Mammalian SWI/SNF Chromatin Remodeling Complexes: Emerging Mechanisms and Therapeutic Strategies.

Small molecule-based targeting of chromatin regulatory factors has emerged as a promising therapeutic strategy in recent years. The development and ongoing clinical evaluation of novel agents targeting a range of chromatin regulatory processes, including DNA or histone modifiers, histone readers, and chromatin regulatory protein complexes, has inspired the field to identify and act upon the full compendium of therapeutic opportunities. Emerging studies highlight the frequent involvement of altered mammalian Switch/Sucrose-Nonfermentable (mSWI/SNF) chromatin-remodeling complexes (also called BAF complexes) in both human cancer and neurological disorders, suggesting new mechanisms and accompanying routes toward therapeutic intervention. Here, we review current approaches for direct targeting of mSWI/SNF complex structure and function and discuss settings in which aberrant mSWI/SNF biology is implicated in oncology and other diseases.

Conditional probability and ratio-based approaches for mapping the coverage of multi-dose vaccines.

Many vaccines are often administered in multiple doses to boost their effectiveness. In the case of childhood vaccines, the coverage maps of the doses and the differences between these often constitute an evidence base to guide investments in improving access to vaccination services and health system performance in low and middle-income countries. A major problem often encountered when mapping the coverage of multi-dose vaccines is the need to ensure that the coverage maps decrease monotonically with successive doses. That is, for doses i $$ i $$ and j $$ j $$ , i < j ⇒ p i ( s ) ≥ p j ( s ) $$ i

Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases.

The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS­ERK signalling pathway. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy. Here we report the discovery of a highly potent (IC50 = 0.071 µM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS­ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.

Domestic and international mobility trends in the United Kingdom during the COVID-19 pandemic: an analysis of facebook data.

BACKGROUND: Since early March 2020, the COVID-19 epidemic across the United Kingdom has led to a range of social distancing policies, which resulted in changes to mobility across different regions. An understanding of how these policies impacted travel patterns over time and at different spatial scales is important for designing effective strategies, future pandemic planning and in providing broader insights on the population geography of the country. Crowd level data on mobile phone usage can be used as a proxy for population mobility patterns and provide a way of quantifying in near-real time the impact of social distancing measures on changes in mobility. METHODS: Here we explore patterns of change in densities, domestic and international flows and co-location of Facebook users in the UK from March 2020 to March 2021. RESULTS: We find substantial heterogeneities across time and region, with large changes observed compared to pre-pademic patterns. The impacts of periods of lockdown on distances travelled and flow volumes are evident, with each showing variations, but some significant reductions in co-location rates. Clear differences in multiple metrics of mobility are seen in central London compared to the rest of the UK, with each of Scotland, Wales and Northern Ireland showing significant deviations from England at times. Moreover, the impacts of rapid changes in rules on international travel to and from the UK are seen in substantial fluctuations in traveller volumes by destination. CONCLUSIONS: While questions remain about the representativeness of the Facebook data, previous studies have shown strong correspondence with census-based data and alternative mobility measures, suggesting that findings here are valuable for guiding strategies.

Structural and Functional Consequences of Three Cancer-Associated Mutations of the Oncogenic Phosphatase SHP2.

The proto-oncogene PTPN11 encodes a cytoplasmic protein tyrosine phosphatase, SHP2, which is required for normal development and sustained activation of the Ras-MAPK signaling pathway. Germline mutations in SHP2 cause developmental disorders, and somatic mutations have been identified in childhood and adult cancers and drive leukemia in mice. Despite our knowledge of the PTPN11 variations associated with pathology, the structural and functional consequences of many disease-associated mutants remain poorly understood. Here, we combine X-ray crystallography, small-angle X-ray scattering, and biochemistry to elucidate structural and mechanistic features of three cancer-associated SHP2 variants harboring single point mutations within the N-SH2:PTP interdomain autoinhibitory interface. Our findings directly compare the impact of each mutation on autoinhibition of the phosphatase and advance the development of structure-guided and mutation-specific SHP2 therapies.

