Saturation genome editing of 11 codons and exon 13 of BRCA2 coupled with chemotherapeutic drug response accurately determines pathogenicity of variants.

The unknown pathogenicity of a significant number of variants found in cancer-related genes is attributed to limited epidemiological data, resulting in their classification as variant of uncertain significance (VUS). To date, Breast Cancer gene-2 (BRCA2) has the highest number of VUSs, which has necessitated the development of several robust functional assays to determine their functional significance. Here we report the use of a humanized-mouse embryonic stem cell (mESC) line expressing a single copy of the human BRCA2 for a CRISPR-Cas9-based high-throughput functional assay. As a proof-of-principle, we have saturated 11 codons encoded by BRCA2 exons 3, 18, 19 and all possible single-nucleotide variants in exon 13 and multiplexed these variants for their functional categorization. Specifically, we used a pool of 180-mer single-stranded donor DNA to generate all possible combination of variants. Using a high throughput sequencing-based approach, we show a significant drop in the frequency of non-functional variants, whereas functional variants are enriched in the pool of the cells. We further demonstrate the response of these variants to the DNA-damaging agents, cisplatin and olaparib, allowing us to use cellular survival and drug response as parameters for variant classification. Using this approach, we have categorized 599 BRCA2 variants including 93-single nucleotide variants (SNVs) across the 11 codons, of which 28 are reported in ClinVar. We also functionally categorized 252 SNVs from exon 13 into 188 functional and 60 non-functional variants, demonstrating that saturation genome editing (SGE) coupled with drug sensitivity assays can enhance functional annotation of BRCA2 VUS.

Injury Length and Arteriole Constriction Shape Clot Growth and Blood-Flow Acceleration in a Mouse Model of Thrombosis.

OBJECTIVE: Quantitative relationships between the extent of injury and thrombus formation in vivo are not well understood. Moreover, it has not been investigated how increased injury severity translates to blood-flow modulation. Here, we investigated interconnections between injury length, clot growth, and blood flow in a mouse model of laser-induced thrombosis. Approach and Results: Using intravital microscopy, we analyzed 59 clotting events collected from the cremaster arteriole of 14 adult mice. We regarded injury length as a measure of injury severity. The injury caused transient constriction upstream and downstream of the injury site resulting in a 50% reduction in arteriole diameter. The amount of platelet accumulation and fibrin formation did not depend on arteriole diameter or deformation but displayed an exponentially increasing dependence on injury length. The height of the platelet clot depended linearly on injury length and the arteriole diameter. Upstream arteriolar constriction correlated with delayed upstream velocity increase, which, in turn, determined downstream velocity. Before clot formation, flow velocity positively correlated with the arteriole diameter. After the onset of thrombus growth, flow velocity at the injury site negatively correlated with the arteriole diameter and with the size of the above-clot lumen. CONCLUSIONS: Injury severity increased platelet accumulation and fibrin formation in a persistently steep fashion and, together with arteriole diameter, defined clot height. Arterial constriction and clot formation were characterized by a dynamic change in the blood flow, associated with increased flow velocity.

Controlled Multifactorial Coagulopathy: Effects of Dilution, Hypothermia, and Acidosis on Thrombin Generation In Vitro.

