Application of RtcB ligase to monitor self-cleaving ribozyme activity by RNA-seq.

Self-cleaving ribozymes are catalytic RNAs and can be found in all domains of life. They catalyze a site-specific cleavage that results in a 5' fragment with a 2',3' cyclic phosphate (2',3' cP) and a 3' fragment with a 5' hydroxyl (5' OH) end. Recently, several strategies to enrich self-cleaving ribozymes by targeted biochemical methods have been introduced by us and others. Here, we develop an alternative strategy in which 5' OH RNAs are specifically ligated by RtcB ligase, which first guanylates the 3' phosphate of the adapter and then ligates it directly to RNAs with 5' OH ends. Our results demonstrate that adapter ligation to highly structured ribozyme fragments is much more efficient using the thermostable RtcB ligase from Pyrococcus horikoshii than the broadly applied Escherichia coli enzyme. Moreover, we investigated DNA, RNA and modified RNA adapters for their suitability in RtcB ligation reactions. We used the optimized RtcB-mediated ligation to produce RNA-seq libraries and captured a spiked 3' twister ribozyme fragment from E. coli total RNA. This RNA-seq-based method is applicable to detect ribozyme fragments as well as other cellular RNAs with 5' OH termini from total RNA.

Multiomics analysis of rheumatoid arthritis yields sequence variants that have large effects on risk of the seropositive subset.

OBJECTIVES: To find causal genes for rheumatoid arthritis (RA) and its seropositive (RF and/or ACPA positive) and seronegative subsets. METHODS: We performed a genome-wide association study (GWAS) of 31 313 RA cases (68% seropositive) and ~1 million controls from Northwestern Europe. We searched for causal genes outside the HLA-locus through effect on coding, mRNA expression in several tissues and/or levels of plasma proteins (SomaScan) and did network analysis (Qiagen). RESULTS: We found 25 sequence variants for RA overall, 33 for seropositive and 2 for seronegative RA, altogether 37 sequence variants at 34 non-HLA loci, of which 15 are novel. Genomic, transcriptomic and proteomic analysis of these yielded 25 causal genes in seropositive RA and additional two overall. Most encode proteins in the network of interferon-alpha/beta and IL-12/23 that signal through the JAK/STAT-pathway. Highlighting those with largest effect on seropositive RA, a rare missense variant in STAT4 (rs140675301-A) that is independent of reported non-coding STAT4-variants, increases the risk of seropositive RA 2.27-fold (p=2.1×10-9), more than the rs2476601-A missense variant in PTPN22 (OR=1.59, p=1.3×10-160). STAT4 rs140675301-A replaces hydrophilic glutamic acid with hydrophobic valine (Glu128Val) in a conserved, surface-exposed loop. A stop-mutation (rs76428106-C) in FLT3 increases seropositive RA risk (OR=1.35, p=6.6×10-11). Independent missense variants in TYK2 (rs34536443-C, rs12720356-C, rs35018800-A, latter two novel) associate with decreased risk of seropositive RA (ORs=0.63-0.87, p=10-9-10-27) and decreased plasma levels of interferon-alpha/beta receptor 1 that signals through TYK2/JAK1/STAT4. CONCLUSION: Sequence variants pointing to causal genes in the JAK/STAT pathway have largest effect on seropositive RA, while associations with seronegative RA remain scarce.

Imaging the fibroblast growth factor receptor network on the plasma membrane with DNA-assisted single-molecule super-resolution microscopy.

Fibroblast growth factor receptors (FGFRs) are a subfamily of receptor tyrosine kinases and central players in health and disease. Following ligand binding and the formation of homo- and heteromeric complexes, FGFRs initiate a cellular response. Challenges in studying FGFR activation are inner-subfamily interactions and a complex heterogeneity of these in the cell membrane, which demand for observation techniques that can resolve individual protein complexes and that are compatible with endogenous protein levels. Here, we established an imaging and analysis pipeline for multiplexed single-molecule localization microscopy (SMLM) of the FGFR network at the plasma membrane. Using DNA-labeled primary antibodies, we visualize all four FGFRs in the same cell with near-molecular spatial resolution. From the super-resolution imaging data, we extract information on FGFR density, spatial distribution, and inner-subfamily colocalization. Our approach is straightforward and easily adaptable to other multiplexed SMLM data of membrane proteins.

