An all-inclusive model for predicting invasive bacterial infection in febrile infants age 7-60 days.

BACKGROUND: Invasive bacterial infections (IBIs) in febrile infants are rare but potentially devastating. We aimed to derive and validate a predictive model for IBI among febrile infants age 7-60 days. METHODS: Data were abstracted retrospectively from electronic records of 37 emergency departments (EDs) for infants with a measured temperature >=100.4 F who underwent an ED evaluation with blood and urine cultures. Models to predict IBI were developed and validated respectively using a random 80/20 dataset split, including 10-fold cross-validation. We used precision recall curves as the classification metric. RESULTS: Of 4411 eligible infants with a mean age of 37 days, 29% had characteristics that would likely have excluded them from existing risk stratification protocols. There were 196 patients with IBI (4.4%), including 43 (1.0%) with bacterial meningitis. Analytic approaches varied in performance characteristics (precision recall range 0.04-0.29, area under the curve range 0.5-0.84), with the XGBoost model demonstrating the best performance (0.29, 0.84). The five most important variables were serum white blood count, maximum temperature, absolute neutrophil count, absolute band count, and age in days. CONCLUSION: A machine learning model (XGBoost) demonstrated the best performance in predicting a rare outcome among febrile infants, including those excluded from existing algorithms. IMPACT: Several models for the risk stratification of febrile infants have been developed. There is a need for a preferred comprehensive model free from limitations and algorithm exclusions that accurately predicts IBIs. This is the first study to derive an all-inclusive predictive model for febrile infants aged 7-60 days in a community ED sample with IBI as a primary outcome. This machine learning model demonstrates potential for clinical utility in predicting IBI.

Helical reconstruction of VP39 reveals principles for baculovirus nucleocapsid assembly.

Baculoviruses are insect-infecting pathogens with wide applications as biological pesticides, in vitro protein production vehicles and gene therapy tools. Its cylindrical nucleocapsid, which encapsulates and protects the circular double-stranded viral DNA encoding proteins for viral replication and entry, is formed by the highly conserved major capsid protein VP39. The mechanism for VP39 assembly remains unknown. We use electron cryomicroscopy to determine a 3.2 Å helical reconstruction of an infectious nucleocapsid of Autographa californica multiple nucleopolyhedrovirus, revealing how dimers of VP39 assemble into a 14-stranded helical tube. We show that VP39 comprises a distinct protein fold conserved across baculoviruses, which includes a Zinc finger domain and a stabilizing intra-dimer sling. Analysis of sample polymorphism shows that VP39 assembles in several closely-related helical geometries. This VP39 reconstruction reveals general principles for baculoviral nucleocapsid assembly.

Prominin 1 and Tweety Homology 1 both induce extracellular vesicle formation.

Prominin-1 (Prom1) is a five-transmembrane-pass integral membrane protein that associates with curved regions of the plasma membrane. Prom1 interacts with membrane cholesterol and actively remodels the plasma membrane. Membrane bending activity is particularly evident in photoreceptors, where Prom1 loss-of-function mutations cause failure of outer segment homeostasis, leading to cone-rod retinal dystrophy (CRRD). The Tweety Homology (Ttyh) protein family has been proposed to be homologous to Prominin, but it is not known whether Ttyh proteins have an analogous membrane-bending function. Here, we characterize the membrane-bending activity of human Prom1 and Ttyh1 in native bilayer membranes. We find that Prom1 and Ttyh1 both induce formation of extracellular vesicles (EVs) in cultured mammalian cells and that the EVs produced are biophysically similar. Ttyh1 is more abundant in EV membranes than Prom1 and produces EVs with membranes that are more tubulated than Prom1 EVs. We further show that Prom1 interacts more stably with membrane cholesterol than Ttyh1 and that this may contribute to membrane bending inhibition in Prom1 EVs. Intriguingly, a loss-of-function mutation in Prom1 associated with CRRD induces particularly stable cholesterol binding. These experiments provide mechanistic insight into Prominin function in CRRD and suggest that Prom and Ttyh belong to a single family of functionally related membrane-bending, EV-generating proteins.