BACKGROUND: Coagulopathy and hemostatic abnormalities remain a challenge in patients following trauma and major surgery. Coagulopathy in this setting has a multifactorial nature due to tissue injury, hemodilution, hypothermia, and acidosis, the severity of which may vary. In this study, we combined computational kinetic modeling and in vitro experimentation to investigate the effects of multifactorial coagulopathy on thrombin, the central enzyme in the coagulation system. METHODS: We measured thrombin generation in platelet-poor plasma from 10 healthy volunteers using the calibrated automated thrombogram assay (CAT). We considered 3 temperature levels (31°C, 34°C, and 37°C), 3 pH levels (6.9, 7.1, and 7.4), and 3 degrees of dilution with normal saline (no dilution, 3-fold dilution, and 5-fold dilution). We measured thrombin-generation time courses for all possible combinations of these conditions. For each combination, we analyzed 2 scenarios: without and with (15 nM) supplementation of thrombomodulin, a key natural regulator of thrombin generation. For each measured thrombin time course, we recorded 5 quantitative parameters and analyzed them using multivariable regression. Moreover, for multiple combinations of coagulopathic conditions, we performed routine coagulation tests: prothrombin time (PT) and activated partial thromboplastin time (aPTT). We compared the experimental results with simulations using a newly developed version of our computational kinetic model of blood coagulation. RESULTS: Regression analysis allowed us to identify trends in our data (P < 10). In both model simulations and experiments, dilution progressively reduced the peak of thrombin generation. However, we did not experimentally detect the model-predicted delay in the onset of thrombin generation. In accord with the model predictions, hypothermia delayed the onset of thrombin generation; it also increased the thrombin peak time (up to 1.30-fold). Moreover, as predicted by the kinetic model, the experiments showed that hypothermia increased the area under the thrombin curve (up to 1.97-fold); it also increased the height of the thrombin peak (up to 1.48-fold). Progressive acidosis reduced the velocity index by up to 24%; acidosis-induced changes in other thrombin generation parameters were much smaller or none. Acidosis increased PT by 14% but did not influence aPTT. In contrast, dilution markedly prolonged both PT and aPTT. In our experiments, thrombomodulin affected thrombin-generation parameters mainly in undiluted plasma. CONCLUSIONS: Dilution with normal saline reduced the amount of generated thrombin, whereas hypothermia increased it and delayed the time of thrombin accumulation. In contrast, acidosis in vitro had little effect on thrombin generation.

Predictive Analysis of Mechanistic Triggers and Mitigation Strategies for Pathological Scarring in Skin Wounds.

Wound fibrosis (i.e., excessive scar formation) is a medical problem of increasing prevalence, with poorly understood mechanistic triggers and limited therapeutic options. In this study, we employed an integrated approach that combines computational predictions with new experimental studies in mice to identify plausible mechanistic triggers of pathological scarring in skin wounds. We developed a computational model that predicts the time courses for six essential cell types, 18 essential molecular mediators, and collagen, which are involved in inflammation and proliferation during wound healing. By performing global sensitivity analyses using thousands of model-simulated wound-healing scenarios, we identified five key processes (among the 90 modeled processes) whose dysregulation may lead to pathological scarring in wounds. By modulating a subset of these key processes, we simulated fibrosis in wounds. Moreover, among the 18 modeled molecular mediators, we identified TGF-ß and the matrix metalloproteinases as therapeutic targets whose modulation may reduce fibrosis. The model predicted that simultaneous modulation of TGF-ß and matrix metalloproteinases would be more effective in treating excessive scarring than modulation of either therapeutic target alone. Our model was validated with previously published and newly generated experimental data, and suggested new in vivo experiments.

Impact of Tissue Factor Localization on Blood Clot Structure and Resistance under Venous Shear.

The structure and growth of a blood clot depend on the localization of tissue factor (TF), which can trigger clotting during the hemostatic process or promote thrombosis when exposed to blood under pathological conditions. We sought to understand how the growth, structure, and mechanical properties of clots under flow are shaped by the simultaneously varying TF surface density and its exposure area. We used an eight-channel microfluidic device equipped with a 20- or 100-µm-long collagen surface patterned with lipidated TF of surface densities ∼0.1 and ∼2 molecules/µm2. Human whole blood was perfused at venous shear, and clot growth was continually measured. Using our recently developed computational model of clot formation, we performed simulations to gain insights into the clot's structure and its resistance to blood flow. An increase in TF exposure area resulted not only in accelerated bulk platelet, thrombin, and fibrin accumulation, but also in increased height of the platelet mass and increased clot resistance to flow. Moreover, increasing the TF surface density or exposure area enhanced platelet deposition by approximately twofold, and thrombin and fibrin generation by greater than threefold, thereby increasing both clot size and its viscous resistance. Finally, TF effects on blood flow occlusion were more pronounced for the longer thrombogenic surface than for the shorter one. Our results suggest that TF surface density and its exposure area can independently enhance both the clot's occlusivity and its resistance to blood flow. These findings provide, to our knowledge, new insights into how TF affects thrombus growth in time and space under flow.