Direct quantification of circulating miRNAs in different stages of nasopharyngeal cancerous serum samples in single molecule level with total internal reflection fluorescence microscopy.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate human gene expression at the post-transcriptional level. Growing evidence indicates that the expression profile of miRNAs is highly correlated with the occurrence of human diseases including cancers. Playing important roles in complex gene regulation processes, the aberrant expression pattern of various miRNAs is implicated in different types and even stages of cancer. Besides localizing in cells, many of these miRNAs are found circulating around the body in a wide variety of fluids such as urine, serum and saliva. Surprisingly, these extracellular circulating miRNAs are highly stable and resistant to degradation, and therefore, are considered as promising biomarkers for early cancer diagnostic via noninvasive extraction from body fluids. Unfortunately, the abundance of these small RNAs is ultralow in the body fluids, making it challenging to quantify them in complex sample matrixes. Establishing a sensitive, specific yet simple assay for an accurate quantification of circulating miRNAs is therefore desirable. Our group previously reported a sensitive and specific detection assay of miRNAs in single molecule level with the aid of total internal reflection fluorescence microscopy. In this work, we advanced the assay to differentiate the expression of a nasopharyngeal carcinoma (NPC) up-regulator hsa-mir-205 (mir-205) in serum collected from patients of different stages of NPC. To overcome the background matrix interference in serum, a locked nucleic acid-modified molecular beacon (LNA/MB) was applied as the detection probe to hybridize, capture and detect target mir-205 in serum matrix with enhanced sensitivity and specificity. A detection limit of 500 fM was achieved. The as-developed method was capable of differentiating NPC stages by the level of mir-205 quantified in serum with only 10 µL of serum and the whole assay can be completed in 1 h. The experimental results agreed well with those previously reported whereas the quantity of miR-205 determined by our assay was found comparable to that of quantitative reverse transcription polymerase chain reaction (qRT-PCR), supporting that this assay can be served as a promising noninvasive detection tool for early NPC diagnosis, monitoring and staging.

Monitoring of DNA-protein interaction with single gold nanoparticles by localized scattering plasmon resonance spectroscopy.

We reported a sensitive detection system for measuring DNA-protein interaction at single plasmonic metal nanoparticles level by Localized Scattering Plasmon Resonance (LSPR) spectroscopy. As a proof of concept, DNA molecules were conjugated to gold nanoparticles (AuNPs) through gold-thiol chemistry and the resulted complex was served as single-particle probes of human topoisomerase I (TOPO). By recording the changes in Rayleigh light scattering signal of the individual nanoparticles upon protein binding, DNA-protein interaction was monitored and measured. The λmax shifts in LSPR spectrum of individual AuNP was found to be highly correlated with the amount of TOPO that bound onto. This technique provides a sensitive and high-throughput platform to screen and monitor accurately the specific biomolecular interactions. It is capable of revealing information such as particle-particle variations that might be buried in conventional bulk measurement.

Repression of PRMT activities sensitize homologous recombination-proficient ovarian and breast cancer cells to PARP inhibitor treatment.

An "induced PARP inhibitor (PARPi) sensitivity by epigenetic modulation" strategy is being evaluated in the clinic to sensitize homologous recombination (HR)-proficient tumors to PARPi treatments. To expand its clinical applications and identify more efficient combinations, we performed a drug screen by combining PARPi with 74 well-characterized epigenetic modulators that target five major classes of epigenetic enzymes. Both type I PRMT inhibitor and PRMT5 inhibitor exhibit high combination and clinical priority scores in our screen. PRMT inhibition significantly enhances PARPi treatment-induced DNA damage in HR-proficient ovarian and breast cancer cells. Mechanistically, PRMTs maintain the expression of genes associated with DNA damage repair and BRCAness and regulate intrinsic innate immune pathways in cancer cells. Analyzing large-scale genomic and functional profiles from TCGA and DepMap further confirms that PRMT1, PRMT4, and PRMT5 are potential therapeutic targets in oncology. Finally, PRMT1 and PRMT5 inhibition act synergistically to enhance PARPi sensitivity. Our studies provide a strong rationale for the clinical application of a combination of PRMT and PARP inhibitors in patients with HR-proficient ovarian or breast cancer.