Helical reconstruction of VP39 reveals principles for baculovirus nucleocapsid assembly.

Baculoviruses are insect-infecting pathogens with wide applications as biological pesticides, in vitro protein production vehicles and gene therapy tools. Its cylindrical nucleocapsid, which encapsulates and protects the circular double-stranded viral DNA encoding proteins for viral replication and entry, is formed by the highly conserved major capsid protein VP39. The mechanism for VP39 assembly remains unknown. We determined a 3.2 Å electron cryomicroscopy helical reconstruction of an infectious nucleocapsid of Autographa californica multiple nucleopolyhedrovirus, revealing how dimers of VP39 assemble into a 14-stranded helical tube. We show that VP39 comprises a unique protein fold conserved across baculoviruses, which includes a Zinc finger domain and a stabilizing intra-dimer sling. Analysis of sample polymorphism revealed that VP39 assembles in several closely-related helical geometries. This VP39 reconstruction reveals general principles for baculoviral nucleocapsid assembly.

Association of In-School and Electronic Bullying with Suicidality and Feelings of Hopelessness among Adolescents in the United States.

BACKGROUND: Suicide-related behaviors increasingly contribute to behavioral health crises in the United States (U.S.) and worldwide. The problem was worsened during the COVID-19 pandemic, especially for youth and young adults. Existing research suggests suicide-related behaviors are a consequence of bullying, while hopelessness is a more distal consequence. This study examines the association of in-school and electronic bullying with suicide-related behavior and feelings of despair among adolescents, adjusted for sociodemographic characteristics, abuse experience, risk-taking behaviors, and physical appearance/lifestyles. METHOD: Using Chi-square, logistic regression, and multinomial logistic regression, we analyzed the US 2019 Youth Risk Behavior Surveillance System (YRBSS) national component. The YRBSS includes federal, state, territorial, and freely associated state, tribal government, and local school-based surveys of representative sample middle and high school students in the US. The 2019 YRBSS participants comprised 13,605 students aged 12 to 18 years and roughly equal proportions of males and females (50.63% and 49.37%, respectively). RESULTS: We observed a significant association (p < 0.05) between being bullied and depressive symptoms, and the association was more vital for youth bullied at school and electronically. Being bullied either at school or electronically was associated with suicidality, with a stronger association for youth who experienced being bullied in both settings. CONCLUSION: Our findings shed light on assessing early signs of depression to prevent the formation of suicidality among bullied youth.

Following Birth Hospitalization: Invasive Bacterial Infections in Preterm Infants Aged 7-90 Days.

OBJECTIVE: To assess the incidence rate of invasive bacterial infections in preterm infants and compare invasive bacterial infection rates and pathogens between preterm and full-term infants at age 7-90 days. STUDY DESIGN: This is a retrospective cohort study of the incidence rate of invasive bacterial infections among all infants born at Kaiser Permanente Northern California (KPNC), with blood and cerebrospinal fluid cultures collected between 7 and 90 days of chronological age from outpatient clinics, from emergency departments, and in the first 24 hours of hospitalization presenting for care between January 1, 2005, and December 31, 2017. Incidence rates of invasive bacterial infection by chronological age and postmenstrual age (PMA) and pathogens were compared between preterm and full-term infants. RESULTS: Between January 1, 2005, and December 31, 2017, a total of 479 729 infants were born at KPNC, including 440 070 full-term infants and 39 659 preterm infants. There were 283 cases of bacteremia in 282 infants. The incidence rate of invasive bacterial infection was significantly higher for preterm infants compared with full-term infants. The highest incidence rates of invasive bacterial infection were in preterm infants at chronological age 7-28 days and/or 37-39 weeks PMA. There was a trend toward lower rates of invasive bacterial infection with increasing PMA in preterm infants aged 61-90 days. Preterm infants aged 29-60 days or at ≥40 weeks PMA and those aged 61-90 days or at ≥43 weeks PMA had a rate of invasive bacterial infection equivalent to the overall rate seen in full-term infants of the same chronological age group. The distribution of pathogens causing bacteremia and meningitis did not differ between preterm and full-term infants. CONCLUSION: PMA and chronological age together were more useful than either alone in informing the incidence rate of invasive bacterial infection in preterm infants during the first 90 days of life.

