ATAC-seq) is a technique widely used to investigate genome-wide chromatin accessibility. The recently published Omni-ATAC-seq protocol substantially improves the signal/noise ratio and reduces the input cell number. High-quality data are critical to ensure accurate analysis. Several tools have been developed for assessing sequencing quality and insertion size distribution for ATAC-seq data; however, key quality control (QC) metrics have not yet been established to accurately determine the quality of ATAC-seq data. Here, we optimized the analysis strategy for ATAC-seq and defined a series of QC metrics for ATAC-seq data, including reads under peak ratio (RUPr), background (BG), promoter enrichment (ProEn), subsampling enrichment (SubEn), and other measurements. We incorporated these QC tests into our recently developed ATAC-seq Integrative Analysis Package (AIAP) to provide a complete ATAC-seq analysis system, including quality assurance, improved peak calling, and downstream differential analysis. We demonstrated a significant improvement of sensitivity (20%–60%) in both peak calling and differential analysis by processing paired-end ATAC-seq datasets using AIAP. AIAP is compiled into Docker/Singularity, and it can be executed by one command line to generate a comprehensive QC report. We used ENCODE ATAC-seq data to benchmark and generate QC recommendations, and developed qATACViewer for the user-friendly interaction with the QC report. The software, source code, and documentation of AIAP are freely available at https://github.com/Zhang-lab/ATAC-seq_QC_analysis.Assay for transposase-accessible chromatin with high-throughput sequencing ( To regulate the transcription of a eukaryotic genome, chromatin must remain in an accessible state to allow binding of transcription factors and initiation of transcription activation The ATAC-seq analysis strategy is primarily adopted from ChIP-seq data analysis. After aligning sequencing reads to the genome, peak calling tools, such as MACS2 qATACViewer, a visualization tool included in AIAP, for user-friendly visualization of QC reports. AIAP is compiled into a Docker/Singularity image to allow maximized compatibility on different operating systems and computing platforms. The software, source code, and documentation are freely available at https://github.com/Zhang-lab/ATAC-seq_QC_analysis.Here, we present ATAC-seq Integrative Analysis Package (AIAP), a software package containing an optimized ATAC-seq data QC and analysis pipeline. Along with general QC metrics, such as library insert fragment size distribution, we specifically introduced a series of QC metrics for ATAC-seq, including reads under peak ratio (RUPr), background (BG), promoter enrichment (ProEn), subsampling enrichment (SubEn), and other measurements. By applying AIAP, we demonstrated a significant improvement in both peak calling and differential analysis by processing the paired-end sequencing data in single-end mode: more than 20% of ATAC-seq peaks can be identified using AIAP, and over 30% more differentially accessible regions (DARs) can be identified by AIAP in downstream analysis. We applied AIAP to reanalyze 70 mouse ENCODE https://github.com/Zhang-lab/ATAC-seq_QC_analysis/.Here, we describe AIAP for processing and analyzing ATAC-seq data. The AIAP workflow typically consists of four steps, as shown in Cutadapt and aligned to the reference genome by BWAmethylQAmethylQA first filters unmapped and low-quality mapped PE reads and then identifies the Tn5 insertion position at each read end by shifting + 4 bp/−5 bp on the positive/negative strands. methylQA further extends 75 bp in both directions around the Tn5 insertion position to create two pseudo single-end (SE) mapped reads with the length of 150 bp in PE as SE (PE-asSE) mode. Next, AIAP compiles different files for downstream analysis (.bed files) and normalized visualization (.bigWig files). The bed file is used to perform peak calling by MACS2 --keep-dup 1000 --nomodel --shift 0 --extsize 150.The data processing step first configures the working path. The ATAC-seq paired-end (PE) raw-read FASTQ files are trimmed by --keep-dup 1000 --nomodel --shift 0 --extsize 0.In PE-noShift mode, AIAP first filters unmapped and low-quality mapped PE reads and then identifies the Tn5 insertion position at each read end by shifting + 4 bp/−5 bp on the positive/negative strands, then isolates the whole fragment between two insertion positions to create one PE mapped fragment for downstream analysis (.bed files) and normalized visualization (.bigWig files). The bed file is used to perform peak calling by MACS2 FastQC to check the sequencing quality, duplication rate, and GC bias before alignment. After alignment, AIAP generates the mapping statistics summary, chromosome distribution of uniquely mapped reads, mitochondrial genome (chrM) contamination rate, library insert fragment size distribution, and library complexity. AIAP also performs a series of post-peak calling quality checks, including peak width distribution, RUPr, BG, ProEN, and SubEn. AIAP also provides saturation analysis, promoter peak distribution, and signal ranking analysis. AIAP reports the quality metrics in a JSON file, which can be visualized using qATACViewer. The default QC standard of AIAP is determined by the QC metrics of mouse ATAC-seq data generated by ENCODE consortium . We defined the key QC metrics below.AIAP performs a series of quality checking steps before and after alignment. AIAP calls preseq (https://github.com/smithlabcode/preseq). 5) To obtain the total number of useful single ends, each end of a non-redundant uniquely mapped read pair will be shifted + 4 bp/−5 bp on the positive/negative strands and then further extended 75 bp in both directions around the Tn5 insertion position.There are 5 QC metrics for alignment. 1) Non-redundant uniquely mapped reads refer to the reads that are uniquely mapped to the reference genome after removing redundancy. 2) The second is chromosome distribution/chrM contamination rate. The former describes the distribution of uniquely mapped reads across all chromosomes, while for the latter, the number of uniquely mapped reads on chrM is used as a QC metric to measure the quality of the ATAC-seq library. 3) Library insert fragment size distribution is measured as the length of DNA fragment defined by non-redundant uniquely mapped read-pairs. 4) Library complexity is estimated in both duplication rate and predicted yield of distinct reads generated by RUPr is defined as the percentage of all useful ends  are randomly selected from the genome outside of ATAC-seq peaks. The ATAC-seq signal in each region is calculated as reads per kilobase per million mapped reads (RPKM). The percentage of all such regions with the ATAC-seq signal over the theoretical threshold (RPKM = 0.377) is considered high-background and used as a QC metric to indicate the background noise.The promoters, the regions +/− 1 kb around transcription start sites (TSSs) of active genes, provide a positive control for OCRs. The ATAC-seq useful ends enriched on detected promoters (ATAC-seq peaks) are used as a QC metric to measure the signal enrichment calculated as follows.The ATAC-seq signal (useful ends) enriched on the detected ATAC-seq peaks is used as a QC metric to measure the signal enrichment at the genome-wide level. To avoid sequencing-depth bias, 10 million useful ends are sampled from the complete dataset, and peak calling is performed to identify the OCRs. SubEn is calculated after 10 million pseudo counts are added into the calculation as background, which can avoid calculation failure caused by the low sequencing depth of testing ATAC-seq library.MACS2 is used to call narrow peaks for a series of subsampling from complete useful ends with a step of 10% of total sequencing depth. The length of identified peaks covering genomic regions at each subsampling are used to calculate the recovery (percentage) of complete peaks covering genomic regions when using complete useful ends.The ATAC-seq peak signals are ranked, and the percentage of promoter peaks in each quantile is determined.R package DESeq2AIAP includes two downstream analysis components: qATACViewer , and listed in Table S1. The ATAC-seq FASTQ files were processed by AIAP as described above. The ChIP-seq FASTQ files were aligned to the mouse genome (mm10 assembly) and were further processed by methylQA. Methylation calling of whole-genome bisulfite sequencing (WGBS) data was downloaded from the ENCODE data portal. The averaged signals of ATAC-seq, ChIP-seq, and WGBS were calculated at 100-bp windows within 5 kb around the center of the ATAC-seq peaks and were plotted in R. The processed DNase I hypersensitive site (DHS) data were downloaded from the ENCODE data portal.The raw data FASTQ files for ATAC-seq and histone ChIP-seq were downloaded from ENCODE data portal  and postnatal day 0 (P0), to evaluate the performance of AIAP. The ATAC-seq peaks generated by AIAP were used as test regions, the read counts were calculated in both PE-asSE and PE-noShift modes, and the DARs were identified as described above with adjusted A). The ATAC-seq peaks are sharper and stronger when the ATAC-seq data have high RUPr and ProEn values, suggesting better signal enrichment and better quality in the ATAC-seq experiments.Conducting QC checks at different steps of data processing and correctly interpreting QC metrics are crucial to ensure a successful and meaningful analysis. Different QC metrics report important information regarding different aspects of genomic data; thus, it is essential to define the key QC metrics for ATAC-seq data before performing an analysis. In addition to the traditional QC metrics shown in Figure 2We further defined BG to directly measure the background noise level in the ATAC-seq experiments. We randomly selected 50,000 genomic regions (size: 500 bp) from regions of the genome that do not overlap with ATAC-seq peaks after peak calling. The ATAC-seq signals over each region are calculated as RPKM, and the regions with an ATAC-seq signal over a theoretical threshold (RPKM = 0.377) are considered high-background regions. The percentage of high-background regions within 50,000 randomly selected genomic regions is used as a QC metric to measure the background noise of the ATAC-seq data. We noticed that different background noise levels were directly reflected by the QC metric of background, especially in the heterochromatin regions, which are enriched with H3K9me3 signals B.To further explore the QC metrics, we used AIAP to process 70 ATAC-seq datasets generated by the ENCODE consortium (Table S2). We specifically checked the key metrics, including RUPr, ProEn, SubEn, and BG. We noticed that these metrics were not dependent on sequencing depth . RUPr waA).To define ATAC-seq peaks, peak calling strategies adopted from ChIP-seq analysis are widely used to analyze ATAC-seq data. However, unlike ChIP-seq data, an ATAC-seq experiment does not have input control and is usually sequenced with the PE sequencing method to profile the size of DNA fragments. The uniquely aligned PE reads have been used to call open chromatin peaks after alignment in many studies Figure 3To validate the sensitivity of our analysis strategy, we downloaded published ATAC-seq data of GM12878 cells generated by the Greenleaf laboratory with the Omni-ATAC-seq protocol ATAC-seq peaks are generally considered regulatory elements that are enriched for specific histone modifications. We downloaded ChIP-seq data of GM12878 histone modifications  that are associated with promoter and enhancer activities to validate the functionality of PE-asSE-specific ATAC-seq peaks. Compared to randomly selected genomic regions, the PE-asSE-specific ATAC-seq peaks were highly enriched in all active histone modifications G–I. ThesA). Correspondingly, only 107 E11.5-specific and 72 P0-specific DARs were found by using only PE-noShift mode. Compared to PE-noShift mode, PE-asSE mode