Decrease of core 2 O-glycans on synovial lubricin in osteoarthritis reduces galectin-3 mediated crosslinking.

The synovial fluid glycoprotein lubricin (also known as proteoglycan 4) is a mucin-type O-linked glycosylated biological lubricant implicated to be involved in osteoarthritis (OA) development. Lubricin's ability to reduce friction is related to its glycosylation consisting of sialylated and unsialylated Tn-antigens and core 1 and core 2 structures. The glycans on lubricin have also been suggested to be involved in crosslinking and stabilization of the lubricating superficial layer of cartilage by mediating interaction between lubricin and galectin-3. However, with the spectrum of glycans being found on lubricin, the glycan candidates involved in this interaction were unknown. Here, we confirm that the core 2 O-linked glycans mediate this lubricin-galectin-3 interaction, shown by surface plasmon resonance data indicating that recombinant lubricin (rhPRG4) devoid of core 2 structures did not bind to recombinant galectin-3. Conversely, transfection of Chinese hamster ovary cells with the core 2 GlcNAc transferase acting on a mucin-type O-glycoprotein displayed increased galectin-3 binding. Both the level of galectin-3 and the galectin-3 interactions with synovial lubricin were found to be decreased in late-stage OA patients, coinciding with an increase in unsialylated core 1 O-glycans (T-antigens) and Tn-antigens. These data suggest a defect in crosslinking of surface-active molecules in OA and provide novel insights into OA molecular pathology.

Competitive Binding Study Revealing the Influence of Fluorophore Labels on Biomolecular Interactions.

Fluorescence methods are important tools in modern biology. Direct labeling of biomolecules with a fluorophore might, however, change interaction surfaces. Here, we introduce a competitive binding assay in combination with fluorescence correlation spectroscopy that reports binding affinities of both labeled and unlabeled biomolecules to their binding target. We investigated how fluorophore labels at different positions of a DNA oligonucleotide affect hybridization to a complementary oligonucleotide and found dissociation constants varying within 2 orders of magnitude. We next demonstrated that placing a fluorophore label at position Leu280 in the protein ligand internalin B does not alter the binding affinity to the MET receptor tyrosine kinase, compared to unlabeled internalin B. Our approach is simple to implement and can be applied to investigate the influence of fluorophore labels in a large variety of biomolecular interactions.

Single-Molecule Super-Resolution Microscopy Reveals Heteromeric Complexes of MET and EGFR upon Ligand Activation.

Receptor tyrosine kinases (RTKs) orchestrate cell motility and differentiation. Deregulated RTKs may promote cancer and are prime targets for specific inhibitors. Increasing evidence indicates that resistance to inhibitor treatment involves receptor cross-interactions circumventing inhibition of one RTK by activating alternative signaling pathways. Here, we used single-molecule super-resolution microscopy to simultaneously visualize single MET and epidermal growth factor receptor (EGFR) clusters in two cancer cell lines, HeLa and BT-20, in fixed and living cells. We found heteromeric receptor clusters of EGFR and MET in both cell types, promoted by ligand activation. Single-protein tracking experiments in living cells revealed that both MET and EGFR respond to their cognate as well as non-cognate ligands by slower diffusion. In summary, for the first time, we present static as well as dynamic evidence of the presence of heteromeric clusters of MET and EGFR on the cell membrane that correlates with the relative surface expression levels of the two receptors.

SPT and Imaging FCS Provide Complementary Information on the Dynamics of Plasma Membrane Molecules.

