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Objectives To assess the prevalence of reported and unreported incidental pulmonary embolism (iPE) in patients with cancer, and to evaluate an artificial intelligence (AI) algorithm for automatic detection of iPE. Methods Retrospective cohort study on patients with cancer with an elective CT study including the chest between 2018-07-01 and 2019-06-30. All study reports and images were reviewed to identify reported and unreported iPE and were processed by the AI algorithm. Results One thousand sixty-nine patients (1892 studies) were included. Per study, iPE was present in 75 studies (4.0%), of which 16 (21.3%) were reported. Unreported iPE had a significantly lower number of involved vessels compared to reported iPE, with a median of 2 (interquartile range, IQR, 1–4) versus 5 (IQR 3–9.75), p < 0.001. There were no significant differences in age, cancer type, or attenuation of the main pulmonary artery. The AI algorithm correctly identified 68 of 75 iPE, with 3 false positives (sensitivity 90.7%, specificity 99.8%, PPV 95.6%, NPV 99.6%). False negatives occurred in cases with 1–3 involved vessels. Of the unreported iPE, 32/59 (54.2%) were proximal to the subsegmental arteries. Conclusion In patients with cancer, the prevalence of iPE was 4.0%, of which only 21% were reported. Greater than 50% of unreported iPE were proximal to the subsegmental arteries. The AI algorithm had a very high sensitivity and specificity with only three false positives, with the potential to increase the detection rate of iPE. Key Points • In a retrospective single-center study on patients with cancer, unreported iPE were common, with the majority lying proximal to the subsegmental arteries. • The evaluated AI algorithm had a very high sensitivity and specificity, so has the potential to increase the detection rate of iPE.

Escherichia coli coordinates replication and division cycles by initiating replication at a narrow range of cell sizes. By tracking replisomes in individual cells through thousands of division cycles in wild-type and mutant strains, we were able to compare the relative importance of previously described control systems. We found that accurate triggering of initiation does not require synthesis of new DnaA. The initiation size increased only marginally as DnaA was diluted by growth after dnaA expression had been turned off. This suggests that the conversion of DnaA between its active ATP- and inactive ADP-bound states is more important for initiation size control than the total free concentration of DnaA. In addition, we found that the known ATP/ADP converters DARS and datA compensate for each other, although the removal of them makes the initiation size more sensitive to the concentration of DnaA. Only disruption of the regulatory inactivation of DnaA mechanism had a radical impact on replication initiation. This result was corroborated by the finding that termination of one round of replication correlates with the next initiation at intermediate growth rates, as would be the case if RIDA-mediated conversion from DnaA-ATP to DnaA-ADP abruptly stops at termination and DnaA-ATP starts accumulating.

Severe alpha-1-antitrypsin deficiency (AATD), phenotype PiZZ, was associated with venous thromboembolism (VTE) in a case-control study.This study aimed to determine the genetic variation in the SERPINA1 gene and a possible thrombotic risk of these variants in a population-based cohort study.The coding sequence of SERPINA1 was analyzed for the Z (rs28929474), S (rs17580), and other qualifying variants in 28,794 subjects without previous VTE (born 1923-1950, 60% women), who participated in the Malmö Diet and Cancer study (1991-1996). Individuals were followed from baseline until the first event of VTE, death, or 2018.Resequencing the coding sequence of SERPINA1 identified 84 variants in the total study population, 21 synonymous, 62 missense, and 1 loss-of-function variant. Kaplan-Meier analysis showed that homozygosity for the Z allele increased the risk of VTE whereas heterozygosity showed no effect. The S (rs17580) variant was not associated with VTE. Thirty-one rare variants were qualifying and included in collapsing analysis using the following selection criteria, loss of function, in frame deletion or non-benign (PolyPhen-2) missense variants with minor allele frequency (MAF)0.1%. Combining the rare qualifying variants with the Z variant showed that carrying two alleles (ZZ or compound heterozygotes) showed increased risk. Cox regression analysis revealed an adjusted hazard ratio of 4.5 (95% confidence interval 2.0-10.0) for combinations of the Z variant and rare qualifying variants. One other variant (rs141620200; MAF = 0.002) showed an increased risk of VTE.The SERPINA1 ZZ genotype and compound heterozygotes for severe AATD are rare but associated with VTE in a population-based Swedish study.