Computational analysis identifies putative prognostic biomarkers of pathological scarring in skin wounds.

BACKGROUND: Pathological scarring in wounds is a prevalent clinical outcome with limited prognostic options. The objective of this study was to investigate whether cellular signaling proteins could be used as prognostic biomarkers of pathological scarring in traumatic skin wounds. METHODS: We used our previously developed and validated computational model of injury-initiated wound healing to simulate the time courses for platelets, 6 cell types, and 21 proteins involved in the inflammatory and proliferative phases of wound healing. Next, we analysed thousands of simulated wound-healing scenarios to identify those that resulted in pathological (i.e., excessive) scarring. Then, we identified candidate proteins that were elevated (or decreased) at the early stages of wound healing in those simulations and could therefore serve as predictive biomarkers of pathological scarring outcomes. Finally, we performed logistic regression analysis and calculated the area under the receiver operating characteristic curve to quantitatively assess the predictive accuracy of the model-identified putative biomarkers. RESULTS: We identified three proteins (interleukin-10, tissue inhibitor of matrix metalloproteinase-1, and fibronectin) whose levels were elevated in pathological scars as early as 2 weeks post-wounding and could predict a pathological scarring outcome occurring 40 days after wounding with 80% accuracy. CONCLUSION: Our method for predicting putative prognostic wound-outcome biomarkers may serve as an effective means to guide the identification of proteins predictive of pathological scarring.

Wound healing in Mac-1 deficient mice.

Mac-1 (CD11b/CD18) is a macrophage receptor that plays several critical roles in macrophage recruitment and activation. Because macrophages are essential for proper wound healing, the impact of Mac-1 deficiency on wound healing is of significant interest. Prior studies have shown that Mac-1-/- mice exhibit deficits in healing, including delayed wound closure in scalp and ear wounds. This study examined whether Mac-1 deficiency influences wound healing in small excisional and incisional skin wounds. Three millimeter diameter full thickness excisional wounds and incisional wounds were prepared on the dorsal skin of Mac-1 deficient (Mac-1-/- ) and wild type (WT) mice, and wound healing outcomes were examined. Mac-1 deficient mice exhibited a normal rate of wound closure, generally normal levels of total collagen, and nearly normal synthesis and distribution of collagens I and III. In incisional wounds, wound breaking strength was similar for Mac-1-/- and WT mice. Wounds of Mac-1 deficient mice displayed normal total macrophage content, although macrophage phenotype markers were skewed as compared to WT. Interestingly, amounts of TGF-ß1 and its downstream signaling molecules, SMAD2 and SMAD3, were significantly decreased in the wounds of Mac-1 deficient mice compared to WT. The results suggest that Mac-1 deficiency has little impact on the healing of small excisional and incisional wounds. Moreover, the findings demonstrate that the effect of single genetic deficiencies on wound healing may markedly differ among wound models. These conclusions have implications for the interpretation of the many prior studies that utilize a single model system to examine wound healing outcomes in genetically deficient mice.

Computational Study of Thrombus Formation and Clotting Factor Effects under Venous Flow Conditions.

A comprehensive understanding of thrombus formation as a physicochemical process that has evolved to protect the integrity of the human vasculature is critical to our ability to predict and control pathological states caused by a malfunctioning blood coagulation system. Despite numerous investigations, the spatial and temporal details of thrombus growth as a multicomponent process are not fully understood. Here, we used computational modeling to investigate the temporal changes in the spatial distributions of the key enzymatic (i.e., thrombin) and structural (i.e., platelets and fibrin) components within a growing thrombus. Moreover, we investigated the interplay between clot structure and its mechanical properties, such as hydraulic resistance to flow. Our model relied on the coupling of computational fluid dynamics and biochemical kinetics, and was validated using flow-chamber data from a previous experimental study. The model allowed us to identify the distinct patterns characterizing the spatial distributions of thrombin, platelets, and fibrin accumulating within a thrombus. Our modeling results suggested that under the simulated conditions, thrombin kinetics was determined predominantly by prothrombinase. Furthermore, our simulations showed that thrombus resistance imparted by fibrin was ∼30-fold higher than that imparted by platelets. Yet, thrombus-mediated bloodflow occlusion was driven primarily by the platelet deposition process, because the height of the platelet accumulation domain was approximately twice that of the fibrin accumulation domain. Fibrinogen supplementation in normal blood resulted in a nonlinear increase in thrombus resistance, and for a supplemented fibrinogen level of 48%, the thrombus resistance increased by ∼2.7-fold. Finally, our model predicted that restoring the normal levels of clotting factors II, IX, and X while simultaneously restoring fibrinogen (to 88% of its normal level) in diluted blood can restore fibrin generation to ∼78% of its normal level and hence improve clot formation under dilution.