Using AAP Guidelines for Managing Febrile Infants Without C-Reactive Protein and Procalcitonin.

BACKGROUND AND OBJECTIVES: In 2021, the American Academy of Pediatrics (AAP) published the Clinical Practice Guideline (CPG) for management of well-appearing, febrile infants 8 to 60 days old. For older infants, the guideline relies on several inflammatory markers, including tests not rapidly available in many settings like C-reactive protein (CRP) and procalcitonin (PCT). This study describes the performance of the AAP CPG for detecting invasive bacterial infections (IBI) without using CRP and PCT. METHODS: This retrospective cohort study included infants aged 8 to 60 days old presenting to Kaiser Permanente Northern California emergency departments between 2010 and 2019 with temperatures ≥38°C who met AAP CPG inclusion criteria and underwent complete blood counts, blood cultures, and urinalyses. Performance characteristics for detecting IBI were calculated for each age group. RESULTS: Among 1433 eligible infants, there were 57 (4.0%) bacteremia and 9 (0.6%) bacterial meningitis cases. Using absolute neutrophil count >5200/mm3 and temperature >38.5°C as inflammatory markers, 3 (5%) infants with IBI were misidentified. Sensitivities and specificities for detecting infants with IBIs in each age group were: 8 to 21 days: 100% (95% confidence interval [CI] 83.9%-100%) and 0% (95% CI 0%-1.4%); 22 to 28 days: 88.9% (95% CI 51.8%-99.7%) and 40.4% (95% CI 33.2%- 48.1%); and 29 to 60 days: 93.3% (95% CI 77.9%-99.2%) and 32.1% (95% CI 29.1%- 35.3%). Invasive interventions were recommended for 100% of infants aged 8 to 21 days; 58% to 100% of infants aged 22 to 28 days; and 0% to 69% of infants aged 29 to 60 days. CONCLUSIONS: When CRP and PCT are not available, the AAP CPG detected IBI in young, febrile infants with high sensitivity but low specificity.

Clinical management and outcomes for febrile infants 29-60 days evaluated in community emergency departments.

Objective: Describe emergency department (ED) management and patient outcomes for febrile infants 29-60 days of age who received a lumbar puncture (LP), with focus on timing of antibiotics and type of physician performing LP. Methods: Retrospective observational study of 35 California EDs from January 1, 2010 through December 31, 2019. Primary analysis was among patients with successful LP and primary outcome was hospital length of stay (LOS). Logistic regression analysis included variables associated with LOS of at least 2 days. Secondary outcomes were bacterial meningitis, hospital admission, length of antibiotics, and readmission. Results: Among 2569 febrile infants (median age 39 days), 667 underwent successful LP and 633 received intravenous antibiotics. Most infants (n = 559, 88.3%) had their LP before intravenous antibiotic administration. Pediatricians performed 54% of LPs and emergency physicians 34%. Sixteen infants (0.6% of 2569) were diagnosed with bacterial meningitis, and none died. Five hundred and fifty-eight (88%) infants receiving an LP were hospitalized. Among patients receiving an LP and antibiotics (n = 633), 6.5% were readmitted within 30 days. Patients receiving antibiotics before LP had a longer length of antibiotics (+ 7.9 hours, 95% confidence interval [CI] 3.8-13.4). Primary analysis found no association between timing of antibiotics and LOS (odds ratio [OR] 0.67, 95% CI 0.34-1.30), but shorter LOS when emergency physicians performed the LP (OR 0.66, 95% CI 0.45-0.97). Conclusions: Febrile infants in the ED had no deaths and few cases of bacterial meningitis. In community EDs, where a pediatrician is often not available, successful LP by emergency physician was associated with reduced inpatient LOS.

