Activation-induced cytidine deaminase causes recurrent splicing mutations in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma. A major mutagenic process in DLBCL is aberrant somatic hypermutation (aSHM) by activation-induced cytidine deaminase (AID), which occurs preferentially at RCH/TW sequence motifs proximal to transcription start sites. Splice sequences are highly conserved, rich in RCH/TW motifs, and recurrently mutated in DLBCL. Therefore, we hypothesized that aSHM may cause recurrent splicing mutations in DLBCL. In a meta-cohort of > 1,800 DLBCLs, we found that 77.5% of splicing mutations in 29 recurrently mutated genes followed aSHM patterns. In addition, in whole-genome sequencing (WGS) data from 153 DLBCLs, proximal mutations in splice sequences, especially in donors, were significantly enriched in RCH/TW motifs (p < 0.01). We validated this enrichment in two additional DLBCL cohorts (N > 2,000; p < 0.0001) and confirmed its absence in 12 cancer types without aSHM (N > 6,300). Comparing sequencing data from mouse models with and without AID activity showed that the splice donor sequences were the top genomic feature enriched in AID-induced mutations (p < 0.0001). Finally, we observed that most AID-related splice site mutations are clonal within a sample, indicating that aSHM may cause early loss-of-function events in lymphomagenesis. Overall, these findings support that AID causes an overrepresentation of clonal splicing mutations in DLBCL.

Probing the Cellular Fate of the Protein Corona around Nanoparticles with Nanofocused X-ray Fluorescence Imaging.

X-ray fluorescence imaging (XRF-imaging) with subcellular resolution is used to study the intracellular integrity of a protein corona that was pre-formed around gold nanoparticles (AuNP). Artificial proteins engineered to obtain Gd coordination for detection by XRF-imaging were used to form the corona. Indications about the degradation of this protein corona at a cellular and subcellular level can be observed by following the Au and Gd quantities in a time and spatial-dependent manner. The extended acquisition times necessary for capturing individual XRF-imaging cell images result in relatively small sample populations, stressing the need for faster image acquisition strategies in future XRF-imaging-based studies to deal with the inherent variability between cells. Still, results obtained reveal degradation of the protein corona during cellular trafficking, followed by differential cellular processing for AuNP and Gd-labelled proteins. Overall, this demonstrates that the dynamic degradation of the protein corona can be tracked by XRF-imaging to a certain degree.

X-Ray Photon Correlation Spectroscopy Towards Measuring Nanoparticle Diameters in Biological Environments Allowing for the In Situ Analysis of their Bio-Nano Interface.

X-ray photon correlation spectroscopy (XPCS), a synchrotron source-based technique to measure sample dynamics, is used to determine hydrodynamic diameters of gold nanoparticles (Au NPs) of different sizes in biological environments. In situ determined hydrodynamic diameters are benchmarked with values obtained by dynamic light scattering. The technique is then applied to analyze the behavior of the Au NPs in a biological environment. First, a concentration-dependent agglomeration in the presence of NaCl is determined. Second, concentration-dependent increase in hydrodynamic diameter of the Au NPs upon the presence of proteins is determined. As X-rays in the used energy range are barely scattered by biological matter, dynamics of the Au NPs can be also detected in situ in complex biological environments, such as blood. These measurements demonstrate the possibility of XPCS for in situ analytics of nanoparticles (NPs) in biological environments where similar detection techniques based on visible light would severely suffer from scattering, absorption, and reflection effects.

Probing the Effect of Rigidity on the Cellular Uptake of Core-Shell Nanoparticles: Stiffness Effects are Size Dependent.

Nanoparticles are well established vectors for the delivery of a wide range of biomedically relevant cargoes. Numerous studies have investigated the impact of size, shape, charge, and surface functionality of nanoparticles on mammalian cellular uptake. Rigidity has been studied to a far lesser extent, and its effects are still unclear. Here, the importance of this property, and its interplay with particle size, is systematically explored using a library of core-shell spherical PEGylated nanoparticles synthesized by RAFT emulsion polymerization. Rigidity of these particles is controlled by altering the intrinsic glass transition temperature of their constituting polymers. Three polymeric core rigidities are tested: hard, medium, and soft using two particle sizes, 50 and 100 nm diameters. Cellular uptake studies indicate that softer particles are taken up faster and threefold more than harder nanoparticles with the larger 100 nm particles. In addition, the study indicates major differences in the cellular uptake pathway, with harder particles being internalized through clathrin- and caveolae-mediated endocytosis as well as macropinocytosis, while softer particles are taken up bycaveolae- and non-receptormediated endocytosis. However, 50 nm derivatives do not show any appreciable differences in uptake efficiency, suggesting that rigidity as a parameter in the biological regime may be size dependent.