There are many problems in biochemistry that are difficult to study experimentally. Simulation methods are appealing due to direct availability of atomic coordinates as a function of time. However, direct molecular simulations are challenged by the size of systems and the time scales needed to describe relevant motions. In theory, enhanced sampling algorithms can help to overcome some of the limitations of molecular simulations. Here, we discuss a problem in biochemistry that offers a significant challenge for enhanced sampling methods and that could, therefore, serve as a benchmark for comparing approaches that use machine learning to find suitable collective variables. More in particular, we study the transitions LacI undergoes upon moving between being non-specifically and specifically bound to DNA. It is found that many degrees of freedom change during this transition and that the transition does not occur reversibly in simulations if only a subset of these degrees of freedom are biased. We also explain why this problem is so important to biologists and the transformative impact that a simulation of it would have on the understanding of DNA regulation.

The rise of antibiotic-resistant bacterial infections poses a global threat. Antibiotic resistance development is generally studied in batch cultures which conceals the heterogeneity in cellular responses. Using single-cell imaging, we studied the growth response of Escherichia coli to sub-inhibitory and inhibitory concentrations of nine antibiotics. We found that the heterogeneity in growth increases more than what is expected from growth rate reduction for three out of the nine antibiotics tested. For two antibiotics (rifampicin and nitrofurantoin), we found that sub-populations were able to maintain growth at lethal antibiotic concentrations for up to 10 generations. This perseverance of growth increased the population size and led to an up to 40-fold increase in the frequency of antibiotic resistance mutations in gram-negative and gram-positive species. We conclude that antibiotic perseverance is a common phenomenon that has the potential to impact antibiotic resistance development across pathogenic bacteria.

Background The protein C (PC) anticoagulant system has a key role in maintaining hemostatic balance. One missense (Ser219Gly) variant in the PC receptor (PROCR) was associated with venous thromboembolism (VTE) in genome-wide association studies. Objectives This study aimed to determine the thrombotic risk of rare and common PROCR variants in a large population-based cohort of middle-aged and older adults. Methods The exonic sequence of PROCR was analyzed for the Ser219Gly variant and other qualifying variants in 28,794 subjects (born 1923–1950, 60% women) without previous VTE, who participated in the Malmö Diet and Cancer study (1991–1996). Incidence of VTE was followed up until 2018. Qualifying variants were defined as loss-of-function or nonbenign (PolyPhen-2) missense variants with minor allele frequencies (MAFs) Results Re-sequencing identified 36 PROCR variants in the study population (26,210 non-VTE exomes and 2,584 VTE exomes), 11 synonymous, 22 missense, and three loss-of-function variants. Kaplan–Meier analysis of the known Ser219Gly variant (rs867186) showed that homozygosity for this variant increased the risk of disease, whereas heterozygosity showed no effect. Cox multivariate regression analysis revealed an adjusted hazard ratio (HR) of 1.5 (95% confidence interval [CI]: 1.1–2.0). Fifteen rare variants were classified as qualifying and were included in collapsing analysis (burden test and SKAT-O). They did not contribute to risk. However, a Arg113Cys missense variant (rs146420040; MAF = 0.004) showed an increased VTE risk (HR = 1.3; 95% CI: 1.0–1.9). Conclusion Homozygosity for the Ser219Gly variant and a previously identified functional PROCR variant (Arg113Cys) was associated with VTE. Other variants did not contribute to VTE.