The dynamics of biomolecules in the plasma membrane is of fundamental importance to understanding cellular processes. Cellular signaling often starts with extracellular ligand binding to a membrane receptor, which then transduces an intracellular signal. Ligand binding and receptor-complex activation often involve a complex rearrangement of proteins in the membrane, which results in changes in diffusion properties. Two widely used methods to characterize biomolecular diffusion are single-particle tracking (SPT) and imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS). Here, we compare the results of recovered diffusion coefficients and mean-square displacements of the two methods by simulations of free, domain-confined, or meshwork diffusion. We introduce, to our knowledge, a new method for the determination of confinement radii from ITIR-FCS data. We further establish and demonstrate simultaneous SPT/ITIR-FCS for direct comparison within living cells. Finally, we compare the results obtained by SPT and ITIR-FCS for the receptor tyrosine kinase MET. Our results show that SPT and ITIR-FCS yield complementary information on diffusion properties of biomolecules in cell membranes.

Results of mammary glands topometry among yakut women with focus on age and importance of age in augmentation mammoplasty.

OBJECTIVE: Introduction: In connection with the development of plastic and aesthetic surgery, there is a steady growth in aesthetic mammoplasty. Modern aesthetics require an emphasis on and accentuation of mammary glands. For our region it is obvious that the physical and sexual development of girls living in the harsh continental climate of Yakutia has its own characteristics. In this regard, the issues of defining a clear standard diagnosis of the regional norm of mammary gland and adjacent topographic layers of the chest wall, as well as its shape, are becoming increasingly relevant. The aim: To identify the individual typological variability of the shape, size, and topometric characteristics of mammary glands of Yakut women with focus on age. PATIENTS AND METHODS: Materials and Methods: Morphometry of the mammary glands was performed in 72 Yakut women. The examined women were divided into the following age groups: group I - IV ( ages 20 -40). The measurements were performed using the Body Logic (Mentor Medical Systems BV - USA) System, where the following indicators were recorded anthropometric characteristics of the body, topometric and organometric characteristics of mammary glands women. RESULTS: Results: The analysis of the obtained data showed that the bodyweight of the women being examined increases by ages 35-40. Dimensional parameters of the transverse diameter of the chest at the level of the submammary fold and at the level of the nipples were also greater in the older age group. Visual asymmetry MG relative to right and left sides is observed in the first age group (ages 26-35). The thickness of the dermal-glandular fold at the level of the lateral, medial and upper poles also tends to increase with age, with higher values on the left side in all groups. Submammary fold as anatomical structure is the key structure that determines the aesthetics of the mammary gland during its augmentation and mastopexy, it is the foundation on which the designs of mammoplasty are based. Its asymmetry is less noticeable in the older age group. When analyzing the size of the areola along the vertical and transverse lines, a pronounced tendency to increase in size with age is observed relative to the right and left sides. CONCLUSION: Conclusions: Thus, as a result of the study, we determined the topo-morphometric parameters of the mammary glands of Yakut women of different age groups. It was observed that with sufficient symmetry of the mammary gland shape in most women in the study groups, there is an asymmetry in the structure of the shape of the chest, probably due to rickets-like conditions widespread in our region. The growth in the thickness of the skin-glandular fold is more pronounced in the fourth age group (ages 36-40), which indicates the hypertrophy of the MG tissues, which is an important factor when calculating the volume of the future implant. The index of extensibility in the lower pole of theMG is important for planning surgical intervention, since it indicates the state of the skin pocket for the implant. It is only natural that with age, MG tissues become susceptibleto natural gravitational ptosis, the same happens to the nipple-areolar complex, as evidenced by its largest transverse and vertical dimensions in the fourth age group. Whenplanning the intervention, these dimensions can be reduced using periareolar mastopexy.

Measuring health literacy - the Swedish Functional Health Literacy scale.

