states in the United States around this time. Analysis was also performed using date of symptom onset instead of diagnosis. Evidence of exposure to SARS-CoV-2 was recorded. To assess whether exposure to SARS-CoV-2 may impact subsequent JIIM development, exposure prior to reported symptom onset was used. Exposure was considered known if the patient had a positive polymerase chain reaction (PCR) to SARS-CoV-2 and historical if a household member had confirmed COVID-19 infection but the patient was not tested. Albert Einstein College of Medicine Institutional Review Board (IRB) approval was obtained  with waiver of informed consent.Statistical analysis was performed using STATA software, version 17.0. All variables were examined to identify distribution of continuous variables, missing data, outliers, and potential data entry errors. Descriptive statistics were applied to evaluate baseline characteristics, which were summarized as mean and standard deviation for normally distributed continuous variables, median and interquartile range (IQR) for non-normally distributed continuous variables, and frequencies and percentages for categorical variables. To compare JIIM pre- and post-pandemic, bivariate associations were explored. After checking assumptions of all statistical tests, non-parametric testing was performed using the Mann-Whitney U test for continuous independent variables and Fisher’s exact test for categorical independent variables. Two-tailed alpha of 0.05 was used to determine statistical significance.The initial ICD-10 code-based search of Epic identified 797 patients. Manual review resulted in removal of 746 patients who did not meet JIIM criteria. Seven patients who were not identified by ATLAS but who were in DMW’s JIIM patient cohort were added to the remaining 51 patients. Of these 58 JIIM patients, 7 patients were excluded due to missing baseline visit information; 51 patients diagnosed with JIIM from January 1, 2008 to July 15, 2022 were included in the final analysis , were female , and were non-white . The median age at diagnosis was 8.1 years . While the median time between symptom onset and diagnosis was 3.5 months , 30% of patients experienced delays in diagnosis of 6 months or greater. Most patients had at least 1 autoantibody, with 63% of patients with myositis-specific antibodies (MSA) and 37% with myositis-associated antibodies (MAA) (Table p = 0.45).The distribution of new JIIM diagnoses is depicted in Fig. p = 0.52) and were non-white . Compared to patients diagnosed pre-pandemic, those diagnosed post-pandemic did not have significantly different CMAS scores (p = 0.97) or degree of weakness (p = 0.92), nor was there significantly different absence of autoantibodies (p = 0.45) or presence of abnormal muscle enzymes (p = 0.27), MSA (p = 0.51), or MAA (p > 0.999). Though not statistically significant, post-pandemic, patients seemed to have fewer delays in diagnosis , were less likely to be male , were older at diagnosis , and had more non-specific cutaneous manifestations . None of the patients diagnosed post-pandemic developed lipodystrophy (p > 0.999) or had evidence of GI (p = 0.56), cardiac (p > 0.999), or pulmonary involvement (p = 0.32), and only one patient (8%) developed cutaneous ulceration compared to 12 patients (35%) pre-pandemic (p = 0.08) -γ-positive dermatomyositis patients and SARS-CoV-2 . In our Our study had several notable strengths. It was performed in the Bronx, New York, which has a large Black and Hispanic/Latino population, thus making it generalizable to a broader range of patients from various racial and ethnic backgrounds. Despite the relatively small sample size, to our knowledge it is by far the largest study to evaluate new diagnosis of JIIM since onset of the COVID-19 pandemic. It is also the first study to statistically compare baseline clinical features of JIIM pre- and post-pandemic. We used clear diagnostic, inclusion, and exclusion criteria, in contrast to many other studies exploring the association of SARS-CoV-2 with IIM . FinallyDespite these strengths, our study had the limitations intrinsically seen with cross-sectional study designs. This was a single-center study with a relatively small sample size, which likely limited the power to detect statistically significant differences pre- and post-pandemic. Given that this was a single-center study, results may not be generalizable to all centers, despite our relatively racially/ethnically diverse sample. The lack of a control group limited our ability to calculate true incidence rates despite similar incidence patterns pre- and post-pandemic in our study. The ATLAS-based search did not identify 7 JIIM patients; however, we relied on multiple supplementary means of identifying JIIM patients at our center, including querying individual providers and referencing provider JIIM patient lists to minimize the likelihood of missing JIIM patients. Furthermore, due to several patients being diagnosed shortly before data collection and analysis, we limited our comparison to baseline features. Therefore, it remains unknown if manifestations later in disease course, prognosis, and/or outcomes differ in patients diagnosed pre- and post-pandemic. The use of January 1, 2020 as the cutoff date for pre- and post-pandemic may mean that patients without exposure to SARS-CoV-2 were included in the post-pandemic group; while this may have attenuated differences between the groups, this date was chosen to avoid the possibility of inaccurately including patients with exposure in the pre-pandemic group. Finally, although we did have information on exposure to SARS-CoV-2 in all 10 patients with onset of symptoms post-pandemic, not all patients had documented infection. Despite evidence of preceding SARS-CoV-2 infection in 50% and high suspicion of preceding infection in 10% of our post-pandemic patients, we cannot determine if and how SARS-CoV-2 infection impacted the onset or manifestations of subsequent JIIM, nor if this differs from the suspected effects of other viruses, due to the observational nature of the study. Many SARS-CoV-2 infections can be asymptomatic or mildly symptomatic, therefore confirmation of infection can only be obtained with objective testing, which was not possible given the retrospective nature of the study.In conclusion, our exploratory study did not find significant differences in JIIM diagnosed pre- and post-pandemic, though non-statistically significant trends emerged. However, larger prospective multicenter studies are warranted to fully evaluate this association and explore whether any differences emerge in disease features or outcomes over time. A larger sample size would also yield greater power to detect differences and allow for better understanding of whether our non-significant but lower rates of delays in diagnosis post-pandemic are significant and, if so, consider why this differs from many pediatric studies demonstrating the opposite. The mechanism by which SARS-CoV-2 impacts JIIM, and if and how this differs from other viruses, also warrants further study.