Background: Current clinical decision rules to exclude deep vein thrombosis (DVT) are underused partly because of their complexity. A simplified rule that can be easily applied would be more appealing to use in clinical practice. Methods: We used individual patient data from prospective diagnostic studies of patients suspected of DVT to develop a new clinical decision rule. The primary outcome was presence of DVT either at initial testing or during follow-up. DVT was considered safely excluded if the upper 95% confidence interval (CI) of DVT prevalence was

Mapping a genetic perturbation to a change in phenotype is at the core of biological research. Advances in microscopy have transformed these studies, but they have largely been confined to examining a few strains or cell lines at a time. In parallel, there has been a revolution in creating synthetic libraries of genetically altered cells with relative ease. Here we describe methods that combine these powerful tools to perform live-cell imaging of pool-generated strain libraries for improved biological discovery.

Metelev et al. use single-molecule tracking to study kinetics of translation directly in E. coli cells, and how it is affected by translation inhibitors and rRNA mutations. Their results support widespread 70S re-initiation on mRNAs. Ribosome mediated mRNA translation is central to life. The cycle of translation, however, has been characterized mostly using reconstituted systems, with only few techniques applicable for studies in the living cell. Here we describe a live-cell ribosome-labeling method, which allows us to characterize the whole processes of finding and translating an mRNA, using single-molecule tracking techniques. We find that more than 90% of both bacterial ribosomal subunits are engaged in translation at any particular time, and that the 30S and 50S ribosomal subunits spend the same average time bound to an mRNA, revealing that 30S re-initiation on poly-cistronic mRNAs is not prevalent in E. coli. Instead, our results are best explained by substantial 70S re-initiation of translation of poly-cistronic mRNAs, which is further corroborated by experiments with translation initiation inhibitors. Finally, we find that a variety of previously described orthogonal ribosomes, with altered anti-Shine-Dalgarno sequences, show significant binding to endogenous mRNAs.

Background: A previous individual participant data (IPD) meta-analysis showed that the Wells rule and D-dimer testing cannot exclude suspected deep vein thrombosis (DVT) in cancer patients. Objectives: To explore reasons for this reduced diagnostic accuracy and to optimize the diagnostic pathway for cancer patients suspected of DVT. Patients and Methods: Using IPD from 13 studies in patients with suspected DVT, DVT prevalence and the predictive value of the Wells rule items and D-dimer testing were compared between patients with and without cancer. Next, we developed a prediction model with five variables selected from all available diagnostic predictors. Results: Among the 10 002 suspected DVT patients, there were 834 patients with cancer. The median prevalence of DVT in these patients with cancer was 37.5% (interquartile range [IQR], 30.8-45.5), whereas it was 15.1% (IQR, 11.5-16.7) in patients without cancer. Diagnostic performance of individual Wells rule items and D-dimer testing was similar across patients with and without cancer, except “immobility” and “history of DVT.” The newly developed rule showed a pooled c-statistic 0.80 (95% confidence interval [CI], 0.75-0.83) and good calibration. However, using this model, still only 4.3% (95% CI, 3.0-5.7) of the suspected patients with cancer could be identified with a predicted DVT posttest probability of

INTRODUCTION: Venous thromboembolism (VTE) is a complex disease that aggregates in families. Both acquired and genetic risk factors are important. Proper recognition and management of high-risk individuals are important. AREAS COVERED: The genetic risk factors for VTE, the clinical consequences, and future perspectives are summarized. Classical thrombophilia i.e. factor V Leiden (rs6025), the prothrombin G20210A mutation (rs1799963), deficiencies of antithrombin, protein C, and protein S and the recent findings from genome wide association studies (GWAS), transcriptome-wide association studies (TWAS), genetic risk score (GRS), VTE candidate genes, expression studies, animal studies, studies using next generation sequencing, pathway analysis, and clinical implications are discussed. EXPERT OPINION: Screening of inherited thrombophilia should be performed in special cases. Identification of strong risk variants might affect the management. The increasing number of genetic risk variants is likely to change management of VTE.