BACKGROUND: The benefits of health promotion efforts vary due to a complexity of reasons. One possible reason for an absence of effects is the level of functional health literacy among the individuals that participate in the interventions. Thus, valid and reliable instruments that capture these kinds of skills are needed. The aim of this study was to develop a Swedish Functional Health Literacy scale to use in health promotion and health prevention by translating the Japanese Functional Health Literacy scale into Swedish and testing some aspects of its validity and test-retest reliability. METHODS: The research project comprised six phases including translation and back translation, validity tests of the two first versions of the instrument based on interviews with professionals and representatives for the target group of the instrument, and a test-retest of the first version among target groups. RESULTS: The items in the first two versions were experienced as unclear, which led to improvements of the next version. The final version of the translated instrument (the Swedish Functional Literacy scale) showed evidence of content validity, and the test-retest confirmed that the instrument had stability over time with a percentage agreement for the items ranging from 63% to 92% (M = 77.2%). CONCLUSION: The items in the Swedish version of the scale are equivalent to the original Japanese scale in terms of language and contents and cover the major aspects of functional health literacy as it is defined in the literature. The translated instrument shows stability over time, that is, reliability, at least for a part of the Swedish population. More validity tests of the Swedish Functional Health Literacy scale based on a broader population are needed.

The communicative and critical health literacy scale--Swedish version.

BACKGROUND: Health literacy (HL) is an important determinant for health and a valuable health indicator within public health. As such, it is a significant outcome variable of health promotion efforts. Valid and reliable instruments are needed to evaluate health promotion interventions and to assess levels of HL in a population. One of the few measurements of communicative and critical HL is the Japanese Communicative and Critical Health Literacy scale (C & C HL scale). To make it possible to use this instrument in Sweden, the C & C HL scale was translated into Swedish and different aspects of validity, including test-retest reliability, of the translated version were tested. METHODS: After translation and back-translation, The Swedish C & C HL scale was tested for content validity and test-retest reliability. Data were collected from a committee consisting of public health experts and bilingual people, and from a test group of 35 persons. RESULTS: The Swedish C & C HL scale was understandable and showed evidence of content validity. The test-retest confirmed that it was stable over time, percentage agreements for the items ranging from 66% to 89% (M = 74%). CONCLUSIONS: The Swedish C & C HL scale is equivalent to the Japanese C & C HL scale in terms of language and content. The items cover the major aspects of communicative and critical HL and are understandable and stable over time, i.e., reliable.

Comorbid physical health burden of serious mental health disorders in 32 European countries.

BACKGROUND: Mental health disorders (MHDs) are associated with physical health disparities, but underlying excess risk and health burden have not yet been comprehensively assessed. OBJECTIVE: To assess the burden of comorbid physical health conditions (PHCs) across serious MHDs in Europe. METHODS: We estimated the relative prevalence risk of PHCs associated with alcohol use disorders (AUD), bipolar disorder (BD), depressive disorders (DD) and schizophrenia (SZ) across working-age populations of 32 European countries in 2019 based on a targeted literature review. Excess physical health burden was modelled using population-attributable fractions and country-level prevalence data. FINDINGS: We screened 10 960 studies, of which 41 were deemed eligible, with a total sample size of over 18 million persons. Relative prevalence of PHCs was reported in 54%, 20%, 15%, 5% and 7% of studies, respectively, for SZ, DD, BD, AUD or mixed. Significant relative risk estimates ranged from 1.44 to 3.66 for BD, from 1.43 to 2.21 for DD, from 0.81 to 1.97 for SZ and 3.31 for AUD. Excess physical health burden ranged between 27% and 67% of the total, corresponding to 84 million (AUD), 67 million (BD), 66 million (DD) and 5 million (SZ) PHC diagnoses in Europe. A 1% reduction in excess risk assuming causal inference could result in two million fewer PHCs across investigated MHDs. CONCLUSIONS: This is the first comprehensive study of the physical health burden of serious MHDs in Europe. The methods allow for updates, refinement and extension to other MHDs or geographical areas. CLINICAL IMPLICATIONS: The results indicate potential population health benefits achievable through more integrated mental and physical healthcare and prevention approaches.

Influence of circadian rhythm on effects induced by mechanical strain in periodontal ligament cells.