Most clinicians recognize the importance of reducinglow-density lipoprotein cholesterol (LDL-C) and, therefore, address thistherapeutic need to decrease cardiovascular disease risk. In addition to thecritical role that LDL-C plays, recent studies have shown the contribution ofother lipid fractions, such as high-density lipoprotein cholesterol (HDL-C)and triglycerides (TG), to overall cardiovascular health. Managed careinitiatives to reduce cardiovascular risk typically focus on highly effectivestatin therapies, which are primarily LDL-C-lowering agents and have lesserTG-lowering and HDL-C-raising effects. However, clinical and epidemiologicdata illustrate the need to expand the scope of therapies to reduce theresidual cardiovascular risk associated with low HDL-C levels and elevatedTG levels, even when LDL-C is managed successfully.To address the value of treating beyond LDL-C level to improvepatient health outcomes and reduce health care-related costs.Several large trials and meta-analyses have investigated the effects of lipid-lowering statin therapy and have consistently demonstrated that statin therapy significantly reduces LDL-C levels and incidence of cardiovascular events. In spite of the efficacy of statin therapy in these studies, statins did not eliminate cardiovascular risk. Rather, significant residual cardiovascular risk remains after treatment with statins, especially in high-risk patients such as those with diabetes. Residual cardiovascular risk stems, at least partially, from low HDL-C and elevated TG. Low HDL-Clevels have been identified as a significant, independent predictor of cardiovascular risk, and increases in HDL-C are associated with reductions in cardiovascular events. High TG levels are a significant risk factor for cardiovascular disease and are a marker for atherogenic remnant lipoproteins, such as very low-density lipoprotein cholesterol (VLDL-C). Additionally, with elevated TG levels, a combination of LDL-C with VLDL-Cin the measure of non-HDL-C may be a better predictor of cardiovascular risk than LDL-C alone. Recent national treatment guidelines suggest that combination therapymay be necessary to address multiple lipid targets ; adding niacin or a fibrate to a statin is a therapeutic option that should be considered. As monotherapy agents, fibrates and niacin havebeen demonstrated to alter several lipid parameters and reduce cardiovascular events. Niacin appears to exert the greatest beneficial effects on the widest range of lipoprotein abnormalities, in addition to possessing an established safety profile. Moreover, niacin/statin combination therapy may provide greater benefits, as manifested through a correction of atherogenic lipid abnormalities, a slowing of atherosclerosis progression in coronaryheart disease (CHD) patients, and a reduction of residual cardiovascular risk. Pharmacoeconomic modeling studies have been used to describe the potential effects on both cardiovascular events and health care costs by the achievement of, or failure to achieve, combined optimal lipid values(OLVs). Achievement of OLVs is predicted to be associated with a reduced risk of cardiovascular events, in which greater magnitudes of risk reduction accompany the achievement of a greater number of lipid goals. Basedon patient baseline lipid values and product labeling information, mathematical models estimate that OLVs are achieved more frequently with extended-release niacin (niacin ER)/simvastatin combination therapy thanwith other high-potency agents. These modeling estimates were maintained in different patient groups, including those with diabetes or the metabolicsyndrome. Finally, these modeling studies estimated that a fixed-doseniacin ER/simvastatin combination therapy would reduce direct medical costs of CHD events more effectively than would high-dose simvastatin monotherapy.Statins are highly effective for lowering LDL-C levels and,consequently, cardiovascular event rates. However, statins do not eliminate cardiovascular risk. Even in the presence of tightly controlled LDL-C levels,evidence indicates that high TG and low HDL-C levels are independent cardiovascular risk factors. Treating lipid parameters beyond LDL-C may require the addition of niacin or a fibrate to statin therapy. Niacin is themost effective agent for raising HDL-C levels, and pharmacoeconomic modeling suggests that niacin ER/statin combination therapy may promote the cost-effective achievement of OLVs in several at-risk patientpopulations.