Computational Identification of Mechanistic Factors That Determine the Timing and Intensity of the Inflammatory Response.

Timely resolution of inflammation is critical for the restoration of homeostasis in injured or infected tissue. Chronic inflammation is often characterized by a persistent increase in the concentrations of inflammatory cells and molecular mediators, whose distinct amount and timing characteristics offer an opportunity to identify effective therapeutic regulatory targets. Here, we used our recently developed computational model of local inflammation to identify potential targets for molecular interventions and to investigate the effects of individual and combined inhibition of such targets. This was accomplished via the development and application of computational strategies involving the simulation and analysis of thousands of inflammatory scenarios. We found that modulation of macrophage influx and efflux is an effective potential strategy to regulate the amount of inflammatory cells and molecular mediators in both normal and chronic inflammatory scenarios. We identified three molecular mediators - tumor necrosis factor-α (TNF-α), transforming growth factor-ß (TGF-ß), and the chemokine CXCL8 - as potential molecular targets whose individual or combined inhibition may robustly regulate both the amount and timing properties of the kinetic trajectories for neutrophils and macrophages in chronic inflammation. Modulation of macrophage flux, as well as of the abundance of TNF-α, TGF-ß, and CXCL8, may improve the resolution of chronic inflammation.

Computational approach to characterize causative factors and molecular indicators of chronic wound inflammation.

Chronic inflammation is rapidly becoming recognized as a key contributor to numerous pathologies. Despite detailed investigations, understanding of the molecular mechanisms regulating inflammation is incomplete. Knowledge of such critical regulatory processes and informative indicators of chronic inflammation is necessary for efficacious therapeutic interventions and diagnostic support to clinicians. We used a computational modeling approach to elucidate the critical factors responsible for chronic inflammation and to identify robust molecular indicators of chronic inflammatory conditions. Our kinetic model successfully captured experimentally observed cell and cytokine dynamics for both acute and chronic inflammatory responses. Using sensitivity analysis, we identified macrophage influx and efflux rate modulation as the strongest inducing factor of chronic inflammation for a wide range of scenarios. Moreover, our model predicted that, among all major inflammatory mediators, IL-6, TGF-ß, and PDGF may generally be considered the most sensitive and robust indicators of chronic inflammation, which is supported by existing, but limited, experimental evidence.

A Step Toward Balance: Thrombin Generation Improvement via Procoagulant Factor and Antithrombin Supplementation.