PURPOSE: The aim of this study was to investigate the influence of mechanical strain on clock gene function in periodontal ligament (PDL) cells. Furthermore, we wanted to analyze whether effects induced by mechanical stress vary in relation to the circadian rhythm. METHODS: Human PDL fibroblasts were synchronized in their circadian rhythm with dexamethasone and stretched over 24 h. Unstretched cells served as controls. Gene expression of the core clock genes were analyzed at 4 h intervals by quantitative real-time polymerase chain reaction (qRT-PCR). Time points 0 h (group SI1) and 12 h (group SI2) after synchronization served as starting points of a 4 h force application period. Collagen-1α (COL-1α/Col-1α), interleukin-1ß (IL1-ß), and runt-related transcription factor 2 (RUNX2/Runx2) were assessed by qRT-PCR and enzyme-linked immunosorbent assay (ELISA) after 2 and 4 h. Statistical analysis comprised one-way analysis of variance (ANOVA) and post hoc tests. RESULTS: After synchronization, the typical pattern for clock genes was visible in control cells over the 24 h period. This pattern was significantly altered by mechanical strain. Under tensile stress, ARNTL gene expression was reduced, while Per1 and 2 gene expression were upregulated. In addition, mechanical stress had a differential effect on the expression of Col-1α and IL1­ß depending on its initiation within the circadian rhythm (group SI1 vs group SI2). For RUNX2, no significant differences in the two groups were observed. CONCLUSION: Our results suggest that mechanical stress affects the molecular peripheral oscillator of PDL cells. Vice versa, the circadian rhythm also seems to partially influence the effects that mechanical stress exerts on PDL cells.

Quantitative visually lossless compression ratio determination of JPEG2000 in digitized mammograms.

The current study presents a quantitative approach towards visually lossless compression ratio (CR) threshold determination of JPEG2000 in digitized mammograms. This is achieved by identifying quantitative image quality metrics that reflect radiologists' visual perception in distinguishing between original and wavelet-compressed mammographic regions of interest containing microcalcification clusters (MCs) and normal parenchyma, originating from 68 images from the Digital Database for Screening Mammography. Specifically, image quality of wavelet-compressed mammograms (CRs, 10:1, 25:1, 40:1, 70:1, 100:1) is evaluated quantitatively by means of eight image quality metrics of different computational principles and qualitatively by three radiologists employing a five-point rating scale. The accuracy of the objective metrics is investigated in terms of (1) their correlation (r) with qualitative assessment and (2) ROC analysis (A z index), employing pooled radiologists' rating scores as ground truth. The quantitative metrics mean square error, mean absolute error, peak signal-to-noise ratio, and structural similarity demonstrated strong correlation with pooled radiologists' ratings (r, 0.825, 0.823, -0.825, and -0.826, respectively) and the highest area under ROC curve (A z , 0.922, 0.920, 0.922, and 0.922, respectively). For each quantitative metric, the highest accuracy values of corresponding ROC curves were used to define metric cut-off values. The metrics cut-off values were subsequently used to suggest a visually lossless CR threshold, estimated to be between 25:1 and 40:1 for the dataset analyzed. Results indicate the potential of the quantitative metrics approach in predicting visually lossless CRs in case of MCs in mammography.

Targeting climate adaptation to safeguard and advance the Sustainable Development Goals.

The international community has committed to achieve 169 Sustainable Development Goal (SDG) targets by 2030 and to enhance climate adaptation under the Paris Agreement. Despite the potential for synergies, aligning SDG and climate adaptation efforts is inhibited by an inadequate understanding of the complex relationship between SDG targets and adaptation to impacts of climate change. Here we propose a framework to conceptualise how ecosystems and socio-economic sectors mediate this relationship, which provides a more nuanced understanding of the impacts of climate change on all 169 SDG targets. Global application of the framework reveals that adaptation of wetlands, rivers, cropland, construction, water, electricity, and housing in the most vulnerable countries is required to safeguard achievement of 68% of SDG targets from near-term climate risk by 2030. We discuss how our framework can help align National Adaptation Plans with SDG targets, thus ensuring that adaptation advances, rather than detracts from, sustainable development.

Truncated lubricin glycans in osteoarthritis stimulate the synoviocyte secretion of VEGFA, IL-8, and MIP-1α: Interplay between O-linked glycosylation and inflammatory cytokines.