resulted in an ∼ 35% increase in the number of DARs identified. We further tested AIAP on other tissues at two developmental stages and found that AIAP identified 32%–168% more DARs in different tissues (Table S5).Chromatin accessibility is dynamically associated with cellular responses to developmental cues, disease progression, and environmental stimuli. The identification of DARs has become an important approach to monitor the activity changes of regulatory elements Figure 4We examined the genomic distribution of DARs and noticed that the distribution of PE-asSE-specific DARs had a similar distribution as shared DARs, that is, most DARs were located in intergenic and intronic regions, which is consistent with their potential enhancer functionality B. BecausAIAP is a new tool to perform quality assurance and downstream analysis of ATAC-seq data. Comparing with other tools (Table S6), AIAP provides a rapid and reliable data processing solution for ATAC-seq data. Using public datasets, we systematically tested the QC metrics of ATAC-seq data and established key QC metrics of ATAC-seq data. We determined that RUPr, ProEn, and BG were important measurements to estimate the quality of ATAC-seq data. All three QC metrics directly reflect the quality of library preparation, and the failure of these QC metrics reflect the low-quality of the ATAC-seq data, which cannot be corrected by merely increasing sequencing depth. We found that the RUPr and ProEn reflect the ATAC-seq signal enrichment, and BG indicates the overall background noise of the data. By combining these QC metrics, we obtained an accurate estimation of the quality of ATAC-seq data. We used AIAP to process 54 mouse ATAC-seq datasets to test and evaluate the QC metrics and generate the range of the QC metrics (Table S2). These ranges of QC metrics can be used as a reference to evaluate the success of ATAC-seq experiments.We optimized the widely used classic analysis methodology and specifically used PE-asSE mode to process the PE sequenced ATAC-seq data. AIAP aligns the PE ATAC-seq data in PE mode to increase the alignment accuracy, and the BAM file is further processed in SE mode for downstream analysis. In PE-asSE mode, AIAP doubles the sequencing depth and dramatically increases the sensitivity of OCR identification. In our test, AIAP identified 20%–40% more ATAC-seq peaks than the widely used classic analysis methods. We further used corresponding DHS data and histone modification data to validate the specificity of newly identified ATAC-seq peaks by AIAP; most of the novel ATAC-seq peaks identified by AIAP were independently validated with DHSs and enriched for active histone modifications. Such a result indicates the high true positive rate resulting from the AIAP analysis strategy.We also suggest that the PE-asSE strategy can improve the sensitivity of discovering DARs, which are wildly used to measure chromatin dynamics Finally, we compiled AIAP into a Docker/Singularity image to facilitate the easy operation of AIAP on high-performance computing clusters. AIAP can complete QC checking and file processing in ∼ 2 h for one typical ATAC-seq dataset, of 37 million PE reads (Table S7). Besides the high performance, AIAP also has a much better sensitivity and higher specificity F when coCode availability.https://github.com/Zhang-lab/ATAC-seq_QC_analysis.The software, source code, and documentation of AIAP are freely available at Competing interests.The authors have declared no competing interests.Shaopeng Liu: Methodology, Software, Formal analysis, Resources, Writing – original draft. Daofeng Li: Methodology, Software, Visualization. Cheng Lyu: Methodology, Software, Formal analysis. Paul M. Gontarz: Formal analysis, Resources. Benpeng Miao: Investigation, Software, Formal analysis. Pamela A.F. Madden: Supervision, Project administration. Ting Wang: Supervision, Project administration, Writing – original draft, Writing – review & editing. Bo Zhang: Conceptualization, Methodology, Software, Formal analysis, Investigation, Supervision, Project administration, Writing – original draft, Writing – review & editing, Visualization.


For accurate and sensitive chimerism analysis, a set of KMR kits , based on detection of insertions/deletions (indels) by qPCR, have been developed. Here, we investigated informativity and validated the accuracy of KMR kits in Japanese donor/recipient pairs and virtual samples of DNA mixtures representative of Japanese genetic diversity. We found that at least one recipient-specific marker among 39 KMR-kit markers was informative in all of 65 Japanese donor/recipient pairs. Moreover, the percentage of recipient chimerism estimated by KMRtrack correlated well with ratios of mixed DNA in virtual samples and with the percentage of chimerism in HSCT recipients estimated by STR-PCR/in-house SNP-qPCR. Moreover, KMRtrack showed better sensitivity with high specificity when compared to STR-PCR to detect recipient chimerism. Chimerism analysis with KMR kits can be a standardized, sensitive, and highly informative method to evaluate the graft status of HSCT recipients.Chimerism analysis is a surrogate indicator of graft rejection or relapse after allogeneic hematopoietic stem cell transplantation (HSCT). Although short tandem repeat PCR (STR-PCR) is the usual method, limited sensitivity and technical variability are matters of concern. Quantitative PCR-based methods to detect single nucleotide polymorphisms (SNP-qPCR) are more sensitive, but their informativity and quantitative accuracy are highly variable Sufficient hematopoietic and immune reconstitution by replacement of recipient-derived cells with donor cells is critical for successful allo-HSCT3.Allogeneic hematopoietic stem cell transplantation , including peripheral blood (PB) stem cell transplantation (PBSCT), bone marrow (BM) transplantation (BMT), and cord blood (CB) transplantation (CBT), can be a curative intervention for various hematologic diseases. However, allo-HSCTs often show comorbidity or mortality due to relapse of the hematologic disease, infections, graft-versus-host disease (GVHD), and graft failure associated with graft rejection or poor graft function5. The major techniques of chimerism analysis are PCR for short tandem repeat (STR) profiles of DNA (STR-PCR), and detection of single nucleotide polymorphisms (SNPs) with real-time quantitative PCR (qPCR)-based methods (SNP-qPCR)7. Although STR-PCR is the predominant method, technical variability among laboratories is pronounced, and the detection limit for recipient chimerism is not very low, ranging from 1 to 10% among studies13. SNP-qPCR is a fast and sensitive method to detect recipient chimerism below 1%, but there are issues such as limited informativity, inconsistent quantitative accuracy, false positive results, and technical variations12. Thus, chimerism analysis lacks methodological standardization and might not discriminate between donor and recipient cells with sufficient reliability13.Chimerism analysis assesses proportions of hematopoietic cells from the donor (donor chimerism) and recipient (recipient chimerism), and it is used as a confirmation of engraftment and as a surrogate indicator for graft rejection or relapseRecently, KMR kits  have been developed as a set of standardized products prepared for chimerism analysis using a real-time qPCR-based assay. Appropriate markers can be screened from among ready-to-use primers for 39 different chromosomal locations in the KMRtype Core and Extended kits, followed by post-allo-HSCT chimerism monitoring with KMRtrack using the primer(s) selected by the KMRtype kits. The 39 KMR markers, which are commercially available as CE-IVD markers, detect specific insertions/deletions (indels) that consist of multiple nucleotides instead of single nucleotides as in SNP-qPCR. As for methodologies and standardization in chimerism analysis, genetic diversity and distribution of indels may differ among ethnic groups of donors and recipients, for which reason KMR kits should be validated in specific contexts. Here, we assessed the informativity and accuracy of chimerism monitoring using KMR kits on samples drawn from an ethnic Japanese cohort.15.Genomic DNA was extracted using the QuickGene DNA whole blood kit S  or the SMITEST EX-R&D  from 130 individuals comprising 65 Japanese donor/recipient pairs who underwent allo-HSCT at Fukushima Medical University Hospital between January 2009 and March 2021 . DNA samples with > 1.8 260/280 nm ratio were mixed carefully for generation of virtual samples. We also tested post-allo-HSCT DNA samples from recipient PB or BM cells. In these samples, values of recipient chimerism obtained by KMRtrack (KMR recipient chimerism) were compared with those of STR-PCR or in-house SNP-qPCR, which had been performed as routine examinations for clinical practice.To screen for informative markers that can distinguish donor and recipient cells, pre-allo-HSCT DNA samples purified from pairs of donors  and recipients  were used in KMRtype Core and, as necessary, KMRtype Extended kits (GenDx). Virtual samples of DNA mixtures with known concentrations obtained from healthy Japanese volunteers were used to validate KMRtrack (GenDx), a tool intended for chimerism monitoring. Concentration of DNA was measured by the absorbance at 260 nm in a spectrophotometer , which is guided by local policy, national law, and the Declaration of Helsinki.17. In-house SNP-qPCR was performed with the sets of primers and probes specific for 5 SNPs 18, using a QuantStudio 3, followed by analysis with QuantStudio Design & Analysis Software v1.4.3 (Thermo Fisher Scientific).For STR-PCR, 20 STR markers  were amplified using a GeneAmp PCR System 9700  as described previouslyAnalysis with KMR kits, which are ready-to-use products prepared for real-time qPCR-based assay, was performed using 10 ng aliquots of DNA from each sample according to manufacturer’s instructions. These kits consist of KMRtype and KMRtrack for pre-allo-HSCT screening and chimerism monitoring after allo-HSCT, respectively. KMRtype Core contains 10 multiplexed primer mixes for 30 markers to distinguish donor-derived and recipient-derived cells for pre-allo-HSCT screening; KMRtype Extended contains 9 additional markers for cases without an appropriate marker in the KMRtype Core set  without any recipient-specific marker in the KMRtype Core kit, resulting in 100% informativity of the KMR kits with recipient-specific markers for the 65 Japanese donor/recipient pairs. However, even with both KMRtype Core and Extend kits, 6 pairs had only one recipient-specific marker  could distinguish 62 of 65 pairs (95.4%). With these 7, we tested the accuracy and reproducibility of KMR markers for estimating ratios of recipient cells with the KMRtrack kit, using virtual samples with known concentrations of mixed DNA from 2 individuals. For samples with ≥ 1% of virtual recipient DNA, values of KMR chimerism determined by these markers correlated highly with ratios of DNA concentrations from 2 individuals, mixed to simulate recipient chimerism  and 21 with mixed chimerism according to STR-PCR or in-house SNP-qPCR with recipient-specific markers previously tested in our laboratory. The values of KMR recipient chimerism correlated well with those of STR-PCR/in-house SNP-qPCR recipient chimerism  had ≤ 2 recipient-specific markers, whereas in our cohort, 6 of 65 donor/recipient pairs (9.2%) had ≤ 2 recipient-specific markers from the KMRtype Core kit. Our results suggest that KMR kit markers enable us to perform appropriate chimerism analysis in a wide variety of donor/recipient pairs. A limitation is that although use of multiple recipient-specific markers is recommended for chimerism monitoring, one unrelated and 5 related pairs had only one recipient-specific marker by using both KMRtype Core and Extended kits. This may be consistent with a recent study that showed at least 40 markers are required to distinguish a large number of the pairs26. There