BACKGROUND: The use of prothrombin complex concentrates in trauma- and surgery-induced coagulopathy is complicated by the possibility of thromboembolic events. To explore the effects of these agents on thrombin generation (TG), we investigated combinations of coagulation factors equivalent to 3- and 4-factor prothrombin complex concentrates with and without added antithrombin (AT), as well as recombinant factor VIIa (rFVIIa), in a dilutional model. These data were then used to develop a computational model to test whether such a model could predict the TG profiles of these agents used to treat dilutional coagulopathy. METHODS: We measured TG in plasma collected from 10 healthy volunteers using Calibrated Automated Thrombogram. TG measurements were performed in undiluted plasma, 3-fold saline-diluted plasma, and diluted plasma supplemented with the following factors: rFVIIa (group rFVIIa); factors (F)II, FIX, FX, and AT (group "combination of coagulation factors" [CCF]-AT); or FII, FVII, FIX, and FX (group CCF-FVII). We extended an existing computational model of TG to include additional reactions that impact the Calibrated Automated Thrombogram readout. We developed and applied a computational strategy to train the model using only a subset of the obtained TG data and used the remaining data for model validation. RESULTS: rFVIIa decreased lag time and the time to thrombin peak generation beyond their predilution levels (P < 0.001) but did not restore normal thrombin peak height (P < 0.001). CCF-FVII supplementation decreased lag time (P = 0.034) and thrombin peak time (P < 0.001) and increased both peak height (P < 0.001) and endogenous thrombin potential (P = 0.055) beyond their predilution levels. CCF-AT supplementation in diluted plasma resulted in an improvement in TG without causing the exaggerated effects of rFVIIa and CCF-FVII supplementation. The differences between the effects of CCF-AT and supplementation with rFVIIa and CCF-FVII were significant for lag time (P < 0.001 and P = 0.005, respectively), time to thrombin peak (P < 0.001 and P = 0.004, respectively), velocity index (P < 0.001 and P = 0.019, respectively), thrombin peak height (P < 0.001 for both comparisons), and endogenous thrombin potential (P = 0.034 and P = 0.019, respectively). The computational model generated subject-specific predictions and identified typical patterns of TG improvement. CONCLUSIONS: In this study of the effects of hemodilution, CCF-AT supplementation improved the dilution-impaired plasma TG potential in a more balanced way than either rFVIIa alone or CCF-FVII supplementation. Predictive computational modeling can guide plasma dilution/supplementation experiments.

Mechanistic Modeling of the Effects of Acidosis on Thrombin Generation.

BACKGROUND: Acidosis, a frequent complication of trauma and complex surgery, results from tissue hypoperfusion and IV resuscitation with acidic fluids. While acidosis is known to inhibit the function of distinct enzymatic reactions, its cumulative effect on the blood coagulation system is not fully understood. Here, we use computational modeling to test the hypothesis that acidosis delays and reduces the amount of thrombin generation in human blood plasma. Moreover, we investigate the sensitivity of different thrombin generation parameters to acidosis, both at the individual and population level. METHODS: We used a kinetic model to simulate and analyze the generation of thrombin and thrombin-antithrombin complexes (TAT), which were the end points of this study. Large groups of temporal thrombin and TAT trajectories were simulated and used to calculate quantitative parameters, such as clotting time (CT), thrombin peak time, maximum slope of the thrombin curve, thrombin peak height, area under the thrombin trajectory (AUC), and prothrombin time. The resulting samples of parameter values at different pH levels were compared to assess the acidosis-induced effects. To investigate intersubject variability, we parameterized the computational model using the data on clotting factor composition for 472 subjects from the Leiden Thrombophilia Study. To compare acidosis-induced relative parameter changes in individual ("virtual") subjects, we estimated the probabilities of relative change patterns by counting the pattern occurrences in our virtual subjects. Distribution overlaps for thrombin generation parameters at distinct pH levels were quantified using the Bhattacharyya coefficient. RESULTS: Acidosis in the range of pH 6.9 to 7.3 progressively increased CT, thrombin peak time, AUC, and prothrombin time, while decreasing maximum slope of the thrombin curve and thrombin peak height (P < 10). Acidosis delayed the onset and decreased the amount of TAT generation (P < 10). As a measure of intrasubject variability, maximum slope of the thrombin curve and CT displayed the largest and second-largest acidosis-induced relative changes, and AUC displayed the smallest relative changes among all thrombin generation parameters in our virtual subject group (1-sided 95% lower confidence limit on the fraction of subjects displaying the patterns, 0.99). As a measure of intersubject variability, the overlaps between the maximum slope of the thrombin curve distributions at acidotic pH levels with the maximum slope of the thrombin curve distribution at physiological pH level systematically exceeded analogous distribution overlaps for CT, thrombin peak time, and prothrombin time. CONCLUSIONS: Acidosis affected all quantitative parameters of thrombin and TAT generation. While maximum slope of the thrombin curve showed the highest sensitivity to acidosis at the individual-subject level, it may be outperformed by CT, thrombin peak time, and prothrombin time as an indicator of acidosis at the subject-group level.

Mismatch repair protein MLH1 suppresses replicative stress in BRCA2-deficient breast tumors.