The primary aim of the study was to identify inflammatory markers relevant for osteoarthritis (OA)-related systemic (plasma) and local (synovial fluid, SF) inflammation. From this, we looked for inflammatory markers that coincided with the increased amount of O-linked Tn antigen (GalNAcα1-Ser/Thr) glycan on SF lubricin. Inflammatory markers in plasma and SF in OA patients and controls were measured using a 44-multiplex immunoassay. We found consistently 29 markers detected in both plasma and SF. The difference in their concentration and the low correlation when comparing SF and plasma suggests an independent inflammatory environment in the two biofluids. Only plasma MCP-4 and TARC increased in our patient cohort compared to control plasma. To address the second task, we concluded that plasma markers were irrelevant for a direct connection with SF glycosylation. Hence, we correlated the SF-inflammatory marker concentrations with the level of altered glycosylation of SF-lubricin. We found that the level of SF-IL-8 and SF-MIP-1α and SF-VEGFA in OA patients displayed a positive correlation with the altered lubricin glycosylation. Furthermore, when exposing fibroblast-like synoviocytes from both controls and OA patients to glycovariants of recombinant lubricin, the secretion of IL-8 and MIP-1α and VEGFA were elevated using lubricin with Tn antigens, while lubricin with sialylated and nonsialylated T antigens had less or no measurable effect. These data suggest that truncated glycans of lubricin, as found in OA, promote synovial proinflammatory cytokine production and exacerbate local synovial inflammation.

Loss of the WNT9a ligand aggravates the rheumatoid arthritis-like symptoms in hTNF transgenic mice.

Agonists and antagonists of the canonical Wnt signaling pathway are modulators of pathological aspects of rheumatoid arthritis (RA). Their activity is primarily modifying bone loss and bone formation, as shown in animal models of RA. More recently, modulation of Wnt signaling by the antagonist Sclerostin has also been shown to influence soft-tissue-associated inflammatory aspects of the disease pointing towards a role of Wnt signaling in soft-tissue inflammation as well. Yet, nothing is known experimentally about the role of Wnt ligands in RA. Here we provide evidence that altering Wnt signaling at the level of a ligand affects all aspects of the rheumatoid arthritic disease. WNT9a levels are increased in the pannus tissue of RA patients, and stimulation of synovial fibroblasts (SFB) with tumor necrosis factor (TNF) leads to increased transcription of Wnt9a. Loss of Wnt9a in a chronic TNF-dependent RA mouse model results in an aggravation of disease progression with enhanced pannus formation and joint destruction. Yet, loss of its activity in the acute K/BxN serum-transfer induced arthritis (STIA) mouse model, which is independent of TNF signaling, has no effect on disease severity or progression. Thus, suggesting a specific role for WNT9a in TNF-triggered RA. In synovial fibroblasts, WNT9a can activate the canonical Wnt/ß-catenin pathway, but it can also activate P38- and downregulate NFκB signaling. Based on in vitro data, we propose that loss of Wnt9a creates a slight proinflammatory and procatabolic environment that boosts the TNF-mediated inflammatory response.

Only the Co-Transcriptional Activity of ß-Catenin Is Required for the Local Regulatory Effects in Hypertrophic Chondrocytes on Developmental Bone Modeling.

In hypertrophic chondrocytes, ß-catenin has two roles. First, it locally suppresses the differentiation of osteoclasts at the chondro-osseous junction by maintaining the pro-osteoclastic factor receptor activator of NF-κB ligand (RANKL) at low levels. Second, it promotes the differentiation of osteoblast-precursors from chondrocytes. Yet, ß-catenin is a dual-function protein, which can either participate in cell-cell adherens junctions or serve as a transcriptional co-activator in canonical Wnt signaling interacting with T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors. Hence, whenever studying tissue-specific requirements of ß-catenin using a conventional conditional knockout approach, the functional mechanisms underlying the defects in the conditional mutants remain ambiguous. To decipher mechanistically which of the two molecular functions of ß-catenin is required in hypertrophic chondrocytes, we used different approaches. We analyzed the long bones of newborn mice carrying either the null-alleles of Lef1 or Tcf7, or mice in which Tcf7l2 was conditionally deleted in the hypertrophic chondrocytes, as well as double mutants for Lef1 and Tcf7l2, and Tcf7 and Tcf7l2. Furthermore, we analyzed Ctnnb1 mutant newborns expressing a signaling-defective allele that retains the cell adhesion function in hypertrophic chondrocytes. None of the analyzed Tcf/Lef single or double mutants recapitulated the previously published phenotype upon loss of ß-catenin in hypertrophic chondrocytes. However, using this particular Ctnnb1 allele, maintaining cell adhesion function, we show that it is the co-transcriptional activity of ß-catenin, which is required in hypertrophic chondrocytes to suppress osteoclastogenesis and to promote chondrocyte-derived osteoblast differentiation. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