is another issue that some markers frequently showed atypical results which could not determine chimerism.The numbers of recipient-specific, donor-specific, and overall total of informative markers in the KMRtype Core kit were significantly greater for unrelated pairs than for related pairs, probably reflecting smaller disparities within families than among unrelated members of a general population. This was even the case for unrelated pairs with matched HLA compared to related HLA-haploidentical pairs with greater disparities in their HLA alleles. High genetic similarity outside the HLA region in related HLA-haploidentical pairs might contribute to successful HLA-haploidentical allo-HSCT7. For donor/recipient pairs with only one marker, we should be aware that accuracy is an important but challenging characteristic.Next, assessment of the KMRtrack kit using virtual pairs consisting of DNA from 2 different individuals showed good correlation between measured and simulated degrees of chimerism, with sensitivity high enough to detect thresholds of 0.1%-0.3% of an amount as small as 10 ng of DNA. Likewise, values of KMR recipient chimerism correlated well with values from previous chimerism analyses performed with STR-PCR or in-house SNP-qPCR. Although KMR recipient chimerism values tended to be lower than the simulated recipient chimerism values of virtual samples or STR-PCR/in-house SNP-qPCR chimerism in post-allo-HSCT samples, KMRtrack detected minor recipient chimerism even in post-allo-HSCT samples for which STR-PCR did not detect recipient chimerism. This indicates better sensitivity of KMRtrack versus STR-PCR, together with the assessment of virtual samples, described above. In contrast, STR-PCR did not detect recipient chimerism in the post-allo-HSCT samples that were negative for KMR recipient chimerism. These findings suggest sufficient quality, sensitivity, and specificity of the KMRtrack kit for chimerism monitoring. However, a limitation is that positivity of the KMRtrack kit does not distinguish between homozygous and heterozygous state of the marker. In addition, there was a certain variability among KMR markers in chimerism monitoring  using KMRtrack. The results of chimerism by qPCR and cdPCR highly correlated with each other. They also found that chimerism results of a next generation sequencing method highly correlated with those of qPCR with KMRtrack10. Our data and these findings together indicate the excellent utility of KMR kits for chimerism analysis with various qPCR and digital PCR technologies. For indel based chimerism analysis, including qPCR and digital PCR with KMR markers, as well as next-generation sequencing, use of multiple markers is recommended to increase accuracy of the tests, regarding the examples like chromosomal abnormalities coinciding with the markers’ position in relapses.The requirement of only 10 ng DNA for sensitive chimerism analysis may be an advantage of the KMRtrack kit, as demonstrated herein. It is possible that higher doses of DNA would further increase sensitivity of the kit’s qPCR reaction, since various other qPCR-based methods usually need volumes of DNA over 100 ng to obtain higher sensitivity than STR-PCRIn conclusion, chimerism analysis with KMR kits is standardized, sensitive, and highly informative to detect recipient chimerism in allo-HSCT recipients. Therefore, this method could harmonize chimerism analysis for detection of recipient cells and serve as a surrogate marker for graft rejection relapse prediction in hematologic malignancies.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.


Neighborhood environments are increasingly recognized as associated with mental well-being among older adults. However, their underlying mechanisms remain unclear. This study investigated mediating effects of cognitive and structural social capital (SC) in relationships between neighborhood environments and mental well-being among older adults.We conducted a cross-sectional analysis of 1,277 community-dwellers aged 60 years and older in Hong Kong in 2021. The Warwick–Edinburgh Mental Well-being Scale assessed mental well-being. Perceived age-friendly environment was assessed. Objective neighborhood environment was measured by the number of neighborhood facilities  within 200-m and 500-m buffer areas from respondents’ residences. Structural equational modeling was used.Perceived age-friendly environment regarding community and health support had a protective role on mental well-being. More community centers were directly associated with better affective-emotional well-being, while more passive leisure facilities directly lowered psychological-functioning well-being. Cognitive SC outweighed structural SC in mediating relationships of neighborhood environment on mental well-being. Our findings advance the ecological model of aging by providing evidence for cognitive and structural SC as mediators to explain the relationship between neighborhood environment and mental well-being. Policy implications for optimizing mental well-being in aging societies are discussed. Translational Significance: The mechanisms of how neighborhood environments influence older adults’ mental well-being remain unclear. The study found that perceived age-friendly environment regarding community and health support had a protective role on mental well-being. More community centers led to better affective-emotional well-being, while more passive leisure facilities lowered psychological-functioning well-being. Cognitive social capital outweighed structural ones in mediating relationships of neighborhood environment on mental well-being. Ensuring more community centers are available for older adults and tailoring the parks and public transportation to older adults’ needs/preferences are beneficial for their well-being.Mental well-being includes the presence of hedonic and eudaimonic well-being . HedonicSocial capital (SC) has emerged as a modifiable factor for promoting older adults’ mental well-being. There is no consensus on how to define SC. One of the widely used definitions is “features of social organization, such as trust, norms, and networks that can improve the efficiency of society by facilitating coordinated actions” , p. 3. SIncreasing attention has been paid to understanding how neighborhood design suppresses and enhances opportunities for improving older adults’ SC and mental health . The ecoNevertheless, most studies exploring how neighborhood environment influences older adults’ mental well-being rely on subjective measures of neighborhood environment . Few incMore importantly, theoretical understanding of how neighborhood environment, SC, and mental well-being are meaningfully linked among older adults remains limited. To better understand pathways in relationships between subjective and objective attributes of neighborhood environment and mental well-being among older adults, the current study aimed to test cognitive and structural SC as mediators.According to ETA , neighboThe meaning-based process emphasizes that neighborhood environments create belonging, trust, and reciprocity, leading to better mental well-being. Theories about place attachment emphasize the transformation of “space” into “place” and the emotional bonding between individuals and their living place  and descIn sum, although relationships between neighborhood environment and SC can be conceptualized through two separate pathways, few have linked SC theory to the ETA and investigated these two pathways simultaneously in relationships between neighborhood environment and mental well-being in old age. Understanding pathways between neighborhood environment and mental well-being may provide an opportunity to develop policy interventions to optimize mental well-being for aging societies. This study addresses the following research questions: (1) how are neighborhood environments associated with mental well-being in later life? (2) Do meaning-based and network-based processes mediate effects of neighborhood environment on late-life mental well-being? We included the perceived age-friendly environment (subjective attributes) and the availability of neighborhood facilities (objective attributes) that are policy-relevant. We conceptualized cognitive and structural SC as mediators. We formulated following hypotheses: (H1) perceived age-friendly environment and the availability of neighborhood facilities are associated with better mental well-being among older adults; (H2) cognitive and structural SC mediate effects of perceived age-friendly environment and the availability of neighborhood facilities on mental well-being. The conceptual framework is presented in Hong Kong is undergoing rapid population aging: the percentage of the population aged 65 and older is projected to double from 18% in 2019 to 35% in 2069 . As a hiThis study used purposive and snowball sampling to recruit participants. Participants were eligible if they were aged 18 or older and able to understand and communicate in Cantonese. Data were collected between June and October 2021 through telephone interviews by trained research assistants. This study was approved by the Human Research Ethics Committee of the University of Hong Kong. All participants provided informed consent. To align with our research objectives, we included 1,304 respondents aged 60 years old out of 1,700 respondents aged 18 and older. Of these, 97.9% provided their home address. The final sample for data analysis was 1,277.Mental well-being was assessed by the seven-item short Warwick–Edinburgh Mental Well-being Scale  in a list of seven categorizations of organizations based on previous studies  and whether they participated in social activities/services in the past 3 months  (cipation . Respond studies  and cult1 = Yes) . SupplemWe assessed subjective attributes of neighborhood environment using perceived age-friendless of neighborhood environment to align with the concepts of age-friendly cities proposed by WHO . Eight iWe geocoded respondents’ residential addresses and used road-network-based service area buffers adjusted by terrain and slope from each building block to define the scope of neighborhoods to measure the objective indicators of neighborhood environment. ArcGIS version 10.8.2 was used to perform geocoding and spatial buffering analysis. We created the 200-m and 500-m buffers to extract the number of neighborhood facilities. The 500-m buffer was selected as it reflects the maximum comfortable walking distance from residences to basic facilities for older adults, while the 200-m buffer was considered because it represents a short walking distance (less than 5 min) for older adults experiencing some functional decline . The accSociodemographic characteristics included age (years), gender, marital status (married vs single/widowed/divorced/separated), education , and subjective financial sufficiency , years of residency, and self-rated health .p value < .05 (two-tailed) were interpreted as statistically significant. We used Mplus 8 software . Most respondents were female (78.9%), married (51.1%), with primary school level education or below (51.2%), and lived in their neighborhoods for over 30 years. Less than half had good self-rated health (41.95%) but reported financial sufficiency (3.04 ± 0.62). They had good affective-emotional and psychological-functioning well-being. The mean scores of “trust,” “reciprocity,” and “sense of community” ranged from 3.41 to 3.77, indicating respondents’ possession of relatively high cognitive SC. Respondents had more than one organizational membership, high levels of social participation, but low levels of volunteering. Respondents perceived neighborhood environment as age-friendly in all domains. p < .001) and psychological-functioning well-being . Structural SC was associated with better affective-emotional  and psychological-functioning well-being . We only found significant and direct associations between age-friendly neighborhood