The Escherichia coli chromosome moves to the replisome.

In Escherichia coli, it is debated whether the two replisomes move independently along the two chromosome arms during replication or if they remain spatially confined. Here, we use high-throughput fluorescence microscopy to simultaneously determine the location and short-time-scale (1 s) movement of the replisome and a chromosomal locus throughout the cell cycle. The assay is performed for several loci. We find that (i) the two replisomes are confined to a region of ~250 nm and ~120 nm along the cell's long and short axis, respectively, (ii) the chromosomal loci move to and through this region sequentially based on their distance from the origin of replication, and (iii) when a locus is being replicated, its short time-scale movement slows down. This behavior is the same at different growth rates. In conclusion, our data supports a model with DNA moving towards spatially confined replisomes at replication.

Association between changed self-rated health and the risk of venous thromboembolism in Malmö Preventive Program: a cohort study.

Poor self-rated health (SRH) is associated with incident arterial cardiovascular disease in both sexes. Studies on the association between SRH and incident venous thromboembolism (VTE) show divergent results in women and no association in men. This study focuses on the association between change in SRH and incident VTE in a cohort of 11,558 men and 6682 women who underwent a baseline examination and assessment of SRH between 1974 and 1992 and a re-examination in 2002-2006. To investigate if changes in SRH over time affect the risk of incident VTE in men and women. During a follow-up time from the re-examination of more than 16 years, there was a lower risk for incident VTE among women if SRH changed from poor at baseline to very good/excellent (HR 0.46, 95% CI 0.28; 0.74) at the re-examination. Stable good SRH (good to very good/excellent at the re-examination, HR 0.60, 95% CI 0.42; 0.89), or change from good SRH at baseline into poor/fair at the re-examination (HR 0.68, 95% CI 0.51; 0.90) were all significantly associated with a reduced risk for VTE. All comparisons were done with the group with stable poor SRH. This pattern was not found among men. Regardless of a decreased or increased SRH during life, having an SRH of very good/excellent at any time point seems to be associated with a decreased risk of VTE among women.

Proteomic Characterization of Plasma in Lemierre's Syndrome.

BACKGROUND: The underlying mechanisms of thrombosis in Lemierre's syndrome and other septic thrombophlebitis are incompletely understood. Therefore, in this case control study we aimed to generate hypotheses on its pathogenesis by studying the plasma proteome in patients with these conditions. METHODS: All patients with Lemierre's syndrome in the Skåne Region, Sweden, were enrolled prospectively during 2017 to 2021 as cases. Age-matched patients with other severe infections were enrolled as controls. Patient plasma samples were analyzed using label-free data-independent acquisition liquid chromatography tandem mass spectrometry. Differentially expressed proteins in Lemierre's syndrome versus other severe infections were highlighted. Functions of differentially expressed proteins were defined based on a literature search focused on previous associations with thrombosis. RESULTS: Eight patients with Lemierre's syndrome and 15 with other severe infections were compared. Here, 20/449 identified proteins were differentially expressed between the groups. Of these, 14/20 had functions previously associated with thrombosis. Twelve of 14 had a suggested prothrombotic effect in Lemierre's syndrome, whereas 2/14 had a suggested antithrombotic effect. CONCLUSION: Proteins involved in several thrombogenic pathways were differentially expressed in Lemierre's syndrome compared to other severe infections. Among identified proteins, several were associated with endothelial damage, platelet activation, and degranulation, and warrant further targeted studies.

Label-free deep learning-based species classification of bacteria imaged by phase-contrast microscopy.

Reliable detection and classification of bacteria and other pathogens in the human body, animals, food, and water is crucial for improving and safeguarding public health. For instance, identifying the species and its antibiotic susceptibility is vital for effective bacterial infection treatment. Here we show that phase contrast time-lapse microscopy combined with deep learning is sufficient to classify four species of bacteria relevant to human health. The classification is performed on living bacteria and does not require fixation or staining, meaning that the bacterial species can be determined as the bacteria reproduce in a microfluidic device, enabling parallel determination of susceptibility to antibiotics. We assess the performance of convolutional neural networks and vision transformers, where the best model attained a class-average accuracy exceeding 98%. Our successful proof-of-principle results suggest that the methods should be challenged with data covering more species and clinically relevant isolates for future clinical use.