Background: To increase the clinical usefulness of the D-dimer test in diagnosis of deep vein thrombosis (DVT), two strategies have been proposed: the age-adjusted, and the clinical pre-test probability (CPTP) adjusted interpretation. However, it is not known which of these strategies is superior. Objective: To conduct an individual patient data (IPD) meta-analysis that compares the sensitivity, specificity, negative predictive value (NPV), and utility (the proportion of all patients who have a negative D-dimer test) when the two strategies are used to interpret D-dimer results. Methods: Using an established IPD database, we conducted a meta-analysis to compare the two strategies. A bivariate random effects regression model was used to estimate and compare the pooled sensitivity and specificity simultaneously. The pooled NPV and utility of the two strategies was compared using a univariate random effects model. Results: Four studies were eligible for this analysis, with a total of 2554 patients. Overall prevalence of DVT was 12% with substantial heterogeneity between studies (P value

Our ability to connect genotypic variation to biologically important phenotypes has been seriously limited by the gap between live-cell microscopy and library-scale genomic engineering. Here, we show how in situ genotyping of a library of strains after time-lapse imaging in a microfluidic device overcomes this problem. We determine how 235 different CRISPR interference knockdowns impact the coordination of the replication and division cycles of Escherichia coli by monitoring the location of replication forks throughout on average >500 cell cycles per knockdown. Subsequent in situ genotyping allows us to map each phenotype distribution to a specific genetic perturbation to determine which genes are important for cell cycle control. The single-cell time-resolved assay allows us to determine the distribution of single-cell growth rates, cell division sizes and replication initiation volumes. The technology presented in this study enables genome-scale screens of most live-cell microscopy assays.

The protein C anticoagulant system plays a key role in maintaining the hemostatic balance. Although several studies have identified thrombomodulin gene (THBD) variants among venous thromboembolism (VTE) patients, the role of THBD in relation to VTE in humans remains to be clarified.This study aimed to determine the thrombotic risk of rare and common THBD variants in a large population-based cohort of middle-aged and older adults.The exome sequence of THBD was analyzed for qualifying variants in 28,794 subjects (born 1923-1950, 60% women), who participated in the Malmö Diet and Cancer study (1991-1996). Patients were followed from baseline until the first event of VTE, death, or 2018. Qualifying variants were defined as loss-of-function or non-benign (PolyPhen-2) missense variants with minor allele frequency0.1%.The single common coding variant rs1042579 was not associated with incident VTE. Sixteen rare variants were classified as qualifying and included in collapsing analysis. Seven individuals with VTE compared to 24 individuals without VTE carried one qualifying variant. Cox multivariate regression analysis adjusted for age, sex, body mass index, systolic blood pressure, smoking and alcohol consumption, rs6025, rs1799963, and the top two eigenvectors from a principal components analysis showed a hazard ratio of 3.0 (95% confidence interval 1.4-6.3) for the rare qualifying variants. The distributions of qualifying variants in THBD showed a difference for individuals with and without incident VTE indicating a possible position effect.Rare qualifying THBD variants were associated with VTE, suggesting that rare variants in THBD contribute to development of VTE.

Venous thromboembolism (VTE) and cardiovascular disease (CVD) share some risk factors such as smoking, obesity, and dietary habits. Poor self-rated health (SRH) has been shown to be a predictor of arterial CVD and mortality for both men and women. The association between SRH and VTE has only been investigated in one previous Swedish study with a cohort that just contained women. This Swedish study did not show any significant associations between poor SRH and VTE in women.A cohort of 22,444 men and 10,902 women in the Malmö Preventive Program was followed for a period of 44 years. All participants in the baseline screening with measurements including SRH were traced in national registers. Data on VTE events were collected from national hospital registries. Cox proportional regression analysis was used to calculate the association between SRH and time to VTE.During a follow-up time of 44.31 years, a total of 2612 individuals were affected by VTE. Good SRH was associated with a lower risk for VTE in women both in the univariate model (HR = 0.75, CI = 0.65-0.85) and after adjustments for age, smoking, BMI and varicose veins (HR = 0.81, CI 0.70-0.93). SRH was not a predictor for VTE in men, neither in the unadjusted (HR = 1.05, CI 0.90-1.13) nor in the fully adjusted model (HR = 1.00, CI = 0.88-1.14).In this cohort study, SRH was associated with VTE in women but not among men. The association was significant even when adjusting for well-known risk factors such as varicose veins, BMI and smoking.