Loss of BRCA2 (breast cancer 2) is lethal for normal cells. Yet it remains poorly understood how, in BRCA2 mutation carriers, cells undergoing loss of heterozygosity overcome the lethality and undergo tissue-specific neoplastic transformation. Here, we identified mismatch repair gene mutL homolog 1 (MLH1) as a genetic interactor of BRCA2 whose overexpression supports the viability of Brca2-null cells. Mechanistically, we showed that MLH1 interacts with Flap endonuclease 1 (FEN1) and competes to process the RNA flaps of Okazaki fragments. Together, they restrained the DNA2 nuclease activity on the reversed forks of lagging strands, leading to replication fork (RF) stability in BRCA2-deficient cells. In these cells, MLH1 also attenuated R-loops, allowing the progression of stable RFs, which suppressed genomic instability and supported cell viability. We demonstrated the significance of their genetic interaction by the lethality of Brca2-mutant mice and inhibition of Brca2-deficient tumor growth in mice by Mlh1 loss. Furthermore, we described estrogen as inducing MLH1 expression through estrogen receptor α (ERα), which might explain why the majority of BRCA2 mutation carriers develop ER-positive breast cancer. Taken together, our findings reveal a role of MLH1 in relieving replicative stress and show how it may contribute to the establishment of BRCA2-deficient breast tumors.

Computational analysis of the effects of reduced temperature on thrombin generation: the contributions of hypothermia to coagulopathy.

BACKGROUND: Hypothermia, which can result from tissue hypoperfusion, body exposure, and transfusion of cold resuscitation fluids, is a major factor contributing to coagulopathy of trauma and surgery. Despite considerable efforts, the mechanisms of hypothermia-induced blood coagulation impairment have not been fully understood. We introduce a kinetic modeling approach to investigate the effects of hypothermia on thrombin generation. METHODS: We extended a validated computational model to predict and analyze the impact of low temperatures (with or without concomitant blood dilution) on thrombin generation and its quantitative parameters. The computational model reflects the existing knowledge about the mechanistic details of thrombin generation biochemistry. We performed the analysis for an "average" subject, as well as for 472 subjects in the control group of the Leiden Thrombophilia Study. RESULTS: We computed and analyzed thousands of kinetic curves characterizing the generation of thrombin and the formation of the thrombin-antithrombin complex (TAT). In all simulations, hypothermia in the temperature interval 31°C to 36°C progressively slowed down thrombin generation, as reflected by clotting time, thrombin peak time, and prothrombin time, which increased in all subjects (P < 10(-5)). Maximum slope of the thrombin curve was progressively decreased, and the area under the thrombin curve was increased in hypothermia (P < 10(-5)); thrombin peak height remained practically unaffected. TAT formation was noticeably delayed (P < 10(-5)), but the final TAT levels were not significantly affected. Hypothermia-induced fold changes in the affected thrombin generation parameters were larger for lower temperatures, but were practically independent of the parameter itself and of the subjects' clotting factor composition, despite substantial variability in the subject group. Hypothermia and blood dilution acted additively on the thrombin generation parameters. CONCLUSIONS: We developed a general computational strategy that can be used to simulate the effects of changing temperature on the kinetics of biochemical systems and applied this strategy to analyze the effects of hypothermia on thrombin generation. We found that thrombin generation can be noticeably impaired in subjects with different blood plasma composition even in moderate hypothermia. Our work provides mechanistic support to the notion that thrombin generation impairment may be a key factor in coagulopathy induced by hypothermia and complicated by blood plasma dilution.

PKM2 dictates the poised chromatin state of PFKFB3 promoter to enhance breast cancer progression.