environment in domains of community and health services  and psychological-functioning well-being. All eight domains of perceived age-friendly environment were significantly associated with higher levels of cognitive SC. Only perceived age-friendly environment in the volunteering domain  was associated with higher levels of structural SC. The number of community centers was significantly associated with higher levels of structural SC . The number of transportation facilities was significantly associated with lower levels of cognitive  and structural SC , respectively.p < .01) but negatively associated with passive leisure facilities .p = .001), housing , and community and health service  domains by 67% (0.6 = 0.04/0.06), 75%, and 50%, respectively. The total indirect effect of perceived age-friendliness of neighborhood environment on affective-emotional well-being in outdoor spaces/buildings , social participation , respect , volunteering , and information/communication  on affective-emotional well-being were significant and mainly mediated by cognitive SC, although their total effects were not significant. Structural SC only significantly mediated associations between affective-emotional well-being and age-friendly environment in the volunteering domain . The total indirect effect of the availability of community centers was significant, although its total effect was not significant. The negative association between affective-emotional well-being and the number of transportation facilities was significantly mediated by cognitive SC .p < .01) and community/health service  domains by 60% and 50%, respectively. The total indirect effects of perceived age-friendliness of neighborhood environment on psychological-functioning well-being in the domains of outdoor space/building, transportation, housing, respect, volunteering, and information/communication were significant and mainly mediated by cognitive SC. Structural SC only significantly mediated associations between psychological-functioning well-being and age-friendly environment in the volunteering domain. The negative association between psychological-functioning well-being and the number of transportation facilities was significantly mediated by cognitive SC . Most findings related to the indirect associations between the perceived age-friendliness of neighborhood environment and mental well-being were similar for the 200-m and 500-m buffers , with structural SC being the key mediator. Previous studies have found that more community centers can encourage more social networks, interaction, and social participation among older adults because older adults find it convenient to visit community centers in their neighborhoods and maintain their structural SC .Our study expands the existing knowledge by revealing that the meaning-based process played a more important role in facilitating older adults’ mental well-being as more significant mediating effects of cognitive SC were found. It is possible that network-based processes  decrease in importance for aging well when people move into advanced age with varying levels of functional limitations. Older people tend to develop a strong sense of belonging to their neighborhood, trust in their community members, attachment to the place and the neighbors surrounding them .In addition to direct and indirect associations between neighborhood environment and mental well-being, we found discrepancies between two observed areas. For instance, more mediating effects of SC between neighborhood facilities and mental well-being were observed in a 200-m buffer area, while more direct effects of neighborhood facilities were found in a 500-m buffer area. Taking public transportation as an example, a greater number of public transportation facilities located within a 200-m buffer may prohibit social interaction. In comparison, a greater number of public transportation facilities within a 500-m buffer area may potentially threaten older adults’ feelings and mental well-being. Future studies can investigate how different buffer areas shape the opportunities related to older adults’ feelings and actions to maintain their mental well-being.Our study has several strengths. It is theoretically informed, integrating both ETA and SC theories, and offers a new theoretical model to explain relationships between neighborhood environment and mental health in old age. The study included a large sample in a large urban and high-density Asian city, applied both subjective and objective attributes of neighborhood environment, estimated the mediating roles of cognitive and structural SC on the two dimensions of affective-emotional and psychosocial-functioning well-being simultaneously using two buffers, and applying SEM analysis. Our study also has some limitations. First, the cross-sectional nature of the data does not allow us to draw causal associations between neighborhood environment and mental well-being. Second, the study did not draw on a random sample. Thus, our findings may not be generalizable to other populations of older people . Future studies may consider including older adults with and without disabilities. Third, we only included individual-level SC without considering multilevel SC and its various types . Future studies that apply both individual and multilevel measures of SC and various forms of SC to capture comprehensive SC profiles are needed to better understand the effects of neighborhood environment on mental well-being. Last, the respondents in our sample have a variety of residence years, ranging from 1 to 91. The mediating effects of SC in the model may differ in older adults who have lived in the community for a long time versus those who are newcomers. Future studies should further investigate whether residence years moderate the mediating effects of SC in the relationship between neighborhood environment and well-being among older adults.Our findings can provide practical implications for urban planning strategies and aging-in-place policies. First, we highlight the role of an age-friendly environment in improving better mental well-being among older adults. Pre-coronavirus disease 2019 (COVID-19) research has documented the positive effects of an age-friendly environment on older adults’ mental health . Our stuOur study innovatively applied an integrative theoretical framework to examine the mediating effects of cognitive and structural SC in relationships between perceived age-friendly neighborhood environment and subjective measures of neighborhood facilities and mental well-being. Both meaning-based and network-based processes were key pathways between neighborhood environment and mental well-being. We expanded the ecological model of aging by linking it to SC theory, thus providing evidence of the importance of an age-friendly environment in enhancing SC and, thus, mental well-being in later life. In Asian societies with rapidly aging populations, it is imperative to improve neighborhood age-friendliness and ensure the availability of community centers to optimize large-scale mental well-being improvement strategies.igac070_suppl_Supplementary_MaterialClick here for additional data file.


N-heterocyclic carbene (NHC) complexes have unique physico-chemical properties as catalysts and a huge potential in medicinal chemistry and pharmacology, exhibiting a variety of notable biological activities. In this review, the most recent studies on ruthenium NHC complexes are summarized, focusing specifically on antimicrobial and antiproliferative activities. Ruthenium NHC complexes are generally active against Gram-positive bacteria, such as Bacillus subtilis, Staphylococcus aureus, Micrococcus luteus, Listeria monocytogenes and are seldom active against Gram-negative bacteria, including Salmonella typhimurium, Pseudomonas aeruginosa and Escherichia coli and fungal strains of Candida albicans. The antiproliferative activity was tested against cancer cell lines of human colon, breast, cervix, epidermis, liver and rat glioblastoma cell lines. Ruthenium NHC complexes generally demonstrated cytotoxicity higher than standard anticancer drugs. Further studies are needed to explore the mechanism of action of these interesting compounds.Ruthenium  N-heterocyclic carbenes (NHCs), cycloruthenated and half-sandwiched compounds ruthenium(II) hexafluorophosphate (16) and bis-ruthenium(II) hexafluorophosphate (12). Inhibition of cell proliferation was studied against human colon carcinoma cell lines (HCT15) and human epidermoid cancer cells (Hep2) by the standard MTT assay. Complex 16 was more active than complex 17 against both cancer cell lines. DNA binding and cleavage studies demonstrated that these complexes may induce cancer cell apoptosis. Moreover, complex 16 showed antimicrobial activities against Gram-positive (Staphylococcus epidermidis NCIM2493) and Gram-negative bacteria (Pseudomonas aeruginosa ATCC 27853), and antimycotic properties against Candida albicans SJ11 (unicellular fungus) by targeting their cell wall and DNA or plasmid inside the cell.Roymahapatra et al., 2015,  describeBacillus subtilis 168 DSM402; Staphylococcus aureus DSM 20231 and ATCC 43300) and Gram-negative  bacteria and the cytotoxic activity against MCF-7 human breast cancer and HT-29 colon adenocarcinoma cells. Complexes 18 and 19 showed activity only against Gram-positive bacteria, while showing no activity against Gram-negative . Compound 19 also showed cytotoxicity against the two cell lines used. The results were compared to cisplatin or 5-fluorouracil . DNA-binding studies were also carried out by recording the circular dichroism of B-DNA and c-Kit2 G4 in the presence of increasing amounts of 19, highlighting a strong binding of 19 to the B-DNA model and suggesting intercalation as a mode of connection. Further studies carried out by the same research group 2 complexes. The antibacterial activity was tested against three Gram-positive , and three Gram-negative  bacteria, using the well diffusion method. Ampicillin was used as a standard control drug . The compounds 26 and 27 demonstrated effective antibacterial activity against the tested bacteria; particularly, the compound 26 showed excellent activity against S. typhimurium, being more active than the reference drug. Moreover, the compound 26, from a concentration of 1 mg/mL, demonstrated a very similar scavenging activity to that of the two controls, butylated hydroxytoluene (BHT) and gallic acid (GA), as determined via DPPH assay. Finally, both 26 and 27 showed cytotoxic activity against human breast cancer cell lines MCF-7 and MDA-MB-231.Slimani et al., 2020,  studied p-cymene)(NHC)Ru(II)Cl2 as antibacterials against Gram-positive  and Gram-negative  strains in a microtiter plate assay, according to CLSI guidelines, using ciprofloxacin as a reference drug. The complex -dichlorido-(η6-p-cymene)ruthenium(II) 28 was the most active compound against Gram-positive bacteria, while it showed low activity against Gram-negative bacteria. This compound showed moderate inhibition of bacterial TrxR (E. coli). Thus, the inhibition of TrxR is unlikely a major mechanism of the antibacterial activity of the ruthenium NHC complexes.Burmeister et al., 2021,  studied NHCs have been recognized as a class of strong donating ligands, which may stabilize diverse metal complexes of catalytic importance. Among these, the NHC ruthenium complexes have been, and still are, a fruitful research, filed for various applications: catalytic, photochemical and biological. This review provides an overview of the most recent studies on ruthenium NHCs complexes with biological activities, focusing on the antimicrobial and antiproliferative properties. Most of these compounds showed interesting antimicrobial activity against Gram-positive bacteria and some are also active against Gram-negative bacteria and fungi. The antiproliferative activity was also demonstrated for several compounds belonging to this class against several types of cancer cell lines.