Single-Cell Chemistry of Photoactivatable Platinum Anticancer Complexes.

The Pt(IV) prodrug trans, trans, trans-[Pt(pyridine)2(N3)2(OH)2] (Pt1) and its coumarin derivative trans, trans, trans-[Pt(pyridine)2(N3)2(OH)(coumarin-3-carboxylate)] (Pt2) are promising agents for photoactivated chemotherapy. These complexes are inert in the dark but release Pt(II) species and radicals upon visible light irradiation, resulting in photocytotoxicity toward cancer cells. Here, we have used synchrotron techniques to investigate the in-cell behavior of these prodrugs and visualize, for the first time, changes in cellular morphology and Pt localization upon treatment with and without light irradiation. We show that photoactivation of Pt2 induces remarkable cellular damage with extreme alterations to multiple cellular components, including formation of vacuoles, while also significantly increasing the cellular accumulation of Pt species compared to dark conditions. X-ray absorption near-edge structure (XANES) measurements in cells treated with Pt2 indicate only partial reduction of the prodrug upon irradiation, highlighting that phototoxicity in cancer cells may involve not only Pt(II) photoproducts but also photoexcited Pt(IV) species.

Comparative Study of the Cellular Uptake and Intracellular Behavior of a Library of Cyclic Peptide-Polymer Nanotubes with Different Self-Assembling Properties.

Particle shape has been described as a key factor in improving cell internalization and biodistribution among the different properties investigated for drug-delivery systems. In particular, tubular structures have been identified as promising candidates for improving drug delivery. Here, we investigate the influence of different design elements of cyclic peptide-polymer nanotubes (CPNTs) on cellular uptake including the nature and length of the polymer and the cyclic peptide building block. By varying the composition of these cyclic peptide-polymer conjugates, a library of CPNTs of lengths varying from a few to over a 150 nm were synthesized and characterized using scattering techniques (small-angle neutron scattering and static light scattering). In vitro studies with fluorescently labeled CPNTs have shown that nanotubes comprised of a single polymer arm with a size between 8 and 16 nm were the most efficiently taken up by three different mammalian cell lines. A mechanistic study on multicellular tumor spheroids has confirmed the ability of these compounds to penetrate to their core. Variations in the proportion of paracellular and transcellular uptake with the self-assembling potential of the CPNT were also observed, giving key insights about the behavior of CPNTs in cellular systems.

Tracking Reactions of Asymmetric Organo-Osmium Transfer Hydrogenation Catalysts in Cancer Cells.

Most metallodrugs are prodrugs that can undergo ligand exchange and redox reactions in biological media. Here we have investigated the cellular stability of the anticancer complex [OsII [(η6 -p-cymene)(RR/SS-MePh-DPEN)] [1] (MePh-DPEN=tosyl-diphenylethylenediamine) which catalyses the enantioselective reduction of pyruvate to lactate in cells. The introduction of a bromide tag at an unreactive site on a phenyl substituent of Ph-DPEN allowed us to probe the fate of this ligand and Os in human cancer cells by a combination of X-ray fluorescence (XRF) elemental mapping and inductively coupled plasma-mass spectrometry (ICP-MS). The BrPh-DPEN ligand is readily displaced by reaction with endogenous thiols and translocated to the nucleus, whereas the Os fragment is exported from the cells. These data explain why the efficiency of catalysis is low, and suggests that it could be optimised by developing thiol resistant analogues. Moreover, this work also provides a new way for the delivery of ligands which are inactive when administered on their own.

X-ray tomography of cryopreserved human prostate cancer cells: mitochondrial targeting by an organoiridium photosensitiser.