Absence of Cardiolipin From the Outer Leaflet of a Mitochondrial Inner Membrane Mimic Restricts Opa1-Mediated Fusion.

Cardiolipin is a tetra-acylated di-phosphatidylglycerol lipid enriched in the matrix-facing (inner) leaflet of the mitochondrial inner membrane. Cardiolipin plays an important role in regulating mitochondria function and dynamics. Yet, the mechanisms connecting cardiolipin distribution and mitochondrial protein function remain indirect. In our previous work, we established an in vitro system reconstituting mitochondrial inner membrane fusion mediated by Opa1. We found that the long form of Opa1 (l-Opa1) works together with the proteolytically processed short form (s-Opa1) to mediate fast and efficient membrane fusion. Here, we extend our reconstitution system to generate supported lipid bilayers with asymmetric cardiolipin distribution. Using this system, we find the presence of cardiolipin on the inter-membrane space-facing (outer) leaflet is important for membrane tethering and fusion. We discuss how the presence of cardiolipin in this leaflet may influence protein and membrane properties, and future applications for this approach.

Roseville Protocol for the Management of Febrile Infants 7-60 Days.

OBJECTIVES: The Roseville Protocol modifies the Rochester Protocol by adding a high-risk temperature criterion of >38.5°C for infants 7 to 28 days old and by allowing febrile infants 29 to 60 days old with abnormal urinalysis but reassuring complete blood cell counts to be discharged home on oral antibiotics without receiving a lumbar puncture (LP). In this study, we define the Roseville Protocol test characteristics to detect invasive bacterial infection (IBI) and retrospectively compare its performance to that of the Rochester, Philadelphia, and Boston protocols. METHODS: In this retrospective study, we examine all cases of fever in infants aged 7 to 60 days presenting to a large health maintenance organization from 2007 to 2016 and having requisite laboratory tests for protocol analysis. The 4 protocols were retrospectively applied to this cohort to calculate each protocol's sensitivity and specificity to detect IBI. Protocols were compared regarding recommended LPs, admissions, and parenteral antibiotics. RESULTS: In 627 infants 7 to 28 days old, the Roseville Protocol had a sensitivity of 96.7% and a negative predictive value of 99.5%. It identified 2 IBIs missed by the Rochester Protocol but recommended an absolute increase of 19% in LPs and admissions. In 1176 infants 29 to 60 days old, the Roseville Protocol had a sensitivity of 91.4% and a negative predictive value of 99.6%. There was an absolute reduction in LPs by 18% to 44% compared to the Rochester Protocol and by 74% to 100% compared to the Philadelphia and Boston protocols. There was an absolute reduction in admissions by 18% to 44% compared to the Rochester Protocol, by 25% to 51% compared to the Philadelphia Protocol, and by 10% to 36% compared to the Boston Protocol. CONCLUSIONS: The Roseville Protocol has sensitivity and specificity comparable to that of existing protocols for IBI in febrile infants 7 to 60 days old, while allowing for fewer invasive procedures and hospitalizations in infants ≥29 days old.

TNNT2 mutations in the tropomyosin binding region of TNT1 disrupt its role in contractile inhibition and stimulate cardiac dysfunction.