Escherichia coli coordinates replication and division cycles by initiating replication at approximately the same size per chromosome at all growth rates. By tracking replisomes in individual cells through thousands of division cycles, we have dissected the mechanism behind this precise process. We have characterized wild-type cells grown under different conditions and also many mutants related to the expression and binding states of the initiator protein DnaA. This rich data set allowed us to compare the relative importance of all previously described control systems. We found that the replication initiation size regulation is not strongly dependent on the absolute concentration of DnaA, nor does it depend on active dnaA expression. Replication initiation is also not consistently triggered by cell division or replication termination. In contrast, some of the factors that convert DnaA between its ATP- and ADP-bound states have a strong effect on initiation size. We suggest a plausible model for DnaA-ATP mediated triggering of initiation at fast growth, where regulatory inactivation of DnaA (RIDA) is the main system for monitoring the number of chromosomes during active replication.

Antimicrobial resistance is an increasing problem globally. Rapid antibiotic susceptibility testing (AST) is urgently needed in the clinic to enable personalized prescription in high-resistance environments and limit the use of broad-spectrum drugs. Previously we have described a 30 min AST method based on imaging of individual bacterial cells. However, current phenotypic AST methods do not include species identification (ID), leaving time-consuming plating or culturing as the only available option when ID is needed to make the sensitivity call. Here we describe a method to perform phenotypic AST at the single-cell level in a microfluidic chip that allows subsequent genotyping by in situ FISH. By stratifying the phenotypic AST response on the species of individual cells, it is possible to determine the susceptibility profile for each species in a mixed infection sample in 1.5 h. In this proof-of-principle study, we demonstrate the operation with four antibiotics and a mixed sample with four species.

Sequence-specific binding of proteins to DNA is essential for accessing genetic information. Here, we derive a simple equation for target-site recognition, which uncovers a previously unrecognized coupling between the macroscopic association and dissociation rates of the searching protein. Importantly, this relationship makes it possible to recover the relevant microscopic rates from experimentally determined macroscopic ones. We directly test the equation by observing the binding and unbinding of individual lac repressor (LacI) molecules during target search. We find that LacI dissociates from different target sequences with essentially identical microscopic dissociation rates. Instead, sequence specificity is determined by the efficiency with which the protein recognizes different targets, effectively reducing its risk of being retained on a non-target sequence. Our theoretical framework also accounts for the coupling between off-target binding and unbinding of the catalytically inactive Cas9 (dCas9), showing that the binding pathway can be obtained from macroscopic data.One Sentence SummaryAssociation and dissociation rates are anti-correlated for reactions that include a nonspecific probing step.

In the past decades, advances in microscopy have made it possible to study the dynamics of individual biomolecules in vitro and resolve intramolecular kinetics that would otherwise be hidden in ensemble averages. More recently, single-molecule methods have been used to image, localize and track individually labeled macromolecules in the cytoplasm of living cells, allowing investigations of intermolecular kinetics under physiologically relevant conditions. In this review, we illuminate the particular advantages of single-molecule techniques when studying kinetics in living cells and discuss solutions to specific challenges associated with these methods., Accepted for publication in Annual Review of Biochemistry