The organoiridium complex Ir[(C,N)2(O,O)] (1) where C, N = 1-phenylisoquinoline and O,O = 2,2,6,6-tetramethyl-3,5-heptanedionate is a promising photosensitiser for Photo-Dynamic Therapy (PDT). 1 is not toxic to cells in the dark. However, irradiation of the compound with one-photon blue or two-photon red light generates high levels of singlet oxygen (1O2) (in Zhang et al. Angew Chem Int Ed Engl 56 (47):14898-14902 https://doi.org/10.1002/anie.201709082,2017), both within cell monolayers and in tumour models. Moreover, photo-excited 1 oxidises key proteins, causing metabolic alterations in cancer cells with potent antiproliferative activity. Here, the tomograms obtained by cryo-Soft X-ray Tomography (cryo-SXT) of human PC3 prostate cancer cells treated with 1, irradiated with blue light, and cryopreserved to maintain them in their native state, reveal that irradiation causes extensive and specific alterations to mitochondria, but not other cellular components. Such new insights into the effect of 1O2 generation during PDT using iridium photosensitisers on cells contribute to a detailed understanding of their cellular mode of action.

Metallation-Induced Heterogeneous Dynamics of DNA Revealed by Single-Molecule FRET.

The metallation of nucleic acids is key to wide-ranging applications, from anticancer medicine to nanomaterials, yet there is a lack of understanding of the molecular-level effects of metallation. Here, we apply single-molecule fluorescence methods to study the reaction of an organo-osmium anticancer complex and DNA. Individual metallated DNA hairpins are characterised using Förster resonance energy transfer (FRET). Although ensemble measurements suggest a simple two-state system, single-molecule experiments reveal an underlying heterogeneity in the oligonucleotide dynamics, attributable to different degrees of metallation of the GC-rich hairpin stem. Metallated hairpins display fast two-state transitions with a two-fold increase in the opening rate to ≈2 s-1 , relative to the unmodified hairpin, and relatively static conformations with long-lived open (and closed) states of 5 to ≥50 s. These studies show that a single-molecule approach can provide new insight into metallation-induced changes in DNA structure and dynamics.

Recent Developments in the Design of Non-Biofouling Coatings for Nanoparticles and Surfaces.

Biofouling is a major issue in the field of nanomedicine and consists of the spontaneous and unwanted adsorption of biomolecules on engineered surfaces. In a biological context and referring to nanoparticles (NPs) acting as nanomedicines, the adsorption of biomolecules found in blood (mostly proteins) is known as protein corona. On the one hand, the protein corona, as it covers the NPs' surface, can be considered the biological identity of engineered NPs, because the corona is what cells will "see" instead of the underlying NPs. As such, the protein corona will influence the fate, integrity, and performance of NPs in vivo. On the other hand, the physicochemical properties of the engineered NPs, such as their size, shape, charge, or hydrophobicity, will influence the identity of the proteins attracted to their surface. In this context, the design of coatings for NPs and surfaces that avoid biofouling is an active field of research. The gold standard in the field is the use of polyethylene glycol (PEG) molecules, although zwitterions have also proved to be efficient in preventing protein adhesion and fluorinated molecules are emerging as coatings with interesting properties. Hence, in this review, we will focus on recent examples of anti-biofouling coatings in three main areas, that is, PEGylated, zwitterionic, and fluorinated coatings.

Assembly and Degradation of Inorganic Nanoparticles in Biological Environments.

In solution, nanoparticles may be conceptually compartmentalized into cores and engineered surface coatings. Recent advances allow for simple and accurate characterization of nanoparticle cores and surface shells. After introduction into a complex biological environment, adsorption of biological molecules to the nanoparticle surface as well as a loss of original surface components occur. Thus, colloidal nanoparticles in the context of the biological environment are hybrid materials with complex structure, which may result in different chemical, physical, and biological outcomes as compared to the original engineered nanoparticles. In this review, we will discuss building up an engineered inorganic nanoparticle from its inside core to its outside surface and following its degradation in a biological environment from its outside to its inside. This will involve the way to synthesize selected inorganic nanoparticles. Then, we will discuss the environmental changes upon exposure of these nanoparticles to biological media and their uptake by cells. Next, the intracellular fate of nanoparticles and their degradation will be discussed. Based on these examples, the need to see nanoparticles in the context of the biological environment as dynamic hybrid materials will be highlighted.