Muscle contraction is regulated by the movement of end-to-end-linked troponin-tropomyosin complexes over the thin filament surface, which uncovers or blocks myosin binding sites along F-actin. The N-terminal half of troponin T (TnT), TNT1, independently promotes tropomyosin-based, steric inhibition of acto-myosin associations, in vitro. Recent structural models additionally suggest TNT1 may restrain the uniform, regulatory translocation of tropomyosin. Therefore, TnT potentially contributes to striated muscle relaxation; however, the in vivo functional relevance and molecular basis of this noncanonical role remain unclear. Impaired relaxation is a hallmark of hypertrophic and restrictive cardiomyopathies (HCM and RCM). Investigating the effects of cardiomyopathy-causing mutations could help clarify TNT1's enigmatic inhibitory property. We tested the hypothesis that coupling of TNT1 with tropomyosin's end-to-end overlap region helps anchor tropomyosin to an inhibitory position on F-actin, where it deters myosin binding at rest, and that, correspondingly, cross-bridge cycling is defectively suppressed under diastolic/low Ca2+ conditions in the presence of HCM/RCM lesions. The impact of TNT1 mutations on Drosophila cardiac performance, rat myofibrillar and cardiomyocyte properties, and human TNT1's propensity to inhibit myosin-driven, F-actin-tropomyosin motility were evaluated. Our data collectively demonstrate that removing conserved, charged residues in TNT1's tropomyosin-binding domain impairs TnT's contribution to inhibitory tropomyosin positioning and relaxation. Thus, TNT1 may modulate acto-myosin activity by optimizing F-actin-tropomyosin interfacial contacts and by binding to actin, which restrict tropomyosin's movement to activating configurations. HCM/RCM mutations, therefore, highlight TNT1's essential role in contractile regulation by diminishing its tropomyosin-anchoring effects, potentially serving as the initial trigger of pathology in our animal models and humans.

Microbiological safety of popular recreation swimming sites in Central California.

The objective of the study was to assess the microbiological safety of popular recreational swimming sites in Central California. Water samples were collected from eleven monitoring sites across the lower reaches of two watersheds for two consecutive swimming seasons (2012-2013), and levels of indicator and pathogenic microorganisms were determined. Data on ambient weather and water chemistry were collected for analyzing their associations with microorganisms in water. All water samples were positive for indicator E. coli with mean concentrations per site ranging from 3.07 to 216.11 MPN/100 ml in 2012 and 13.4 to 226.97 MPN/100 ml in 2013. Mean E. coli concentrations in 27% and 36% samplings sites exceeded the EPA 2012 Recreational Water Quality Criteria recommended mean concentration of ≤ 126 CFU/100 ml of E. coli, in 2012 and 2013, respectively. Cryptosporidium spp. oocysts were detected in all water samples from all sampling sites, with an overall prevalence of 50% and mean concentrations of 0.08 oocysts/l in 2012 and 0.19 oocysts/l in 2013. Giardia spp. cysts were detected at eight sites, with an overall prevalence of 28.8% and mean concentration of 0.2 cysts/l in both years. The majority of the detected Cryptosporidium spp. oocysts and Giardia spp. cysts appeared damaged under microscopy. E. coli O157:H7 was detected in 9% of water samples, with positive samples limited to three sites. Salmonella spp. were detected in all but one site across the two years, with mean concentrations of 0.94 MPN/l in 2012 and 1.85 MPN/l in 2013. Cryptosporidium spp. oocyst concentrations were negatively associated with 30-day mean wind speed and cumulative precipitation and dissolved oxygen in water. Giardia spp. cyst concentrations were positively associated with turbidity and pH of water and negatively associated with E. coli concentrations and 24-h mean air temperature. Salmonella spp. concentrations were positively associated with 30-day mean air temperature. The occurrence of E. coli O157:H7 was positively associated with previous 30-day cumulative precipitation.

An Interscholastic Network To Generate LexA Enhancer Trap Lines in Drosophila.

Binary expression systems like the LexA-LexAop system provide a powerful experimental tool kit to study gene and tissue function in developmental biology, neurobiology, and physiology. However, the number of well-defined LexA enhancer trap insertions remains limited. In this study, we present the molecular characterization and initial tissue expression analysis of nearly 100 novel StanEx LexA enhancer traps, derived from the StanEx1 index line. This includes 76 insertions into novel, distinct gene loci not previously associated with enhancer traps or targeted LexA constructs. Additionally, our studies revealed evidence for selective transposase-dependent replacement of a previously-undetected KP element on chromosome III within the StanEx1 genetic background during hybrid dysgenesis, suggesting a molecular basis for the over-representation of LexA insertions at the NK7.1 locus in our screen. Production and characterization of novel fly lines were performed by students and teachers in experiment-based genetics classes within a geographically diverse network of public and independent high schools. Thus, unique partnerships between secondary schools and university-based programs have produced and characterized novel genetic and molecular resources in Drosophila for open-source distribution, and provide paradigms for development of science education through experience-based pedagogy.