Background: Since its discovery, poly (ADP-ribose) polymerase 1 (PARP-1) has been extensively studied due to its regulatory role in numerous biologically crucial pathways. PARP inhibitors have opened new therapeutic avenues for cancer patients and have gained approval as standalone treatments for certain types of cancer. With continued advancements in the research of PARP inhibitors, we can fully realize their potential as therapeutic targets for various diseases.Purpose: To assess the current understanding of PARP-1 mechanisms in radioprotection and radiotherapy based on the literature.Methods: We searched the PubMed database and summarized information on PARP inhibitors, the interaction of PARP-1 with DNA, and the relationships between PARP-1 and p53/ROS, NF-κB/DNA-PK, and caspase3/AIF, respectively.Results: The enzyme PARP-1 plays a crucial role in repairing DNA damage and modifying proteins. Cells exposed to radiation can experience DNA damage, such as single-, intra-, or inter-strand damage. This damage, associated with replication fork stagnation, triggers DNA repair mechanisms, including those involving PARP-1. The activity of PARP-1 increases 500-fold on DNA binding. Studies on PARP-1-knockdown mice have shown that the protein regulates the response to radiation. A lack of PARP-1 also increases the organism’s sensitivity to radiation injury. PARP-1 has been found positively or negatively regulate the expression of specific genes through its modulation of key transcription factors and other molecules, including NF-κB, p53, Caspase 3, reactive oxygen species (ROS), and apoptosis-inducing factor (AIF).Conclusion: This review provides a comprehensive analysis of the physiological and pathological roles of PARP-1 and examines the impact of PARP-1 inhibitors under conditions of ionizing radiation exposure. The review also emphasizes the challenges and opportunities for developing PARP-1 inhibitors to improve the clinical outcomes of ionizing radiation damage. The PARP-1 nucleoprotein is expressed at high levels in eukaryotic cells, where it serves as an essential component of the DNA base excision-repair (BER) system . Recent PARP-1 inhibitors can be used in combination with ionizing radiation or chemotherapy to enhance the sensitivity of tumor cells to these treatments. They can also serve as standalone treatments that are both effective and relatively non-toxic for cancers with DNA repair gene defects, including those with BRCA-1/2 mutations, such as some triple-negative breast cancers. Most PARP inhibitors developed to date are not specific for PARP-1 and function by interacting with the catalytic NAD-binding domain and thus competing with NAD for PARP-1 binding by simulating the substrate-enzyme interaction and interfering with double-stranded break (DSB) repair . Others The transcription factor NF-κB, associated with inflammation, is typically found in the cytosol bound to the inhibitory protein IκB. When stimulated by intracellular or extracellular stimuli, IκB is degraded, leading to the nuclear translocation of NF-κB, which binds to the promoter regions of specific target genes, upregulating their expression . PARP-1 Cell death facilitated by PARP-1 shares many features with typical apoptosis, necessitating the relocation of the mitochondrial AIF-1 to the nuclear compartment . SphingoExposure to ionizing radiation increases PARP-1 activity levels significantly within one minute. Inhibiting PARP-1 activity can significantly slow the rate of DNA damage repair . PARP-1 PARP inhibitors disrupt DNA repair, providing ample opportunities for radiosensitization, adjuvant treatment, or chemical synergism. Many such inhibitors have been evaluated in various preclinical and clinical studies, demonstrating their primary function of interfering with PARP-1 activities in DNA repair and transcriptional regulation. The PARP substrate 3-aminobenzamide (3-AB) is a first-generation PARP inhibitor lacking in specificity, although it can inhibit a reported 96% of PARP-1 activity in cells, adversely affecting DNA synthesis, glucose metabolism, and overall cellular viability . 3-AB cain vivo patient-derived xenograft (PDX) model of BRCA mutant breast cancer (HBCx-17)  . The invHBCx-17) . When trHBCx-17) . BGP-15 HBCx-17) . E7449 cHBCx-17) . BYK2041HBCx-17) . The PARHBCx-17) . DPQ andHBCx-17) . It has HBCx-17) . Detailsin vivo radiotherapy and chemotherapy in various cancers, including lung cancer, colorectal cancer, glioblastoma, and serous ovarian cancer xenotransplantation ation in regulating DNA repair and DNA metabolism . InvestiIn vivo studies using dual-gene mutant mice have demonstrated higher rates of embryonic developmental defects, apoptosis, and elevated levels of oxidative damage and intracellular phosphorylation in adult tissue samples  can enhance radioresistance. This improvement is mediated by PARP-1-dependent cell death pathways, which involve PARP-1 activation and ROS generation inhibition. The combination of FRT treatment and PARP-1 knockout significantly reduces ROS levels in cells and prevents the mitochondrial translocation of AIF  is an inhibitor of NF-κB signaling that can protect against endotoxin-induced liver damage . OB can Inhibitors of NF-κB can also suppress caspase-3-dependent