Cancer often develops multidrug resistance (MDR) when cancer cells become resistant to numerous structurally and functionally different chemotherapeutic agents. MDR is considered one of the principal reasons for the failure of many forms of clinical chemotherapy. Several factors are involved in the development of MDR including increased expression of efflux transporters, the tumor microenvironment, changes in molecular targets and the activity of cancer stem cells. Recently, researchers have designed and developed a number of small molecule inhibitors and derivatives of natural compounds to overcome various mechanisms of clinical MDR. Unfortunately, most of the chemosensitizing approaches have failed in clinical trials due to non-specific interactions and adverse side effects at pharmacologically effective concentrations. Nanomedicine approaches provide an efficient drug delivery platform to overcome the limitations of conventional chemotherapy and improve therapeutic effectiveness. Multifunctional nanomaterials have been found to facilitate drug delivery by improving bioavailability and pharmacokinetics, enhancing the therapeutic efficacy of chemotherapeutic drugs to overcome MDR. In this review article, we discuss the major factors contributing to MDR and the limitations of existing chemotherapy- and nanocarrier-based drug delivery systems to overcome clinical MDR mechanisms. We critically review recent nanotechnology-based approaches to combat tumor heterogeneity, drug efflux mechanisms, DNA repair and apoptotic machineries to overcome clinical MDR. Recent successful therapies of this nature include liposomal nanoformulations, cRGDY-PEG-Cy5.5-Carbon dots and Cds/ZnS core–shell quantum dots that have been employed for the effective treatment of various cancer sub-types including small cell lung, head and neck and breast cancers. Cancer is a deadly disease characterized by the uncontrolled proliferation of cells. Mutations followed by genetic instabilities result in the initiation, progression and development of tumors . Cancer Multidrug resistance (MDR) mechanisms limit the efficacy of chemotherapy in cancer cells –8 and haThe phenomenon of MDR is a complex and multifactorial process, illustrated in Fig. Dysfunctional apoptotic pathways, increased repair of DNA damage, alterations in the cell cycle, and overexpression of cyclin-dependent kinases (CDKs) contribute to the development of resistance to chemotherapeutic drugs in cancer cells . MoreoveSome of the members of the superfamily of ABC proteins are typically expressed on the plasma membrane. They efflux cytotoxic agents from cells, thereby contributing to clinical MDR –33. ABC Since the early 1990s many drugs have been evaluated for their possible inhibition of ABC efflux transporters. First-generation P-gp inhibitors such as verapamil, cyclosporine A, quinine, and erythromycin were found to be effective in-vitro but showed inadequate pharmacological limitations, adverse side effects and low affinity towards this transporter during in-vivo experiments , 36. To . demonstrated the role of ferulic acid on P-gp modulation to overcome MDR in colchicine-selected KB-ChR-8–5 resistant cells and in the MDR xenograft mouse model via the PI3K/Akt/NF-κB signaling pathway [Fourth-generation inhibitors include natural compounds and several flavonoids with inhibitory effects on ABC efflux pumps. Natural compounds such as curcumin, piperine, tea polyphenol epigallocatechin-3-gallate (EGCG), silibinin, parthenolide, quercetin, capsaicin, carnosic acid, 6-gingerol, procyanidin, limonin, and β-carotene act as inhibitors of P-gp, and can be utilized as chemosensitizing agents to reverse MDR. Natural phytochemicals can sometimes downregulate P-gp expression by modulating different cell signaling pathways. These phytochemicals augment chemotherapy-mediated apoptotic signals in P-gp-overexpressing cells . They ha pathway . These n pathway , 44.. performed an extensive in-vitro study that tested the role of the spleen TKI entospletinib (GS-9973) in the reversal of ABCG2-mediated MDR. Entosletinib was found to reverse resistance to mitoxantrone and doxorubicin in cells overexpressing ABCG2 transporters. The ATPase activity of ABCG2 was enhanced due to the binding of entospletinib at the drug-substrate binding site [. reported that sitravatinib interferes with the tumor microenvironment and immune-checkpoint blockade (PD-1) in many cancer models [More than 50 tyrosine kinase inhibitors (TKIs) have been found to be efficient in clinical research and are approved by the US Food and Drug Administration (FDA) for anticancer therapy , 46. Numing site . Yang etr models . It alsor models , 55. Majr models , 57.MDR1 siRNA plus doxorubicin to overcome P-gp-mediated cancer MDR. The siRNA targeting the MDR1 gene successfully silenced the MDR1 mRNA by approximately 90% and enhanced the accumulation of doxorubicin in drug-resistant cells [Combining gene therapy with chemotherapeutic agents can sometimes improve therapeutic efficacy. Various types of nucleic acid-based molecules such as small interfering RNAs (siRNAs), plasmid DNA, short hairpin loops and circulating miRNAs enable the regulation of specific genes to regulate and reverse MDR in cancer cells , 59. Donnt cells . Major oTo overcome the inadequacies of existing treatment and therapy, nanomedicine offers innovative, robust and flexible drug design and delivery alternatives based on genetic profiling of individual patients to engender personalized treatment of cancer MDR –63. The The delivery of nanomaterials to tumor cells is typically achieved by both active and passive mechanisms. In the active mode of nanoparticle uptake, the surface of nanoparticles is decorated with specific targeting ligands such as antibodies or peptides, cell-specific ligands which facilitate uptake of the nanoparticles via receptor-mediated endocytosis. During passive uptake, the nanomaterials tend to accumulate in the tumor interstitial spaces due to long-circulating systemic properties and are selectively taken up by cells due to leaky vasculature and impaired lymphatic systems . PassiveVarious nanomaterials found successful for drug delivery and targeting tumors are liposomes, polymeric nanoparticles, micelles, dendrimers, metal nanoparticles, mesoporous silica nanoparticles, graphene nanoparticles, quantum dots and siRNA-conjugated nanomaterials, which all help to reverse the MDR in cancer cells. Dual drug delivery via nanoparticle systems was also developed in which combinations of drugs are co-delivered to cells, and the presence of one drug enhances the bioavailability of another drug , 75. Cer. provided an update on several nanoparticles which showed improved therapeutic abilities in clinical studies and listed the approval status of promising nanosystems to improve human health from the early 1990s to 2019 [Furthermore, the combination of chemotherapeutic drugs with gene therapy, specifically siRNA co-delivery via nanoparticles, was found to be more successful in the reversal of cancer MDR by targeting cellular signaling pathways , 82. Nan to 2019 . In rece. reported the efficacy of iRGD functionalized albumin-based synthetic protein nanoparticles (SPNPs) to deliver siRNA specific for STAT3 into intracranial GBM tumors. STAT3 siRNA-loaded SPNPs showed efficient penetration of the BBB, significant downregulation of the STAT3 expression and tumor regression in both GL26 glioma cell and GL26 syngeneic mouse models [ABC transporters are overexpressed by brain endothelial cells that form the blood–brain barrier (BBB) and are involved in the efflux of toxic foreign compounds as well as blood-derived compounds. These transporters prevent chemotherapeutic drugs from reaching their target site of action within the brain , 86. Seve models . The usee models .. demonstrated the role of mitoxantrone loaded poly (ε-caprolactone)-pluronic F68-poly (ε-caprolactone)/PLGA-PEG-PLGA) mixed nanomicelles to reverse MDR in MCF-7/ADR cells under exposure of near-infrared (NIR) light. These nanomicelles upon irradiation with NIR light generate higher levels of ROS, thus decreasing P-gp activity, leading to improved, higher concentrations of intracellular drugs and further cell apoptosis. This approach reverses MDR via nano-mediated PDT [Over the past decade, photodynamic therapy (PDT) has attracted substantial attention as an efficacious alternative treatment approach to overcome MDR. Delivery of photosensitizers and drugs simultaneously is difficult. It was found that PDT could also be improved by employing nanomaterials to mitigate MDR . PDT maiated PDT . Figure l-lysine (PLL), and N-(2-hydroxypropyl)-methacrylamide (HPMA) are commonly used for nanoformulation synthesis and drug delivery [MRP1 via inhibition of NF-κB in retinoblastoma Y79 cells [Polymeric nanomaterials have been found to play a crucial role in the delivery of dual chemotherapeutic drugs for the reversal of MDR. In fact, a polymeric liposome was the first nanoformulation approved by FDA to be used as a nanotechnology-based anticancer therapeutic . These ndelivery . Polymerdelivery . Many podelivery –101. Le delivery . In anot79 cells . Figure Liposomal nanoformulations are spherical vesicles that encompass amphiphilic phospholipids and cholesterol associated with an aqueous lumen. Liposomes can allow the encapsulation of both hydrophobic as well as hydrophilic chemotherapeutic drugs within their cores. A liposomal nanoformulation was the first clinically approved nanosystem for anticancer drug delivery . Table 22k-PLA5k) for co-delivery of doxorubicin with curcumin to reverse MDR via dual-drug based nanomicelles in drug-resistant MCF-7/ADR cells and in a xenograft model [. applied a triblock polymer (NSC-PLL-PA) for co-delivery of si-MDR1 RNA and doxorubicin to resistant HepG2/ADM cells and a xenograft model. Moreover, nanomicelles were observed to accumulate in tumors 24 h post-injection and inhibit tumor growth [Micelles are specialized nanomaterials obtained by self-assembly of hydrophilic and hydrophobic blocks in an aqueous environment with a hydrophobic core. The hydrophobic core has the advantage of being able to entrap hydrophobic drugs within its core. Polymers such as poly (aspartic acid) (PAA), poly (caprolactone) (PCL), poly (lactic-co-glycolide) (PLG) and polyethylene glycol (PEG) are used for micelle formation. Table ft model . Nanomicr growth . Variousr growth .Table 3NNanoemulsions (oil/water) are biocompatible, highly stable nano-size (10–1000 nm) emulsions that are frequently used to entrap and improve the delivery of hydrophobic drugs and pharmaceutically active compounds . Table 3., employed polyamidoamine (PAMAM) dendrimers