Association of Circulating Long Noncoding 7S RNA with Deep Vein Thrombosis.

Mitochondrial dysfunction is a recognized factor in the pathogenesis of deep vein thrombosis (DVT). The role of 7S RNA, a long noncoding RNA that plays an important role in mitochondrial function, in DVT remains unclear. In this study, we aimed to investigate the potential use of 7S RNA as a biomarker in DVT. Plasma samples were obtained from 237 patients (aged 16-95 years) with suspected DVT recruited in a prospective multicenter management study (SCORE) where 53 patients were objectively confirmed with a diagnosis of DVT and the rest were diagnosed as non-DVT. 7S RNA was measured using quantitative real-time polymerase chain reaction in plasma samples. The plasma expression of 7S RNA was significantly lower in DVT compared with non-DVT (0.50 vs. 0.95, p = 0.043). With the linear regression analysis, we showed that the association between the plasma expression of 7S RNA and DVT (ß = -0.72, p = 0.007) was independent of potential confounders. Receiver-operating characteristic curve analysis showed the area under the curve values of 0.60 for 7S RNA. The findings of the present study showed a notable association between 7S RNA and DVT. However, further investigations are needed to fully elucidate the exact role of 7S RNA in the pathophysiology of DVT and its diagnostic value.

Regulatory elements coordinating initiation of chromosome replication to the Escherichia coli cell cycle.

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.

Can molecular dynamics be used to simulate biomolecular recognition?

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. In particular, we study the transitions LacI undergoes upon moving between being non-specifically and specifically bound to DNA. 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.

Unreported incidental pulmonary embolism in patients with cancer: Radiologic natural history and risk of recurrent venous thromboembolism and death.

PURPOSE: To assess the risk of recurrent venous thromboembolism (VTE) and death in patients with unreported cancer-associated incidental pulmonary embolism (iPE). MATERIALS AND METHODS: Matched cohort study on cancer patients with a CT study including the chest between 2014-01-01 and 2019-06-30. Studies were reviewed for unreported iPE, and cases were matched with controls without iPE. Cases and controls were followed for one year, with recurrent VTE and death as outcome events. RESULTS: Of the included 2960 patients, 171 patients had unreported and untreated iPE. While controls had a one-year VTE risk of 8.2 events per 100 person-years, cases with a single subsegmental iPE had a recurrent VTE risk of 20.9 events, and between 52.0 and 72.0 events per 100 person-years for multiple subsegmental iPE and more proximal iPE. In multivariable analysis, multiple subsegmental and more proximal iPE were significantly associated with the risk of recurrent VTE, while single subsegmental iPE was not associated with the risk of recurrent VTE (p = 0.13). In the subgroup of patients (n = 47) with cancer not in the highest Khorana VTE risk category, no metastases and up to three involved vessels, recurrent VTE occurred in two patients (4.7 cases per 100 person-years). There were no significant associations between iPE burden and risk of death. CONCLUSION: In cancer patients with unreported iPE, iPE burden was associated with the risk of recurrent VTE. However, having a single subsegmental iPE was not associated with the risk of recurrent VTE. There were no significant associations between iPE burden and risk of death.

Real-time single-molecule 3D tracking in E. coli based on cross-entropy minimization.

Reaching sub-millisecond 3D tracking of individual molecules in living cells would enable direct measurements of diffusion-limited macromolecular interactions under physiological conditions. Here, we present a 3D tracking principle that approaches the relevant regime. The method is based on the true excitation point spread function and cross-entropy minimization for position localization of moving fluorescent reporters. Tests on beads moving on a stage reaches 67 nm lateral and 109 nm axial precision with a time resolution of 0.84 ms at a photon count rate of 60 kHz; the measurements agree with the theoretical and simulated predictions. Our implementation also features a method for microsecond 3D PSF positioning and an estimator for diffusion analysis of tracking data. Finally, we successfully apply these methods to track the Trigger Factor protein in living bacterial cells. Overall, our results show that while it is possible to reach sub-millisecond live-cell single-molecule tracking, it is still hard to resolve state transitions based on diffusivity at this time scale.