Methylation of the chromatin modifier KMT2D by SMYD2 contributes to therapeutic response in hormone-dependent breast cancer.

Activating mutations in PIK3CA are frequently found in estrogen-receptor-positive (ER+) breast cancer, and the combination of the phosphatidylinositol 3-kinase (PI3K) inhibitor alpelisib with anti-ER inhibitors is approved for therapy. We have previously demonstrated that the PI3K pathway regulates ER activity through phosphorylation of the chromatin modifier KMT2D. Here, we discovered a methylation site on KMT2D, at K1330 directly adjacent to S1331, catalyzed by the lysine methyltransferase SMYD2. SMYD2 loss attenuates alpelisib-induced KMT2D chromatin binding and alpelisib-mediated changes in gene expression, including ER-dependent transcription. Knockdown or pharmacological inhibition of SMYD2 sensitizes breast cancer cells, patient-derived organoids, and tumors to PI3K/AKT inhibition and endocrine therapy in part through KMT2D K1330 methylation. Together, our findings uncover a regulatory crosstalk between post-translational modifications that fine-tunes KMT2D function at the chromatin. This provides a rationale for the use of SMYD2 inhibitors in combination with PI3Kα/AKT inhibitors in the treatment of ER+/PIK3CA mutant breast cancer.

Discovery and In Vivo Efficacy of AZ-PRMT5i-1, a Novel PRMT5 Inhibitor with High MTA Cooperativity.

PRMT5, a type 2 arginine methyltransferase, has a critical role in regulating cell growth and survival in cancer. With the aim of developing MTA-cooperative PRMT5 inhibitors suitable for MTAP-deficient cancers, herein we report our efforts to develop novel "MTA-cooperative" compounds identified through a high-throughput biochemical screening approach. Optimization of hits was achieved through structure-based design with a focus on improvement of oral drug-like properties. Bioisosteric replacement of the original thiazole guanidine headgroup, spirocyclization of the isoindolinone amide scaffold to both configurationally and conformationally lock the bioactive form, and fine-tuning of the potency, MTA cooperativity, and DMPK properties through specific substitutions of the azaindole headgroup were conducted. We have identified an orally available in vivo lead compound, 28 ("AZ-PRMT5i-1"), which shows sub-10 nM PRMT5 cell potency, >50-fold MTA cooperativity, suitable DMPK properties for oral dosing, and significant PRMT5-driven in vivo efficacy in several MTAP-deficient preclinical cancer models.

A liquid chromatography/mass spectrometry-based generic detection method for biochemical assay and hit discovery of histone methyltransferases.

Epigenetic modifications of the genome, such as DNA methylation and posttranslational modifications of histone proteins, contribute to gene regulation. Growing evidence suggests that histone methyltransferases are associated with the development of various human diseases, including cancer, and are promising drug targets. High-quality generic assays will facilitate drug discovery efforts in this area. In this article, we present a liquid chromatography/mass spectrometry (LC/MS)-based S-adenosyl homocysteine (SAH) detection assay for histone methyltransferases (HMTs) and its applications in HMT drug discovery, including analyzing the activity of newly produced enzymes, developing and optimizing assays, performing focused compound library screens and orthogonal assays for hit confirmations, selectivity profiling against a panel of HMTs, and studying mode of action of select hits. This LC/MS-based generic assay has become a critical platform for our methyltransferase drug discovery efforts.

A subnational reproductive, maternal, newborn, child, and adolescent health and development atlas of India.

Understanding the fine scale and subnational spatial distribution of reproductive, maternal, newborn, child, and adolescent health and development indicators is crucial for targeting and increasing the efficiency of resources for public health and development planning. National governments are committed to improve the lives of their people, lift the population out of poverty and to achieve the Sustainable Development Goals. We created an open access collection of high resolution gridded and district level health and development datasets of India using mainly the 2015-16 National Family Health Survey (NFHS-4) data, and provide estimates at higher granularity than what is available in NFHS-4, to support policies with spatially detailed data. Bayesian methods for the construction of 5 km × 5 km high resolution maps were applied for a set of indicators where the data allowed (36 datasets), while for some other indicators, only district level data were produced. All data were summarised using the India district administrative boundaries. In total, 138 high resolution and district level datasets for 28 indicators were produced and made openly available.