apoptotic cell death . ActivatCaspase-3 is a key effector caspase that triggers apoptotic cell death, partly through its ability to cleave substrate proteins, including PARP-1, DNA fragmentation factor (DFF45), and Lamin B . Severe in vitro, suggesting that HMGB1 knockdown can promote caspase-3 upregulation and the cleavage of PARP. Increases in ROS production can promote DNA damage, increasing cell radiosensitivity  Determining an optimal low-intensity dose regimenSince its discovery, PARP-1 has been extensively characterized as a regulator of numerous biologically important pathways. PARP inhibitors provide new therapeutic opportunities to cancer patients, leading to their approval as single-agent treatments for certain cancer types. While the next-generation of these small-molecule inhibitors is in active development, the current versions continue to be applied as radiosensitizing drugs in clinical trials in combination with radiotherapy and chemotherapy. Further advancement in PARP inhibitor research could fully underscore the value of targeting this pathway for treating various diseases, thus providing novel therapeutic options to numerous patients, including those currently without effective treatments.While early experiments showed that single doses of Olaparib, Rucaparib, Niraparib, and Talazoparib were the maximum doses tolerated, it is unclear whether the maximum tolerated dose is appropriate for a tumor-selective drug that is not expected to damage normal tissues. Therefore, opting for the optimal biological dose instead of the maximum tolerated dose might be preferable to improve patients’ quality of life. Further research is needed to determine whether the low-intensity dose regimen, which may be less toxic and more affordable, is as effective as the approved single-dose regimen.(2) Identifying predictive biomarkersToxicity issues and the need for meticulous titration of the cytotoxic agents and PARP inhibitors still constrain the combined use of PARP inhibitors with genotoxic chemotherapy and radiotherapy. A well-designed dose plan will be necessary to optimize the therapeutic index, which may have to be considered on an individual basis. The preclinical and clinical data show that when PARP inhibitors are combined with cytotoxic drugs, curative effects, and tolerance can be achieved with shorter treatment courses and lower dosages. We posit that it is necessary to reclassify PARP inhibitors based on their dual molecular mechanisms to accurately evaluate suitable combination strategies .(2) Iden(3) Understanding the clinical mechanisms that drive resistance to PARP inhibitors in BRCA-mutant cancersA crucial objective, alongside determining the optimum tolerance for combined PARP inhibitor therapy, is the discovery of predictive biomarkers for patient segregation in conjunction with the therapy and understanding the impact of this combination on the resistance mechanisms against PARP inhibitors to achieve a more sustained clinical response. A critical query is whether a combined treatment regimen could alleviate the resistance to PARP inhibitor monotherapy seen in patients with BRCA mutations. It remains uncertain how much the combined administration of PARP inhibitors and other drugs can delay or prevent the onset of PARP inhibitor resistance in these patients and potentially lead to longer-lasting responses. Furthermore, the sequence and timing of administering the therapeutic agents demand careful consideration.The prospect of mitigating resistance to PARP inhibitor monotherapy in patients with BRCA mutations through combination therapy presents an intriguing question. It is unclear whether the simultaneous use of PARP inhibitors and auxiliary agents could impede or prevent the emergence of resistance to PARP inhibitors in these patients, potentially resulting in more enduring responses. Furthermore, the arrangement and timing of administering these therapeutic agents call for meticulous planning and examination.(4) Leveraging biological insights into the response to PARP inhibitors to guide the design of combination therapyThe presence of BRCA mutations does not universally predict the sensitivity of tumors to PARP inhibitors. In fact, PARP inhibitors display a spectrum of sensitivity in BRCA-mutant cancers. For instance, ovarian cancers with BRCA mutations show greater sensitivity than breast, prostate, or pancreatic cancers with the same mutations. Furthermore, cancers with BRCA mutations that are usually unrelated to BRCA carriers are even less sensitive to PARP inhibitors . Genome Enhancing our understanding of the biological responses to PARP inhibitors could significantly contribute to designing effective combination therapies involving PARP inhibitors. Given that PARP inhibitors trigger extreme genomic instability, resulting in cancer cell death, one potential strategy could involve leveraging this phenotype. This could be achieved by pairing PARP inhibitors with agents that amplify cell death processes, such as apoptosis or necrosis, in the context of enhanced genomic instability .The research conducted over the past few decades indicates that the combined use of PARPi holds immense promise for cancer treatment, provided several significant hurdles can be overcome.