A Growth Factor-Free Co-Culture System of Osteoblasts and Peripheral Blood Mononuclear Cells for the Evaluation of the Osteogenesis Potential of Melt-Electrowritten Polycaprolactone Scaffolds.

Scaffolds made of biodegradable biomaterials are widely used to guide bone regeneration. Commonly, in vitro assessment of scaffolds' osteogenesis potential has been performed predominantly in monoculture settings. Hence, this study evaluated the potential of an unstimulated, growth factor-free co-culture system comprised of osteoblasts (OB) and peripheral blood mononuclear cells (PBMC) over monoculture of OB as an in vitro platform for screening of bone regeneration potential of scaffolds. Particularly, this study focuses on the osteogenic differentiation and mineralized matrix formation aspects of cells. The study was performed using scaffolds fabricated by means of a melt electrowriting (MEW) technique made of medical-grade polycaprolactone (PCL), with or without a surface coating of calcium phosphate (CaP). Qualitative results, i.e., cell morphology by fluorescence imaging and matrix mineralization by von Kossa staining, indicated the differences in cell behaviours in response to scaffolds' biomaterial. However, no obvious differences were noted between OB and OB+PBMC groups. Hence, quantitative investigation, i.e., alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP) activities, and gene expression were quantitatively evaluated by reverse transcription-polymerase chain reaction (RT-qPCR), were evaluated only of PCL/CaP scaffolds cultured with OB+PBMC, while PCL/CaP scaffolds cultured with OB or PBMC acted as a control. Although this study showed no differences in terms of osteogenic differentiation and ECM mineralization, preliminary qualitative results indicate an obvious difference in the cell/non-mineralized ECM density between scaffolds cultured with OB or OB+PBMC that could be worth further investigation. Collectively, the unstimulated, growth factor-free co-culture (OB+PBMC) system presented in this study could be beneficial for the pre-screening of scaffolds' in vitro bone regeneration potential prior to validation in vivo.

Cyclic Peptide-Polymer Nanotubes as Efficient and Highly Potent Drug Delivery Systems for Organometallic Anticancer Complexes.

Functional drug carrier systems have potential for increasing solubility and potency of drugs while reducing side effects. Complex polymeric materials, particularly anisotropic structures, are especially attractive due to their long circulation times. Here, we have conjugated cyclic peptides to the biocompatible polymer poly(2-hydroxypropyl methacrylamide) (pHPMA). The resulting conjugates were functionalized with organoiridium anticancer complexes. Small angle neutron scattering and static light scattering confirmed their self-assembly and elongated cylindrical shape. Drug-loaded nanotubes exhibited more potent antiproliferative activity toward human cancer cells than either free drug or the drug-loaded polymers, while the nanotubes themselves were nontoxic. Cellular accumulation studies revealed that the increased potency of the conjugate appears to be related to a more efficient mode of action rather than a higher cellular accumulation of iridium.

Redesigning the Maternal, Infant and Early Childhood Home Visiting Program Performance Measurement System.

Objectives Statute for the Maternal, Infant and Early Childhood Home Visiting (MIECHV) Program requires that states and territories receiving Program funding assess improvements for participating families across six areas that address maternal and child well-being. In 2015, the MIECHV Program performance measurement system was redesigned to allow for national-level analyses and cross-grantee comparisons. The new measures were aligned with other federal performance measures to help ensure context for program analyses. The number of measures was also reduced to lessen reporting burden. This paper describes the redesign process and resulting national performance measures. Methods The redesign process included holding listening sessions with stakeholders and experts; reviewing the findings from other home visiting performance initiatives; consulting with experts; soliciting and responding to public comment on draft measures; seeking clearance from the Office of Management and Budget; and specifying each measure with detailed eligibility criteria, the timing and frequency of assessments, and the window for data collection. Results The redesign resulted in a set of 19 measures that all MIECHV-funded home visiting programs began collecting in 2016. This is nearly half the number of measures that MIECHV awardees had been reporting prior to the redesign. The measures are aligned with other federal measures, including those used in Healthy People 2020 and those used for other maternal and child health programs. Conclusions for Practice Data reported by MIECHV Program awardees will be used to assess their performance, identify areas for targeted technical assistance to support continuous improvement, and ensure meaningful impacts for at-risk families.