BET inhibition targets ABC-DLBCL constitutive B-cell receptor signaling through PAX5.

B-cell receptor (BCR) signaling is essential for the diffuse large B-cell lymphoma (DLBCL) subtype that originates from activated B-cells (ABCs). ABC-DLBCL cells are sensitive to Bruton tyrosine kinase intervention. However, patients with relapsed or refractory ABC-DLBCL had overall response rates from 33% to 37% for Bruton tyrosine kinase inhibitors, suggesting the evaluation of combination-based treatment for improved efficacy. We investigated the efficacy and mechanism of the bromodomain and extraterminal motif (BET) inhibitor AZD5153 combined with the Bruton tyrosine kinase inhibitor acalabrutinib in ABC-DLBCL preclinical models. AZD5153 is a bivalent BET inhibitor that simultaneously engages the 2 bromodomains of BRD4. Adding AZD5153 to acalabrutinib demonstrated combination benefits in ABC-DLBCL cell line and patient-derived xenograft models. Differential expression analyses revealed PAX5 transcriptional activity as a novel downstream effector of this drug combination. PAX5 is a transcription factor for BCR signaling genes and may be critical for perpetually active BCR signaling in ABC-DLBCL. Our analyses further indicated significant alterations in BCR, RELB/alternative NF-κB, and toll-like receptor/interferon signaling. Validation of these results mapped a positive-feedback signaling loop regulated by PAX5. We demonstrated that AZD5153 decreased PAX5 expression, whereas acalabrutinib disruption of BCR signaling inhibited PAX5 activation. Furthermore, several interferon levels were decreased by AZD5153 and acalabrutinib in tumors. Adding interferon-beta1 (IFNß1) to cells treated with acalabrutinib partially rescued PAX5 activation. Our results demonstrate that AZD5153 enhances the efficacy of acalabrutinib through PAX5 and BCR mechanisms that are critical for ABC-DLBCL.

Geospatial Analyses of Recent Household Surveys to Assess Changes in the Distribution of Zero-Dose Children and Their Associated Factors before and during the COVID-19 Pandemic in Nigeria.

The persistence of geographic inequities in vaccination coverage often evidences the presence of zero-dose and missed communities and their vulnerabilities to vaccine-preventable diseases. These inequities were exacerbated in many places during the coronavirus disease 2019 (COVID-19) pandemic, due to severe disruptions to vaccination services. Understanding changes in zero-dose prevalence and its associated risk factors in the context of the COVID-19 pandemic is, therefore, critical to designing effective strategies to reach vulnerable populations. Using data from nationally representative household surveys conducted before the COVID-19 pandemic, in 2018, and during the pandemic, in 2021, in Nigeria, we fitted Bayesian geostatistical models to map the distribution of three vaccination coverage indicators: receipt of the first dose of diphtheria-tetanus-pertussis-containing vaccine (DTP1), the first dose of measles-containing vaccine (MCV1), and any of the four basic vaccines (bacilli Calmette-Guerin (BCG), oral polio vaccine (OPV0), DTP1, and MCV1), and the corresponding zero-dose estimates independently at a 1 × 1 km resolution and the district level during both time periods. We also explored changes in the factors associated with non-vaccination at the national and regional levels using multilevel logistic regression models. Our results revealed no increases in zero-dose prevalence due to the pandemic at the national level, although considerable increases were observed in a few districts. We found substantial subnational heterogeneities in vaccination coverage and zero-dose prevalence both before and during the pandemic, showing broadly similar patterns in both time periods. Areas with relatively higher zero-dose prevalence occurred mostly in the north and a few places in the south in both time periods. We also found consistent areas of low coverage and high zero-dose prevalence using all three zero-dose indicators, revealing the areas in greatest need. At the national level, risk factors related to socioeconomic/demographic status (e.g., maternal education), maternal access to and utilization of health services, and remoteness were strongly associated with the odds of being zero dose in both time periods, while those related to communication were mostly relevant before the pandemic. These associations were also supported at the regional level, but we additionally identified risk factors specific to zero-dose children in each region; for example, communication and cross-border migration in the northwest. Our findings can help guide tailored strategies to reduce zero-dose prevalence and boost coverage levels in Nigeria.