for the co-delivery of ferulic acid and paclitaxel. These dual-drug loaded PAMAM dendrimers were also decorated with arginyl-glycyl-aspartic acid (RGD) to combat MDR mediated by P-gp in drug-resistant KB ChR-8–5 cells. These results revealed the enhanced intracellular accumulation of paclitaxel in cells and also indicated increased pro-apoptotic protein expressions of caspase 3, caspase 9, p53 and Bax [. demonstrated the role of dual-functionalized PAMAM dendrimers in the inhibition of P-gp function in Caco-2 and MDCK/MDR1 cells [Dendrimers are nano-size hyper-branched, spherical polymeric nanomaterials with symmetric core and end groups that facilitate surface conjugation and modification. Anbazhagan et al and Bax . SimilarR1 cells .3O4 NPs) have intrinsic properties that make them ideal nanosystems to facilitate therapies using radiation, photodynamics and hyperthermia. Iron oxide nanoparticles can be utilized as a contrast agent to improve conventional MRI imaging [Several metals and metal oxides have attracted intense biomedical attention for their use as nanomaterials in diagnosis, drug delivery and therapy. Gold (Au) and iron oxide nanoparticles  to reverse P-gp-mediated MDR in K562/A02 leukemia cells. Their findings indicate that the transcriptional downregulation of the MDR1 gene further aids in the reversal of MDR [. evaluated the effect of trimethoxusilylpropyl ethylenediamine triacetic acid (EDT)-coated and doxorubicin-conjugated iron oxide nanoparticles on human glioblastoma U251 cells and a mouse model for reversal of MDR. Their findings indicate that drug-conjugated magnetic nanoformulation activates multiple mechanisms to overcome drug resistance. It inhibited cell proliferation and enhanced apoptotic cell death. Furthermore, downregulation of the DNA repair gene and upregulation of caspase 3 and p53 genes were observed in U251 cells [. conjugated 2-(9-anthracenylmethylene)-hydrazinecarbothioamide (ANS) and 6-mercaptopurine (6-MP) with AuNPs and evaluated the resulting toxicity and drug resistance in MCF-7/ADR cells. Their findings indicated that smaller AuNPs have more efficient binding with P-gp, whereas larger-size nanoparticles avoid effective recognition by P-gp [. demonstrated the exploitation of folate-gold-bilirubin (FGB) nanoconjugates to overcome P-gp-mediated MDR in P-gp-overexpressing KB-ChR-8–5 cells and in a xenograft mouse model. The results indicated the FGB nanoconjugate proved to be a potent inhibitor as compared to bilirubin and AuNPs alone. FGB nanoconjugates also induced intracellular ROS and initiated DNA strand breakage and other apoptotic changes in P-gp-overexpressing cells. The xenograft model treated with FGB nanoconjugates also revealed suppression of tumor growth with pronounced apoptosis [. demonstrated that drug-functionalized gold nanorods (AuNRs) mediated P-gp trafficking in P-gp + J774.2 cells. Treatment with AuNRs containing azithromycin (Azith-AuNRs), clarithromycin (Clarith-AuNRs) and tricyclic ketolide (TriKeto-AuNRs) led to ligand-dependent accumulation and inhibition of the efflux of these nanorods by P-gp. Increased intracellular accumulation of AuNRs was observed for nanorods conjugated with P-gp substrates (Azith-AuNRs and Clarith-AuNRs), while nanorods conjugated with low-affinity P-gp substrates (TriKeto-AuNRs) was unaffected [Several reports indicate that metal nanomaterials are able to interfere with drug efflux transporters and cause the reversal of drug resistance by increasing drug retention and cellular bioavailability , 128–131l of MDR . Noruzi 51 cells . AuNPs h by P-gp . Rathinapoptosis . Deardenaffected .Quantum dots (QDs) are nanosized semiconductor particles with advantageous optical and electrical properties that have been successfully employed in several biomedical applications. QDs generate intracellular ROS, thereby causing cancer cell death through oxidative DNA damage . Further. confirmed that pH-sensitive MSNs co-polymerized with d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) successfully deliver doxorubicin to resistant MCF-7/ADR cells. These MSNs showed clathrin-mediated endocytosis, and higher drug uptake and retention. The TPGS moiety of nanoformulation contributed exclusively to the inhibition of P-gp drug efflux in tumor-bearing SCID mice [Mesoporous silica nanoparticles are nanosize drug carriers that have gained attention as versatile drug delivery vehicles having a large surface area, high stability, negligible toxicity, customized pore size and ease of encapsulating various biogenic molecules. Table CID mice  and demoNumerous engineered nanomaterials have emerged recently with the ability to deliver multiple agents such as chemotherapeutic drugs, adjuvants, and nucleic acids  to overcome MDR. These nanomaterials help to overcome MDR achieved by both efflux pump-mediated and efflux pump-independent mechanisms , 143.. conjugated TPGS-PLGA with doxorubicin and metformin for the co-delivery of P-gp inhibitor and chemotherapeutic drugs to inhibit drug efflux [. conjugated doxorubicin with PLNs and evaluated their potential for drug delivery and P-gp inhibition in MDA435/LCC6/MDR1 and EMT6/AR1 resistant cell lines. The nanoformulations were able to bypass the efflux pumps as they were phagocytized into cells, thereby enhancing doxorubicin accumulation and retention in resistant cells as compared to free doxorubicin [. demonstrated the reversal of hypoxia-mediated drug resistance in resistant A2780/ADR and MCF-7/ADR cell lines as well as in 3D spheroid cultures via co-delivery of doxorubicin and anti-P-gp siRNA (siP-gp)-conjugated PEGylated nanoparticles. The siRNA inhibits MRP1 gene expression under hypoxic conditions, thereby increasing doxorubicin delivery to MDR cells [. demonstrated the role of etoposide-conjugated lipid nanocapsules in the reversal of P-gp-mediated MDR in C6, F98 and 9L glioma cells. Etoposide intracellular efficiency was enhanced by lipid nanocapsules in P-gp-overexpressing MDR cells [g-(PEG-v-PNIPAAm)7/PTX) and doxorubicin (β-CD-g-(PEG-v-PNIPAAm)7/Dox) both in-vitro (HepG2/MDR1 and H460/MDR1 cells) and in-vivo (HepG2/MDR1 bearing xenograft BALB/c mice). These novel nanoparticles facilitate the reversal of cancer MDR by enhancing the cellular uptake of nanoparticulated drugs, intracellular drug retention, and inhibiting pump-mediated drug resistance [. described the application of a PEGylated PLA nanosystem for the combined delivery of cyclosporine A and gefitinib in in-vitro and in-vivo cancer resistance models. Their findings indicated that the nanosystem disrupts EGFR-mediated downstream signaling cascades and eventually inhibits tumor growth and invasion. It also inactivates the function of the signal transducer and the activator of transcription-3 (STAT-3)-mediated signaling [P-gp transporters play a role in the development of clinical MDR in several cancer subtypes . Therefog efflux . Drug deorubicin . Joshi eDR cells . LamprecDR cells . Some stsistance . Cheng asistance . Han et ignaling .. showed that nanodiamonds are an ideal drug delivery system that offers biocompatibility, drug conjugation, controlled release and enhanced aqueous dispersion properties. Nanodiamonds alter the tumor efflux pumps, hence facilitating doxorubicin intracellular retention and pronounced apoptosis in various human and murine breast cancer resistant cells and in a xenograft model [Nanodiamonds are carbon nanoparticles that offer binding sites for certain therapeutic agents. Reversible binding allows sustained release of drug at the target site, thereby achieving excellent biocompatibility. Chow et alft model .2+ ions within mitochondria, which curbs cellular respiration by disturbing mitochondrial calcium ion homeostasis, blocking ATP production and further inhibiting P-gp-mediated cell resistance. Hypoxia conditions generated within cells due to suppressed cellular respiration also downregulate the hypoxia-inducible factor-1 alpha (HIF-1α) gene and inhibit expression of the P-gp efflux transporter. The study revealed that the TCaNG nanosystem inhibits the biosynthesis as well as functional activity of P-gp transporters and facilitates the reversal of tumor drug resistance in MCF-7/ADR resistant cells and nude mice [Calcium phosphate-based nanomaterials are also used for the reversal of P-gp-mediated drug resistance via energy-dependent inhibition of efflux transporters . Calciumude mice . These m. demonstrated the co-delivery of curcumin and paclitaxel via core–shell polymeric NPs in human ovarian cancer SKOV3 and SKOV3-TR30 cells and in tumor-bearing xenograft mice to reverse drug resistance. Their results demonstrated that the NPs are internalized via CD44 receptors present on the surface of ovarian cells. Curcumin was found to efficiently inhibit the P-gp drug efflux transporter, resulting in elevated intracellular paclitaxel retention, inhibition of cellular migration and cytotoxicity and enhanced reduction in tumor growth in a murine model [Certain natural compounds have also been used to inhibit drug efflux transporters. Zhao et alne model .. demonstrated the co-delivery of anti-Pgp antibody and doxorubicin in SWCNTs in efforts to target and eliminate K562R leukemia stem cells. Their results clearly showed inhibition of tumor development and metastases [. conjugated both ABCG2 and ABCB1 sequences onto pH-sensitive carbonate apatite nanoparticles for dual siRNA-mediated targeting of human breast cancer cell lines (MCF-7). This dual targeting approach sensitized the MCF-7 cells and enhanced toxicity by more than 50% when treated with cisplatin, paclitaxel and doxorubicin. While single siRNA targeting resensitized the cells, the dual siRNA targeting approach offered enhanced toxicity [Single-walled carbon nanotubes (SWCNTs) have been reported to efficiently overcome drug resistance in some experimental models. Li et altastases . In an etoxicity .Normal cells employ various repair mechanisms to avoid the replication of mutated DNA and to circumvent malignant transformation. If the damaged DNA is not repaired, the mutated cells are normally eliminated by apoptosis . MDR not. synthesized poly (2-)pyridine-2-yldisulfanyl)ethyl acrylate) (PDS) nanoparticles loaded with doxorubicin and decorated with a cRGD peptide and observed their stability and drug release in both acidic pH and redox potential conditions in colon cancer HCT-116 cells. Their results indicated that these nanoparticles are a promising nanotherapeutic system [. employed a nanomicelle system for the co-delivery of the P-gp inhibitors disulfiram and paclitaxel in PEG-b-PLL/DMA with l-lysine side chains in efforts to reverse drug resistance. The nanomicelles tend to reverse surface charges depending