The hypoxic milieu is a critical modulator of aerobic glycolysis, yet the regulatory mechanisms between the key glycolytic enzymes in hypoxic cancer cells are largely unchartered. In particular, the M2 isoform of pyruvate kinase (PKM2), the rate-limiting enzyme of glycolysis, is known to confer adaptive advantages under hypoxia. Herein, we report that non-canonical PKM2 mediates HIF-1α and p300 enrichment at PFKFB3 hypoxia-responsive elements (HREs), causing its upregulation. Consequently, the absence of PKM2 activates an opportunistic occupancy of HIF-2α, along with acquisition of a poised state by PFKFB3 HREs-associated chromatin. This poised nature restricts HIF-2α from inducing PFKFB3 while permitting the maintenance of its basal-level expression by harboring multiple histone modifications. In addition, the clinical relevance of the study has been investigated by demonstrating that Shikonin blocks the nuclear translocation of PKM2 to suppress PFKFB3 expression. Furthermore, TNBC patient-derived organoids and MCF7 cells-derived xenograft tumors in mice exhibited substantial growth inhibition upon shikonin treatment, highlighting the vitality of targeting PKM2. Conclusively, this work provides novel insights into the contributions of PKM2 in modulating hypoxic transcriptome and a previously unreported poised epigenetic strategy exhibited by the hypoxic breast cancer cells for ensuring the maintenance of PFKFB3 expression.

Sequencing-based functional assays for classification of BRCA2 variants in mouse ESCs.

Sequencing of genes, such as BRCA1 and BRCA2, is recommended for individuals with a personal or family history of early onset and/or bilateral breast and/or ovarian cancer or a history of male breast cancer. Such sequencing efforts have resulted in the identification of more than 17,000 BRCA2 variants. The functional significance of most variants remains unknown; consequently, they are called variants of uncertain clinical significance (VUSs). We have previously developed mouse embryonic stem cell (mESC)-based assays for functional classification of BRCA2 variants. We now developed a next-generation sequencing (NGS)-based approach for functional evaluation of BRCA2 variants using pools of mESCs expressing 10-25 BRCA2 variants from a given exon. We use this approach for functional evaluation of 223 variants listed in ClinVar. Our functional classification of BRCA2 variants is concordant with the classification reported in ClinVar or those reported by other orthogonal assays.

Computational analysis of intersubject variability and thrombin generation in dilutional coagulopathy.

BACKGROUND: Blood dilution is a frequent complication of massive transfusion during trauma and surgery. This article investigates the quantitative effects of blood plasma dilution on thrombin generation in the context of intersubject variability. STUDY DESIGN AND METHODS: A thoroughly validated computational model was used to simulate thrombin generation curves for 472 healthy subjects in the Leiden Thrombophilia Study. Individual thrombin curves were calculated for undiluted blood and for different dilution scenarios. For every such curve, five standard quantitative parameters of thrombin generation were calculated and analyzed. RESULTS: Thrombin generation parameters in diluted blood plasma displayed significant intersubject variability (with a coefficient of variation up to approx. 28%). Nevertheless, dilutional effects in the majority (or all) of the subjects in the study group were characterized by persistent patterns. In particular, the largest dilution-induced change typically occurred in the maximum slope (MS) of the thrombin curve, followed by a change in thrombin peak height (PH), whereas the smallest change often occurred in the area under the curve. The identified patterns demonstrated considerable robustness to variations in dilution scenario and tissue factor concentration. CONCLUSION: Dilutional effects on thrombin generation in a human population can be predicted from trends identified for the "average" subject and then refined by performing an analysis of actual subjects in the study group. The MS and PH are dilution indicators that are both sensitive and reliable across a large subject group and could potentially be used as disease markers in the diagnosis of coagulopathic conditions.

Evolution and dynamics of regulatory architectures controlling polymyxin B resistance in enteric bacteria.

Complex genetic networks consist of structural modules that determine the levels and timing of a cellular response. While the functional properties of the regulatory architectures that make up these modules have been extensively studied, the evolutionary history of regulatory architectures has remained largely unexplored. Here, we investigate the transition between direct and indirect regulatory pathways governing inducible resistance to the antibiotic polymyxin B in enteric bacteria. We identify a novel regulatory architecture -- designated feedforward connector loop -- that relies on a regulatory protein that connects signal transduction systems post-translationally, allowing one system to respond to a signal activating another system. The feedforward connector loop is characterized by rapid activation, slow deactivation, and elevated mRNA expression levels in comparison with the direct regulation circuit. Our results suggest that, both functionally and evolutionarily, the feedforward connector loop is the transitional stage between direct transcriptional control and indirect regulation.