Sex-Specific Risk Factors for Deep Venous Thrombosis and Pulmonary Embolism in a Population-Based Historical Cohort Study of Middle-Aged and Older Individuals.

Background Whether sex-specific differences exist for risk factors for pulmonary embolism (PE) and deep venous thrombosis (DVT), with the exception of pregnancy and estrogen therapy, has been sparsely studied. We aimed to study whether sex-specific differences of risk factors exist for noncancer-related DVT and PE in middle-aged and older individuals without cardiovascular history or previous diagnosis in a population-based historical (retrospective) cohort. Methods and Results Potential venous thromboembolism (VTE) risk factors were registered at baseline in 15 807 women and 9996 men aged 44 to 74 years, who participated in the Malmö Diet and Cancer study (1991-1996). We excluded subjects with a previous history of VTE, cancer, a diagnosis of cardiovascular disease, or a diagnosis of cancer-associated VTE during follow-up. Patients were followed up from baseline until the first event of PE or DVT, death, or December 31, 2018. During the follow-up period, 365 (2.3%) women and 168 (1.7%) men were affected by first DVT, and 309 (2.0%) women and 154 (1.5%) men were affected by first PE. In the multivariable Cox regression models, the anthropometric obesity markers of weight, body mass index, waist and hip circumference, fat percentage, and muscle weight were in a dose-dependent way associated with DVT and PE among women but not men. In an analysis that included patients with cardiovascular disease and cancer-related VTE, the results were similar for women. For men, several obesity measures became significantly associated with PE or DVT but were weaker than in women, especially for DVT. Conclusions Anthropometric obesity measures are more important risk factors for both DVT and PE among women than men, especially for individuals without cardiovascular history or previous diagnosis or cancer-related VTE.

Antibiotic perseverance increases the risk of resistance development.

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.

Incidental pulmonary embolism in patients with cancer: prevalence, underdiagnosis and evaluation of an AI algorithm for automatic detection of pulmonary embolism.

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.

Genetic variation of the blood coagulation regulator tissue factor pathway inhibitor and venous thromboembolism among middle-aged and older adults: A population-based cohort study.

Background: Tissue factor is the main initiator of blood coagulation, and tissue factor pathway inhibitor (TFPI) is the primary inhibitor of the initiation of blood coagulation.The genetic variation of TFPI and the relation to venous thromboembolism (VTE), that is, venous thrombosis and pulmonary embolism, remains to be clarified. This exome sequencing study aimed to determine the molecular epidemiology of the TFPI gene and the relation to VTE in a large population-based cohort of middle-aged and older adults. Methods: The exomes of TFPI were analyzed for variants in 28,794 subjects without previous VTE (born 1923-1950, 60% women), who participated in the Malmö Diet and Cancer Study (1991-1996). Patients were followed until the first event of VTE, death, or 2018. Qualifying variants were defined as loss-of-function or nonbenign (PolyPhen-2) missense variants with minor allele frequency less than 0.1%. Results: No common variant was associated with VTE. Nine rare variants (two loss-of-function and seven nonbenign missense) were classified as qualifying and included in collapsing analysis. Prevalence of qualifying variants was 0.09%. Five individuals with VTE compared to 17 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 ancestry showed a hazard ratio of 2.9 (95% CI, 1.2-7.1) for rare qualifying variants. Conclusion: Rare qualifying TFPI variants were associated with VTE, suggesting that rare variants in TFPI contribute to the development of VTE. The qualifying TFPI gene variants were very rare, suggesting a constrained gene.

Conformational Change of Transcription Factors from Search to Specific Binding: A lac Repressor Case Study.