Systematic Pan-Cancer Characterization of Nuclear Receptors Identifies Potential Cancer Biomarkers and Therapeutic Targets.

The nuclear receptor (NR) superfamily is one of the major druggable gene families, representing targets of approximately 13.5% of approved drugs. Certain NRs, such as estrogen receptor and androgen receptor, have been well demonstrated to be functionally involved in cancer and serve as informative biomarkers and therapeutic targets in oncology. However, the spectrum of NR dysregulation across cancers remains to be comprehensively characterized. Through computational integration of genetic, genomic, and pharmacologic profiles, we characterized the expression, recurrent genomic alterations, and cancer dependency of NRs at a large scale across primary tumor specimens and cancer cell lines. Expression levels of NRs were highly cancer-type specific and globally downregulated in tumors compared with corresponding normal tissue. Although the majority of NRs showed copy-number losses in cancer, both recurrent focal gains and losses were identified in select NRs. Recurrent mutations and transcript fusions of NRs were observed in a small portion of cancers, serving as actionable genomic alterations. Analysis of large-scale CRISPR and RNAi screening datasets identified 10 NRs as strongly selective essential genes for cancer cell growth. In a subpopulation of tumor cells, growth dependencies correlated significantly with expression or genomic alterations. Overall, our comprehensive characterization of NRs across cancers may facilitate the identification and prioritization of potential biomarkers and therapeutic targets, as well as the selection of patients for precision cancer treatment. SIGNIFICANCE: Computational analysis of nuclear receptors across multiple cancer types provides a series of biomarkers and therapeutic targets within this protein family.

Systematic illumination of druggable genes in cancer genomes.

By combining 6 druggable genome resources, we identify 6,083 genes as potential druggable genes (PDGs). We characterize their expression, recurrent genomic alterations, cancer dependencies, and therapeutic potentials by integrating genome, functionome, and druggome profiles across cancers. 81.5% of PDGs are reliably expressed in major adult cancers, 46.9% show selective expression patterns, and 39.1% exhibit at least one recurrent genomic alteration. We annotate a total of 784 PDGs as dependent genes for cancer cell growth. We further quantify 16 cancer-related features and estimate a PDG cancer drug target score (PCDT score). PDGs with higher PCDT scores are significantly enriched for genes encoding kinases and histone modification enzymes. Importantly, we find that a considerable portion of high PCDT score PDGs are understudied genes, providing unexplored opportunities for drug development in oncology. By integrating the druggable genome and the cancer genome, our study thus generates a comprehensive blueprint of potential druggable genes across cancers.

Multifunctional encoded self-assembling protein nanofibrils as platform for high-throughput and multiplexed detection of biomolecules.

A one-dimensional nanofibrillar array formed by the co-assembly of native and biotin-functionalized beta-amyloid (Aß) peptide was developed for biomolecule sensing. With the presence of biotin moiety, a variety of biomolecular probes can be conjugated onto the nanofibrils, thus converting the protein assembly into a miniature biosensor. In this work, DNA probes were immobilized onto the fibril for the detection of cDNA sequences. The as-developed "DNA-nanoarray" achieved a detection limit at subattomole level (183 fM in 10 µL). This highly sensitive, yet simple, assay requires a trace amount of sample consumption (<10 µL) and is pretreatment-free. In addition, we reported the preparation of alternate-segmented amyloid nanofibrils with multifunctionality. The fibrils hereby serve as an encoded template that can be visualized with various fluorescence labeling dyes for barcode recognition purpose, and, hence, multiplex detection of biomolecules was achieved. Regarding that each protein nanofibril represents a single detection platform, a large number of single fibrils simultaneously are monitored with the dual-color TIRFM in a high-throughput manner.