Synchrotron X-Ray Fluorescence Nanoprobe Reveals Target Sites for Organo-Osmium Complex in Human Ovarian Cancer Cells.

A variety of transition metal complexes exhibit anticancer activity, but their target sites in cells need to be identified and mechanisms of action elucidated. Here, it was found that the sub-cellular distribution of [Os(η6 -p-cym)(Azpy-NMe2 )I]+ (p-cym=p-cymene, Azpy-NMe2 =2-(p-[dimethylamino]phenylazo)pyridine) (1), a promising drug candidate, can be mapped in human ovarian cancer cells at pharmacological concentrations using a synchrotron X-ray fluorescence nanoprobe (SXRFN). SXRFN data for Os, Zn, Ca, and P, as well as TEM and ICP analysis of mitochondrial fractions suggest localization of Os in mitochondria and not in the nucleus, accompanied by mobilization of Ca from the endoplasmic reticulum, a signaling event for cell death. These data are consistent with the ability of 1 to induce rapid bursts of reactive oxygen species and especially superoxide formed in the first step of O2 reduction in mitochondria. Such metabolic targeting differs from the action of Pt drugs, offering promise for combatting Pt resistance, which is a current clinical problem.

Frailty and sarcopenia in Bogotá: results from the SABE Bogotá Study.

BACKGROUND: Latin American countries like Colombia are experiencing a unique aging process due to a mixed epidemiological regime of communicable and non-communicable diseases. AIMS: To estimate the prevalence of frailty and sarcopenia among older adults in Colombia and identify variables associated with these conditions. METHODS: Data come from the "Salud Bienestar y Envejecimiento" (SABE) Bogotá Study, a cross-sectional study conducted in 2012 in Bogotá, Colombia. Sociodemographic, health, cognitive and anthropometric measures were collected from 2000 community-dwelling adults aged 60 years and older. Frailty variable was created using the Fried phenotype and sarcopenia following the European Working Group on Sarcopenia in Older People algorithm. Logistic regression analyses were used to identify factors associated with frailty and sarcopenia. RESULTS: A total of 135 older adults are frail (9.4 %), while 166 have sarcopenia (11.5 %). Older age and female gender have a significant association with both conditions (Frailty: Age OR 1.05, 95 % CI 1.03-1.06, Gender OR 1.44, 95 % CI 1.12-1.84; Sarcopenia: Age 1.04, 95 % CI 1.02-1.07, Gender OR 1.51, 95 % CI 1.05-2.17). Depression was also significantly associated with frailty (OR 1.17, 95 % CI 1.12-1.22), while smoking was significantly associated with sarcopenia (OR 2.38, 95 % CI 1.29-4.37). Finally, higher function, measured by independence in IADL (Instrumental Activities of Daily Living) was significantly associated with less frailty (OR 0.74, 95 % CI 0.64-0.86). Education, higher number of comorbidities, better MMSE score, activities of daily living disability and alcohol consumption were not significantly associated with frailty or sarcopenia. CONCLUSIONS: Frailty, sarcopenia and multimorbidity are overlapping, yet distinct conditions in this sample. There are potentially reversible factors that are associated with frailty and sarcopenia in this sample. Future studies need to analyze the best way to prevent these conditions, and examine individuals that have frailty, sarcopenia and comorbidities to design interventions to improve their quality of life.

In-Cell Activation of Organo-Osmium(II) Anticancer Complexes.

The family of iodido OsII arene phenylazopyridine complexes [Os(η6 -p-cym)(5-R1 -pyridylazo-4-R2 -phenyl))I]+ (where p-cym=para-cymene) exhibit potent sub-micromolar antiproliferative activity towards human cancer cells and are active in vivo. Their chemical behavior is distinct from that of cisplatin: they do not readily hydrolyze, nor bind to DNA bases. We report here a mechanism by which they are activated in cancer cells, involving release of the I- ligand in the presence of glutathione (GSH). The X-ray crystal structures of two active complexes are reported, 1-I (R1 =OEt, R2 =H) and 2-I (R1 =H, R2 =NMe2 ). They were labelled with the radionuclide 131 I (ß- /γ emitter, t1/2 8.02 d), and their activity in MCF-7 human breast cancer cells was studied. 1-[131 I] and 2-[131 I] exhibit good stability in both phosphate-buffered saline and blood serum. In contrast, once taken up by MCF-7 cells, the iodide ligand is rapidly pumped out. Intriguingly, GSH catalyzes their hydrolysis. The resulting hydroxido complexes can form thiolato and sulfenato adducts with GSH, and react with H2 O2 generating hydroxyl radicals. These findings shed new light on the mechanism of action of these organo-osmium complexes.