In a process known as facilitated diffusion, DNA-binding proteins find their target sites by combining three-dimensional diffusion and one-dimensional scanning of the DNA. Following the trade-off between speed and stability, agile exploration of DNA requires loose binding, whereas, at the DNA target site, the searching protein needs to establish tight interactions with the DNA. To enable both efficient search and stable binding, DNA-binding proteins and DNA often switch conformations upon recognition. Here, we study the one-dimensional diffusion and DNA binding of the dimeric lac repressor (LacI), which was reported to adopt two different conformations when binding different conformations of DNA. Using coarse-grained molecular dynamic simulations, we studied the diffusion and the sequence-specific binding of these conformations of LacI, as well as their truncated or monomeric variants, with two DNA conformations: straight and bent. The simulations were compared to experimental observables. This study supports that linear diffusion along DNA combines tight rotation-coupled groove tracking and rotation-decoupled hopping, where the protein briefly dissociates and reassociates just a few base pairs away. Tight groove tracking is crucial for target-site recognition, while hopping speeds up the overall search process. We investigated the diffusion of different LacI conformations on DNA and show how the flexibility of LacI's hinge regions ensures agility on DNA as well as faithful groove tracking. If the hinge regions instead form α-helices at the protein-DNA interface, tight groove tracking is not possible. On the contrary, the helical hinge region is essential for tight binding to bent, specific DNA, for the formation of the specific complex. Based on our study of different encounter complexes, we argue that the conformational change in LacI and DNA bending are somewhat coupled. Our findings underline the importance of two distinct protein conformations for facilitated diffusion and specific binding, respectively.

Rapid antibiotic susceptibility testing and species identification for mixed samples.

Antimicrobial resistance is an increasing problem on a global scale. Rapid antibiotic susceptibility testing (AST) is urgently needed in the clinic to enable personalized prescriptions in high-resistance environments and to limit the use of broad-spectrum drugs. Current rapid 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 sample in 2 h. In this proof-of-principle study, we demonstrate the operation with four antibiotics and mixed samples with combinations of seven species.

Association between self-rated health and venous thromboembolism in Malmö Preventive Program: A cohort study.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

Direct measurements of mRNA translation kinetics in living cells.

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.

Thrombotic risk determined by rare and common SERPINA1 variants in a population-based cohort study.

BACKGROUND: Severe alpha-1-antitrypsin deficiency (AATD), phenotype PiZZ, was associated with venous thromboembolism (VTE) in a case-control study. OBJECTIVES: 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. PATIENTS/METHODS: 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. RESULTS: 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. CONCLUSIONS: The SERPINA1 ZZ genotype and compound heterozygotes for severe AATD are rare but associated with VTE in a population-based Swedish study.

Classic Thrombophilias and Thrombotic Risk Among Middle-Aged and Older Adults: A Population-Based Cohort Study.

Background Five classic thrombophilias have been recognized: factor V Leiden (rs6025), the prothrombin G20210A variant (rs1799963), and protein C, protein S, and antithrombin deficiencies. This study aimed to determine the thrombotic risk of classic thrombophilias in a cohort of middle-aged and older adults. Methods and Results Factor V Leiden, prothrombin G20210A and protein-coding variants in the PROC (protein C), PROS1 (protein S), and SERPINC1 (antithrombin) anticoagulant genes were determined in 29 387 subjects (born 1923-1950, 60% women) who participated in the Malmö Diet and Cancer study (1991-1996). The Human Gene Mutation Database was used to define 68 disease-causing mutations. Patients were followed up from baseline until the first event of venous thromboembolism (VTE), death, or Dec 31, 2018. Carriership (n=908, 3.1%) for disease-causing mutations in the PROC, PROS1, and SERPINC1 genes was associated with incident VTE: Hazard ratio (HR) was 1.6 (95% CI, 1.3-1.9). Variants not in Human Gene Mutation Database were not linked to VTE (HR, 1.1; 95% CI, 0.8-1.5). Heterozygosity for rs6025 and rs1799963 was associated with incident VTE: HR, 1.8 (95% CI, 1.6-2.0) and HR, 1.6 (95% CI, 1.3-2.0), respectively. The HR for carrying 1 classical thrombophilia variant was 1.7 (95% CI, 1.6-1.9). HR was 3.9 (95% CI, 3.1-5.0) for carriers of ≥2 thrombophilia variants. Conclusions The 5 classic thrombophilias are associated with a dose-graded risk of VTE in middle-aged and older adults. Disease-causing variants in the PROC, PROS1, and SERPINC1 genes were more common than the rs1799963 variant but the conferred genetic risk was comparable with the rs6025 and rs1799963 variants.