The Cancer Surfaceome Atlas integrates genomic, functional and drug response data to identify actionable targets.

Cell-surface proteins (SPs) are a rich source of immune and targeted therapies. By systematically integrating single-cell and bulk genomics, functional studies and target actionability, in the present study we comprehensively identify and annotate genes encoding SPs (GESPs) pan-cancer. We characterize GESP expression patterns, recurrent genomic alterations, essentiality, receptor-ligand interactions and therapeutic potential. We also find that mRNA expression of GESPs is cancer-type specific and positively correlates with protein expression, and that certain GESP subgroups function as common or specific essential genes for tumor cell growth. We also predict receptor-ligand interactions substantially deregulated in cancer and, using systems biology approaches, we identify cancer-specific GESPs with therapeutic potential. We have made this resource available through the Cancer Surfaceome Atlas ( http://fcgportal.org/TCSA ) within the Functional Cancer Genome data portal.

Direct quantification of single-molecules of microRNA by total internal reflection fluorescence microscopy.

MicroRNAs (miRNAs) express differently in normal and cancerous tissues and thus are regarded as potent cancer biomarkers for early diagnosis. However, the short length and low abundance of miRNAs have brought challenges to the established detection assay in terms of sensitivity and selectivity. In this work, we present a novel miRNA detection assay in single-molecule level with total internal reflection fluorescence microscopy (TIRFM). It is a solution-based hybridization detection system that does not require pretreatment steps such as sample enrichment or signal amplification. The hsa-miR-21 (miR-21) is chosen as target miRNA for its significant elevated content in a variety of cancers as reported previously. Herein, probes of complementary single-stranded oligonucleotide were hybridized in solution to miR-21 and labeled with fluorescent dye YOYO-1. The fluorescent hybrids were imaged by an electron-multiplying charge-coupled device (EMCCD) coupled TIRFM system and quantified by single-molecule counting. This single molecule detection (SMD) assay shows a good correlation between the number of molecules detected and the factual concentration of miRNA. The detection assay is applied to quantify the miR-21 in extracted total RNA samples of cancerous MCF-7 cells, HepG2 cells, and normal HUVEC cells, respectively. The results agreed very well with those from the prevalent real-time polymerase chain reaction (qRT-PCR) analysis. This assay is of high potential for applications in miRNA expression profiling and early cancer diagnosis.

A family of diastereomeric dodecanuclear coordination cages based on inversion of chirality of individual triangular cyclic helicate faces.

The dodecanuclear coordination cage [Cd12(Lnaph)12(Lmes)4](BF4)24 consists of a set of four triangular, trinuclear helical panels {Cd3(µ-Lnaph)3}6+ (based on ditopic bridging ligands Lnaph), which are connected by four tritopic ligands Lmes. The result is that the four triangular helical panels and the four Lmes-capped triangular faces of the cuboctahedral core form two alternating subsets of the eight triangular faces of the cuboctahedron. Crystallographic investigations revealed that the triangular helicate faces can have 'clockwise' (C) or 'anticlockwise' (A) helicity, and that the helicity of each face can vary independently of the others as they are mechanically separated. This generates a set of three diastereoisomers in which all four cyclic helicate faces in the cuboctahedron have the same chirality (AAAA/CCCC enantiomers with T symmetry; AAAC/CCCA enantiomers with C 3 symmetry; and achiral AACC with S 4 symmetry). This mirrors the known behaviour of many simpler M4L6 tetrahedral cages which can likewise exist as T, C 3 or S 4 isomers according to the sense of tris-chelate chirality around each individual metal centre: but here it is translated onto a much larger scale by the four chiral units being entire trinuclear helicate faces rather than single metal centres. 1H NMR spectroscopy confirms the presence of the three diastereoisomers with their different molecular symmetries in a ratio slightly different from what is expected on purely statistical grounds; and 1H NMR measurements on a non-equilibrium sample (enriched by manual crystal-picking before preparing the solution) showed that the distribution does not change over several weeks in solution, indicating the kinetic inertness of the cage assemblies.