on cellular pH conditions. Usually, nanomicelles have negative surface charge densities in neutral plasma circulations (pH 7.4) but they switch to a positive charge in an acidic tumor environment (pH 6.5–6.7). These positive surface charges facilitate their enhanced uptake into cells to overcome the drug resistance in MCF-7/ADR cells [The tumor microenvironment also plays an important role in MDR as well as cancer progression and development. Cancer cells and stromal cells embedded in the extracellular matrix play a crucial role in cancer cell invasion, metastasis and drug sensitivity . Cancer c system . Huo et DR cells . SimilarDR cells .. investigated the role of polymeric nanomaterials that mimic the cancer cell membrane and could be conjugated with haemoglobin and doxorubicin for reversal of drug resistance. The haemoglobin has an oxygen-carrying capacity that suppresses the expression of the HIF-1α factor, further downregulating the MDR1 gene and enhancing cytotoxicity in MCF-7 and MCF-10A cell lines [. demonstrated the application of silver nitrate nanoparticles (AgNPs) to target angiogenesis by downregulating VEGF and GLUT1 gene expression and inhibiting HIF-1α signaling in MCF-7 cells [. evaluated the role of nanomicelles decorated with siRNA specific to silence the HIF-1α gene (siHIF) and doxorubicin in prostate cancer PC3 cells and in a xenograft mice model. Their findings indicated the inhibition of cell proliferation, disturbed angiogenesis and suppressed migration of cells in hypoxic conditions along with tumor growth inhibition in PC3 xenograft mice without elicitation of any immune reaction. siHIF-decorated nanomicelles downregulate MDR1 gene expression and also sensitize the cells to doxorubicin under a hypoxic environment [. demonstrated the co-delivery of siHIF and cisplatin-conjugated chitosan-modified TPGS-b-(PCL-ran-PGA) nanoparticles in nasopharyngeal carcinoma for improved reversal of drug resistance in CNE-2 cells. The observations showed that silencing HIF-1α gene expression eventually inhibits P-gp expression, enhancing the sensitivity of cisplatin in multidrug-resistant cancer cells [. observed that perfluorocarbon nanocarriers supply oxygen targeted to the tumor hypoxic microenvironment in tumor-bearing nude mice for lung re-oxygenation and to overcome drug resistance [. reported co-delivery of sorafenib and CA IX-C4.16 by TPGS nanoparticles in multidrug-resistant cancer cells to overcome hypoxia-mediated MDR. Sorafenib inhibited the p-AKT signaling pathway and upregulated the tumoricidal M1 macrophage by inducing caspase 3/7 apoptotic pathways in experimental human renal cell carcinoma A498/Evr resistant cells and RAW 264.7 macrophages [The tumor microenvironment is also responsible for creating the hypoxic conditions that lead to MDR, as oxygen-deprived cells grow slowly and are less susceptible to conventional chemotherapeutic drugs. Oxidative stress leads to changes in the cancer microenvironment. Increased oxidative stress promotes tumor development and associated drug resistance . Targetill lines . Yang et-7 cells . Liu et ironment . Lian eter cells . Song etsistance . Alsaab rophages .. explains the crucial role of miRNA regulation in different types of cancer [. showed the role of folic acid-conjugated chitosan nanoparticles for co-delivery of anti-CD73 siRNA and dinaciclib to manage tumor growth and to reverse drug resistance in murine breast cancer 4T1 cells, murine colon cancer CT26 cells and in xenograft mice [. demonstrated the co-delivery of mir-34a and doxorubicin in hyaluronic acid-functionalized chitosan nanoparticles to target apoptotic signaling pathways. Their results demonstrated the enhanced delivery of a nanoformulation into tumor cells and inhibition of Bcl-2 expression and Notch-1 signaling pathways in human breast cancer MDA-MB-231 cells and in nude BALB/c mice [. synthesized nanomicelles for co-delivery of gemcitabine and miRNA-205 in pancreatic cancer MIA PaCa-2R and CAPAN-1R cells and nude xenograft mice. Their results showed sustained drug release and miRNA-205-mediated suppression of tumor growth, activation of apoptosis-mediated signaling pathways and reversal of drug resistance [6-methylguanine DNA methyltransferase (MGMT), a DNA repair protein that facilitates acquired drug resistance. In another study, Kato et al. synthesized a novel liposomal nanoformulation known as LipoTrust conjugated with siRNA that is able to silence the gene responsible for MGMT and enhance the sensitivity of glioma cells to treatment. Treatment with the nanoformulation led to a reduction in tumor volume and inhibition of the activity of the MGMT enzyme in a majority of the cells and in a xenograft mouse model [Cell cycle regulation is essential for proper cell division and growth; it is maintained and regulated by cyclins and cyclin-dependent kinases (CDKs). Some chemotherapeutic drugs specifically target different stages of the cell cycle to arrest the cell growth of rapidly dividing cancer cells. The overexpression of CDKs in cancer cells can also account for resistance to conventional chemotherapy . A recenf cancer . Polymeraft mice . Targetiaft mice . Deng etB/c mice . Mittal sistance . Becausese model .. demonstrated that doxorubicin conjugated with an HPMA copolymer has the potential to reverse drug resistance by inhibiting the drug detoxification system, inducing apoptosis by enhancing DNA damage and also suppressing UDP and glutathione expression [. developed organosilicate nanoparticles for co-delivery of ethacrynic acid (EA) and cisplatin to inhibit GST and intracellular GSH detoxification. The EA treatment induced inhibition of GST and enhanced intracellular uptake of cisplatin, synergistically preventing cellular detoxification in A375/DDP cells and suppressing tumor development in a nude xenograft murine model [. demonstrated the co-delivery of buthionine sulfoximine (a GSH inhibitor), celecoxib (a P-gp inhibitor) and doxorubicin in hybrid polymeric nanoparticles. They found that there was enhanced downregulation of GSH and P-gp expression and elevated intracellular doxorubicin retention in MCF-7/ADR cells. This nanomediated drug delivery platform exhibited the improved delivery of multiple target inhibitors and potent chemotherapeutic drugs to efficiently overcome MDR [. developed cisplatin-conjugated 2-dimensional (2-D)-titanium carbide nanomaterials and evaluated their potency in non-small lung carcinoma A549/DDP-resistant cells and nude xenograft mice. Their results showed that the 2D nanomaterials interfered with total glutathione (GSH/GSSG) levels, expression of glutamylcysteine synthetase and glutathione peroxidase in both resistant cell lines and a murine model. The titanium carbide 2D nanomaterials also revealed excellent biocompatibility in a murine model, enhanced intracellular accumulation of cisplatin and suppression of tumor growth [. developed a glucosamine-grafted and doxorubicin-loaded hybrid nanosystem that interacted with GLUT1 receptors to enhance targeted receptor-mediated endocytosis in MCF-7 and MCF-7/ADR cells and tumor bearing nude xenograft mice. Due to elevated levels of GSH within cells, the pluronic L61 entity of the nanosystem induced intracellular ROS generation, release of cytochrome-c and also disruption of mitochondrial respiration. Intracellular doxorubicin accumulation was observed in cells that led to inhibition of cancer cell growth and tumor development, eventually facilitating the reversal of drug resistance [Cytochrome P450 (CYP) superfamily enzymes oxidize fatty acids, steroids, and xenobiotics. They are involved in the clearance of various compounds from cells. In addition to carcinogenesis, CYP2 and CYP3 enzymes also contribute to MDR by activation or degradation of chemotherapeutic agents. There is a significant correlation between the upregulation of CYP enzymes and induction of the efflux transporters involved in the metabolism and detoxification of a wide spectrum of anticancer drugs, leading to MDR in cancer cells , 185. Fopression . Han andpression . Niu et ne model . Wu et acome MDR . Zhu et r growth . Wang etsistance . Wang ansistance .. studied the efficacy of wild-type p53 DNA loaded into PLGA nanoparticles to treat breast cancer, and evaluated their antiproliferative activity. They found the stable and sustained transfer of the wild-type p53 gene into cells with antiproliferative activity to overcome drug resistance [. synthesized solid lipid nanoparticles for gene delivery to overcome drug resistance. Their results indicated efficient delivery of the p53 gene  through nanoparticles in non-small cell lung carcinoma H1299 cells and in a xenograft murine model with improved biodistribution, inhibition of cell growth, suppression of tumor development and upregulated apoptotic pathways [. utilized the co-delivery of Bcl-2-specific siRNA and paclitaxel through a liposomal nanoformulation with the aim of silencing Bcl-2-mediated signaling pathways and suppression of tumor growth in human breast cancer MDA-MB-231 cells and in a 4T1 mouse model to facilitate the reversal of drug resistance [. also demonstrated the co-delivery of doxorubicin and siRNA specific to MRP1 and Bcl-2 mRNA via cationic liposomal nanoformulations to overcome drug resistance not related to drug efflux. Their results showed efficient drug accumulation, inhibition of efflux pumps via MRP1 gene expression and induction of cell death mechanisms in human lung cancer H69AR cells, MCF-7/AD breast cancer cells, HCT15 colon cancer cells and A2780/AD ovarian cancer cells [. reported co-delivery of doxorubicin and Bcl-2 targeting siRNA via mesoporous silica nanoparticles resulting in enhanced cytotoxicity in human A2780/AD ovarian cancer cells. These nanoparticles bypassed efflux pumps and were internalized in perinuclear regions, thereby reversing drug resistance not dependent upon efflux pumps [Downregulation of apoptotic pathways is often observed in cancer cells. Activation of certain signal transduction pathways is known to result in decreased apoptotic cell death in cancer cells . For exasistance . Choi etpathways . Wang etsistance . Saad eter cells . Chen etux pumps . In anotux pumps .. investigated pH-sensitive magnetic iron oxide nanoparticles (PMNs) for the treatment of resistant heterogeneous tumors in-vivo. The PMNs were used for diagnosis of early stage resistant heterogeneous tumors. They allowed dual-modal tumor diagnosis via MRI and fluorescence imaging of tumors with diameters up to 3 mm. PMNs engineered to respond to pH conditions within the tumor could be an efficient treatment strategy to overcome MDR [Tumor heterogeneity is a distinct phenomenon that is a major impediment