Background Lemierre’s syndrome is typically caused by Fusobacterium necrophorum where an oropharyngeal infection is followed by septic internal jugular vein thrombophlebitis with subsequent septic embolization. Yet, the pathogenesis of septic thrombophlebitis, differences dependent on the presence of jugular vein thrombosis, and the role of anticoagulant therapy are insufficiently understood. Methods Patients with invasive infection with F. necrophorum and Lemierre’s syndrome who had been investigated for jugular vein thrombosis were included from a previous population-based observational study in Sweden. Medical records were reviewed and compared in patients with and without jugular vein thrombosis. Then, patients with jugular vein thrombosis were compared by exposure to therapeutic, prophylactic, or no anticoagulation. Outcomes examined were thrombosis progression, early or late peripheral septic complications, chronic major sequelae, 30-day mortality, and major bleeding. Results Fifty-one of 82 (62%) radiologically investigated patients with Lemierre’s syndrome had jugular vein thrombosis. Patients with jugular vein thrombosis had lower platelet levels (median, 76 vs 112 ×109/L; P = .04) on presentation and more days to defervesence (12 vs 7 days; P = .03) yet similar rates of major sequelae and 30-day mortality. No significant differences in outcomes were seen between patients with jugular vein thrombosis exposed to therapeutic, prophylactic, or no anticoagulation therapy, yet study outcomes were rare. Conclusions Patients with Lemierre’s syndrome with jugular vein thrombosis were more severely affected, yet had similar prognosis. Most patients with jugular vein thrombosis recovered well without therapeutic anticoagulation therapy, though adverse events were similarly rare in anticoagulated patients. The observational design and rarity of study outcomes require cautious interpretation.

Many bacterial species and antibiotic classes exhibit heteroresistance, a phenomenon in which a susceptible bacterial isolate harbors a resistant subpopulation that can grow in the presence of an antibiotic and cause treatment failure. The resistant phenotype is often unstable and without antibiotic selection it reverts back to susceptibility. Here we studied the dynamics by which these resistant subpopulations are enriched in the presence of antibiotic and recede back to their baseline frequency in the absence of selection. An increasing understanding of this instability will allow more effective diagnostics and treatment of infections caused by heteroresistant bacteria. We show for clinical isolates of Escherichia coli and Salmonella enterica that different antibiotics at levels below the MIC of the susceptible main population can cause rapid enrichment of resistant subpopulations with increased copy number of genes that cause resistance. Modelling and growth rate measurements of bacteria with increased gene copy number in cultures and by microscopy of single-cells in a microfluidic chip show that the fitness cost of gene amplifications and their intrinsic instability drives their rapid loss in the absence of selection. Using a common antibiotic susceptibility test, we demonstrate that this test strongly underestimates the occurrence of heteroresistance in clinical isolates., Bacterial populations can show heteroresistance, where an antibiotic resistant subpopulation is part of a susceptible one. Pereira et al. show in Escherichia coli and Salmonella enterica that disk diffusion, a common antibiotic susceptibility test, underestimates the occurrence of heteroresistance in clinical isolates.

Although ubiquitously thought of as a simple string of letters, DNA exhibits complex physicochemical properties. As a result, DNA can store information beyond the extensively studied explicit genetic message. The mechanical code of DNA has not been studied systematically in a genome-wide context until recent groundbreaking work by Basu et al.

Homologous recombination (HR) is essential for the accurate repair of double-stranded DNA breaks (DSBs); it begins when the RecBCD2 complex resects the ends of the DSB into 3' single-stranded DNA (ssDNA) on which a RecA filament assembles. HR depends on the ability of this RecA-ssDNA filament to locate the homologous repair template on the sister chromosome. The mechanism by which the homology is located among vast amounts of heterologous DNA is not yet understood, despite a long history of research. Here, we directly visualize the repair of DSBs in hundreds of individual cells, using high-throughput microfluidics and fluorescence microscopy. We find that in E. coli, DSB repair is completed in 15 minutes without fitness loss. We further show that the search takes less than 10 minutes and is mediated by a thin, highly dynamic RecA filament that stretches throughout the cell. We propose a model in which the architecture of the RecA filament effectively reduces search dimensionality to two dimensions. The model is corroborated by the observation that the search time does not depend on the length of the cell or the amount of DNA, and also predicts a search time that is consistent with our measurement. Since the RecA family proteins are conserved in all organisms, our results also translate to other systems that rely on homologous recombination.