Easy To Synthesize, Robust Organo-osmium Asymmetric Transfer Hydrogenation Catalysts.

Asymmetric transfer hydrogenation (ATH) is an important process in organic synthesis for which the Noyori-type Ru(II) catalysts [(arene)Ru(Tsdiamine)] are now well established and widely used. We now demonstrate for the first time the catalytic activity of the osmium analogues. X-ray crystal structures of the 16-electron Os(II) catalysts are almost identical to those of Ru(II). Intriguingly the precursor complex was isolated as a dichlorido complex with a monodentate amine ligand. The Os(II) catalysts are readily synthesised (within 1 h) and exhibit excellent enantioselectivity in ATH reactions of ketones.

A new performance measurement system for maternal and child health in the United States.

OBJECTIVE: The Title V Maternal and Child Health (MCH) Block Grant is the linchpin for US MCH services. The first national performance measures (NPMs) for MCH were instituted in 1997. Changing trends in MCH risk factors, outcomes, health services, data sources, and advances in scientific knowledge, in conjunction with budgetary constraints led the Maternal and Child Health Bureau (MCHB) to design a new performance measurement system. METHODS: A workgroup was formed to develop a new system. The following guiding principles were used: (1) Afford States more flexibility and reduce the overall reporting burden; (2) Improve accountability to better document Title V's impact; (3) Develop NPMs that encompass measures in: maternal and women's health, perinatal health, child health, children with special health care needs, adolescent health, and cross-cutting areas. RESULTS: A three-tiered performance measurement system was proposed with national outcome measures (NOMs), NPMs and evidence-based/informed strategy measures (ESMs). NOMs are the ultimate goals that MCHB and States are attempting to achieve. NPMs are measures, generally associated with processes or programs, shown to affect NOMs. ESMs are evidence-based or informed measures that each State Title V program develops to affect the NPMs. There are 15 NPMs from which States select eight, with at least one from each population area. MCHB will provide the data for the NOMs and NPMs, when possible. CONCLUSIONS: The new performance measurement system increases the flexibility and reduces the reporting burden for States by allowing them to choose 8 NPMs to target, and increases accountability by having States develop actionable ESMs. SIGNIFICANCE: The new national performance measure framework for maternal and child health will allow States more flexibility to address their areas of greatest need, reduce their data reporting burden by having the Maternal and Child Health Bureau provide data for the National Outcome and Performance Measures, yet afford States the opportunity to develop measurable strategies to address their selected performance measures.

A public resource facilitating clinical use of genomes.

Rapid advances in DNA sequencing promise to enable new diagnostics and individualized therapies. Achieving personalized medicine, however, will require extensive research on highly reidentifiable, integrated datasets of genomic and health information. To assist with this, participants in the Personal Genome Project choose to forgo privacy via our institutional review board- approved "open consent" process. The contribution of public data and samples facilitates both scientific discovery and standardization of methods. We present our findings after enrollment of more than 1,800 participants, including whole-genome sequencing of 10 pilot participant genomes (the PGP-10). We introduce the Genome-Environment-Trait Evidence (GET-Evidence) system. This tool automatically processes genomes and prioritizes both published and novel variants for interpretation. In the process of reviewing the presumed healthy PGP-10 genomes, we find numerous literature references implying serious disease. Although it is sometimes impossible to rule out a late-onset effect, stringent evidence requirements can address the high rate of incidental findings. To that end we develop a peer production system for recording and organizing variant evaluations according to standard evidence guidelines, creating a public forum for reaching consensus on interpretation of clinically relevant variants. Genome analysis becomes a two-step process: using a prioritized list to record variant evaluations, then automatically sorting reviewed variants using these annotations. Genome data, health and trait information, participant samples, and variant interpretations are all shared in the public domain-we invite others to review our results using our participant samples and contribute to our interpretations. We offer our public resource and methods to further personalized medical research.