to the treatment of cancer. Clonal and subclonal mutations are mainly responsible for the heterogeneity of tumors. Heterogeneity also occurs due to the self-renewal and differentiation properties of tumors . Geneticcome MDR . Liu andcome MDR .. targeted the Notch signaling pathway, which is a potential regulator of CSCs and facilitates cancer progression. A nanomediated strategy to block the Notch pathway could work efficiently against CSCs. γ-secretase inhibitors (GSIs) conjugated with MSNs have demonstrated significant blocking of the Notch signaling pathway and reduction in tumor growth in in-vivo xenograft model after oral delivery of nanoparticles [Cancer stem cells (CSCs) are groups of cells  within a tumor that are characterized by stem-cell-like properties such as the ability to self-renew and to differentiate, leading to heterogeneity and acquired resistance to chemo- and/or radiotherapy . Most charticles , 213.MDR1 gene knockdown and enhanced intracellular retention of paclitaxel and associated antitumor potency in colon cancer CSCs [Nanoconjugated gene silencing strategies have also been employed to target MDR-specific genes and inhibit CSC-mediated drug resistance. In one study, a lipid-based nanoformulation for co-delivery of paclitaxel and siRNA targeting CD133+ cells was evaluated to target the specific subsets of cells that are responsible for drug resistance and progression of colon cancer. The nanoformulation was evaluated in CHOK1 cells and gene silencing via siMDR1 was performed in CD133+ HT-29 colon cells which exhibited efficient cer CSCs .. targeted the TG2 enzyme via co-delivery of gemcitabine and a siRNA-conjugated liposomal nanoformulation in pancreatic ductal adenocarcinoma (PDAC) nude mice. Their results showed efficient downregulation of endogenous TG2 by siRNA, inhibiting the growth of PDAC and further enhancing therapeutic antitumor activity [Tissue transglutaminase (TG-2) is a multifunctional enzyme and another key regulator that has a crucial role in CSC-mediated cancer progression and drug resistance. Verma et alactivity . Barth aactivity .Nanomaterials offer an extraordinary platform to overcome the limitations imposed by different mechanisms involved in the development of MDR. Combining conventional treatments with current nanotechnology advances might be a promising therapeutic approach to eliminate multidrug-resistant cancer. Nanomaterials are able to block P-gp and ABCG2 pumps and/or bypass the transporters to reverse drug-efflux-mediated MDR. Furthermore, nanomaterials functionalized with different targeting ligands allow therapeutic drugs to reach tumor sites directly via blood circulation. pH-sensitive nanosystems can exploit hypoxic tumor microenvironments, thereby reducing the expression of pro-angiogenic factors via downregulating the expression of HIF-1α. Stimuli-responsive nanosystems take advantage of unique cellular properties including pH variation, redox potential as well as enzymatic activation to overcome the MDR phenomena. Thermal, magnetic and light-based nanosystems have recently been identified for efficient reversal of drug resistance. Antibody-functionalized metal nanosystems can actively target and recognize multidrug-resistant tumor cells. Nanoparticles functionalized with siRNA are able to reprogram the gene expression pattern of resistant cells. Furthermore, nanomaterials improve the therapeutic specificity and enhance the biodistribution and pharmacokinetics of chemotherapeutic drugs. There are currently many nano-based formulations in clinical trials, and some are now used in the clinic. The merits of nanosystems need to be further explored to effectively combat drug-resistant cancer.


A systematic literature search according to PRISMA criteria was made by using two main databases. Only studies published up until to October 2022 in the English language with ≥10 enrolled patients were selected. Seven studies including 326 pts, of whom 201 (61.6%) harboring DDR defects, were selected. The majority of selected papers were retrospective and four out of seven (57.1%) had small sample size (<50 pts). Three out of seven (42.8%) studies reported a more favorable outcome  after therapy with alpha emitters (223Ra-therapy or [225Ac]Ac-PSMA-617) in subjects with DDR defects with respect to those without mutations. In two studies employing alpha or beta emitters ([177Lu]/[225Ac]-PMSA), no significant benefit was registered in pts harboring DDR defects. In all but one paper, no significant difference in response rate was reported among pts with or without DDR mutations. Although preliminary and biased by the retrospective design, preliminary data suggest a trend towards a longer survival in PCa pts harboring DDR defects submitted to radionuclide targeted therapy with alpha emitters.The aim of the present review was to assess the impact of DNA damage repair (DDR) mutations on response and outcome of patients (pts) affected by advanced prostate cancer (PCa) submitted to radionuclide therapies with [ Prostate cancer (PCa) is the second most commonly diagnosed malignancy in men and a cancer-related leading cause of death worldwide . LocalizIn recent years, aside from taxane-based chemotherapy, novel therapeutic options have been introduced for the management of mCRPC, such as androgen-receptor signaling inhibitors (ARSI), including abiraterone, enzalutamide, darolutamide and apalutamide, and immunotherapy with sipuleucel-T or the immune checkpoint blocker pembrolizumab .223Ra]RaCl2 (223Ra-therapy) with respect to those submitted to the best standard of care, the U.S. Food and Drug Administration (FDA) approved XofigoTM , for the targeted alpha therapy (TAT) of mCRPC Ga-PSMA-11), as the gold standard for the staging of high-risk PCa and for the diagnosis of biochemical recurrence (BCR). On the therapeutic side, the international, open-label, phase 3 VISION proved that mCRPC subjects treated with [177Lu]Lu-PSMA-617 had both longer imaging-based progression-free survival and overall survival with respect to subjects submitted to the best standard of care [177Lu]Lu-PSMA-617 (PluvictoTM) has been recently FDA-approved for the radioligand therapy (RLT) of mCRPC, previously tested for PSMA-expression by PSMA PET/CT Ac-PSMA-617. The quality appraisal of the selected studies is represented in The findings of the selected papers for each thematic area are described in the following paragraphs.223Ra-therapy. HR mutations were identified in 10 cases (HR+) while 18 patients did not show any abnormality (HR-). The primary endpoint was to assess whether HR+ patients had a higher clinical benefit from 223Ra-therapy with respect to HR- subjects. Among all the enrolled men, 64% had ALP response within 12 weeks, assessed following PCWG3 criteria, with a significantly higher rate of response in HR+ than in HR- men . In addition, HR+ patients exhibited significantly longer time to ALP progression and prolonged time to next systemic therapy. Notably, HR+ positive patients had a meaningfully more favorable  outcome than HR- ones, with an overall survival (OS) of 36.9 vs. 19.0 months .Isaacsson and coworkers focused their retrospective study on mutations in DDR genes involved in the HR pathway, a mechanism particularly relevant in case of radiation-induced double-stranded DNA (ds-DNA) breaks . The aut223Ra-therapy Ac-PSMA-617 [225Ac]Ac-PSMA-617. Nevertheless, the results obtained in “poorly responders” were not compared with those detectable in good-responders. It is worth mentioning that all the enrolled patients were heavily pre-treated: in particular, 4 had been previously treated with the beta-emitter [177Lu]Lu-PSMA-617, with consequent potentially relevant pre-mutagenic impact. The prevalence of DDR defects was retrospectively assessed by Kratochwil and coworkers in mCRPC patients with visceral and skeletal metastases submitted to TAT with [PSMA-617 . From an177Lu/225Ac]-PSMA-617 or PSMA-I&T) was assessed through a minimum 6 weeks’ follow-up in an overall number of 40 mCRPC patients [68Ga] or [18F]-PSMA and received [177Lu] or [225Ac]-RLT according to radiopharmaceuticals’ availability or physicians’ preference. No significant differences among the two cohorts were found in PSA response (59% in DDR+ vs. 65% in DDR-) or in term of PFS  and OS . In an observational cohort study carried out by Privè and colleagues, the response to PSMA-RLT with beta or alpha emitters  were determined Lu-PSMA-617), submitted to [225Ac]Ac-PSMA-617 RLT: in all cases PSMA-expression was tested with [68Ga]Ga-PSMA-11 PET/CT and on tumor tissue by histochemical analysis before therapy enrollment [177Lu]Lu-PSMA-617 therapy, high PSMA density at histochemical analysis, and the presence of DDR defects. Van der Doelen and coworkers have recently carried out an observational cohort study including 13 consecutive mCRPC patients  and, even more recently, by the implementation of [177Lu]Lu-PSMA-617 for mCRPC Ga-PSMA-11 PET/CT performed 8 weeks after the second cycle of RLT with [177Lu]Lu-PSMA-617 is predictive of OS and progressive disease, while PSA is of limited predictive value Ac-PSMA-617, n = 1), while two of the three papers reporting no clinical benefit in DDR mutated subjects included patients submitted to RLT with [177Lu]Lu-PSMA or [225Ac]Ac-PSMA-617. This issue might have introduced a further bias, partially explaining the discrepancy among studies. A further consideration has to be made concerning the various radiopharmaceuticals employed for the targeted therapy. It has to be underlined, in fact, that the three studies showing a survival benefit in mCRPC patients with DDR defects were all performed with alpha emitters (177Lu or yttrium-90 (90Y), have been widely used in clinical practice [It is worth mentioning, in fact, that radiation-induced damage strictly depends on the energy and type of involved particles. Beta-emitters, such as practice ,41,42: tpractice ,44. On tpractice ,46. In apractice , as showFinally, all the patients included in the selected papers had been heavily pre-treated with various therapeutic regimens before radionuclide targeted therapy. The optimal sequence of the various available therapies for mCRPC has still to be defined yet , howeverDDR defects are frequently detected in patients affected by advanced PCa submitted to targeted radionuclide therapy. Although biased by the retrospective design and the small sample size, preliminary clinical reports suggest that subjects with DDR mutations might have a trend towards a longer survival when submitted to therapy employing alpha-emitters. Further prospective studies with larger series are needed to better define the role of DDR genes in PCa patients treated with alpha-emitters.