CRISPR/Cas12a-mediated labeling of MET receptor enables quantitative single-molecule imaging of endogenous protein organization and dynamics.

Single-molecule localization microscopy (SMLM) reports on protein organization in cells with near-molecular resolution and in combination with stoichiometric labeling enables protein counting. Fluorescent proteins allow stoichiometric labeling of cellular proteins; however, most methods either lead to overexpression or are complex and time demanding. We introduce CRISPR/Cas12a for simple and efficient tagging of endogenous proteins with a photoactivatable protein for quantitative SMLM and single-particle tracking. We constructed a HEK293T cell line with the receptor tyrosine kinase MET tagged with mEos4b and demonstrate full functionality. We determine the oligomeric state of MET with quantitative SMLM and find a reorganization from monomeric to dimeric MET upon ligand stimulation. In addition, we measured the mobility of single MET receptors in vivo in resting and ligand-treated cells. The combination of CRISPR/Cas12a-assisted endogenous protein labeling and super-resolution microscopy represents a powerful tool for cell biological research with molecular resolution.

GA4GH: International policies and standards for data sharing across genomic research and healthcare.

The Global Alliance for Genomics and Health (GA4GH) aims to accelerate biomedical advances by enabling the responsible sharing of clinical and genomic data through both harmonized data aggregation and federated approaches. The decreasing cost of genomic sequencing (along with other genome-wide molecular assays) and increasing evidence of its clinical utility will soon drive the generation of sequence data from tens of millions of humans, with increasing levels of diversity. In this perspective, we present the GA4GH strategies for addressing the major challenges of this data revolution. We describe the GA4GH organization, which is fueled by the development efforts of eight Work Streams and informed by the needs of 24 Driver Projects and other key stakeholders. We present the GA4GH suite of secure, interoperable technical standards and policy frameworks and review the current status of standards, their relevance to key domains of research and clinical care, and future plans of GA4GH. Broad international participation in building, adopting, and deploying GA4GH standards and frameworks will catalyze an unprecedented effort in data sharing that will be critical to advancing genomic medicine and ensuring that all populations can access its benefits.

Quantitative assessment of microcalcification cluster image quality in digital breast tomosynthesis, 2-dimensional and synthetic mammography.

Quantitative assessment of microcalcification (MC) cluster image quality is presented, in terms of cluster signal-difference-to-noise ratio (SDNR) intercomparison among digital breast tomosynthesis (DBT) and 2-dimensional (2D) and synthetic-2-dimensional (s2D) mammography. A phantom that provides realistic appearance of MC clusters located in uniform and nonuniform background was imaged in 2D and DBT, considering various scattering conditions. MC cluster SDNR differentiation is investigated with respect to MC particle size (uniform background) and surrounding parenchyma density (nonuniform background). An accurate MC cluster segmentation method was used to delineate individual MC particles and estimate MC cluster SDNR. Analysis of the uniform part of the phantom indicated higher performance of DBT and 2D over s2D for the smallest cluster size (106-177 µm), no difference among mammographic modes for the largest MC cluster (224-354 µm), and enhanced role of 2D for decreasing cluster size and increasing scattering. Analysis of the nonuniform part of the phantom indicated DBT performed better than 2D and s2D in case of dense parenchyma pattern, while 2D and s2D did not differ across parenchyma density patterns and scattering conditions. The presented MC cluster SDNR analysis was capable of revealing subtle differences among mammographic modes and suggests a methodology for clinical image quality assessment. Graphical abstract.