Comparative Pathogenicity of Wildlife and Bovine Escherichia coli O157:H7 Strains in Experimentally Inoculated Neonatal Jersey Calves.

Shiga toxin-producing Escherichia coli, like E. coli O157:H7, are important human and animal pathogens. Naturally-acquired E. coli O157:H7 infections occur in numerous species but, particularly, cattle have been identified as a significant reservoir for human cases. E. coli O157:H7 are isolated from a number of domestic and wild animals, including rodents that share a living space with cattle. These Shiga toxin-producing E. coli O157:H7 strains can be highly virulent in humans, but little is known about the sequelae of interspecies transfer. In a group of neonatal calves, we determined the differences in colonization patterns and lesions associated with infection using either a wildlife or bovine E. coli O157:H7 strain. In calves challenged with the wildlife E. coli O157:H7 strain, the large (descending) colon was solely colonized, which differed substantially from the calves inoculated with the bovine E. coli O157:H7 strain, where the spiral colon was the principal target of infection. This study also demonstrated that while both interspecies- and intraspecies-derived E. coli O157:H7 can infect young calves, the distribution and severity differs.

The Prevalence of Bacterial Meningitis in Febrile Infants 29-60 Days With Positive Urinalysis.

OBJECTIVES: This study evaluates whether bacterial meningitis prevalence differs by urinalysis result and whether antibiotic treatment of presumed urinary tract infection without cerebrospinal fluid (CSF) culture produces adverse sequelae in febrile infants 29 to 60 days old. METHODS: This retrospective cohort study identified febrile infants 29 to 60 days old presenting to Kaiser Permanente Northern California sites from 2007 to 2015 who underwent urinalysis and blood, urine, and CSF cultures, comparing the prevalence of meningitis among infants with positive versus negative urinalysis results using a two 1-sided test for equivalence. Additionally, febrile infants treated with antibiotics for positive urinalysis results without CSF culture were identified and their charts were reviewed for adverse sequelae. RESULTS: Full evaluation was performed in 833 febrile infants (835 episodes). Three of 337 infants with positive urinalysis (0.9%; 95% confidence interval [CI]: 0.0%-1.9%) and 5 of 498 infants with negative urinalysis (1%; 95% CI: 0.1%-1.9%) had meningitis. These proportions were statistically equivalent within 1%, using two 1-sided test with a P value of .04. There were 341 febrile infants (345 episodes) with positive urinalysis treated with antibiotics without lumbar puncture. Zero cases of missed bacterial meningitis were identified (95% CI: 0%-1.1%). Zero cases of severe sequelae (sepsis, seizure, neurologic deficit, intubation, PICU admission, death) were identified (95% CI: 0%-1.1%). CONCLUSIONS: The prevalence of bacterial meningitis does not differ by urinalysis in febrile infants 29 to 60 days old. Antibiotic treatment of infants with positive results for urinalysis without lumbar puncture may be safe in selected cases.

Identification of quantitative trait loci controlling gene expression during the innate immunity response of soybean.

Microbe-associated molecular pattern-triggered immunity (MTI) is an important component of the plant innate immunity response to invading pathogens. However, most of our knowledge of MTI comes from studies of model systems with relatively little work done with crop plants. In this work, we report on variation in both the microbe-associated molecular pattern-triggered oxidative burst and gene expression across four soybean (Glycine max) genotypes. Variation in MTI correlated with the level of pathogen resistance for each genotype. A quantitative trait locus analysis on these traits identified four loci that appeared to regulate gene expression during MTI in soybean. Likewise, we observed that both MTI variation and pathogen resistance were quantitatively inherited. The approach utilized in this study may have utility for identifying key resistance loci useful for developing improved soybean cultivars.

Enzymatic role for soybean ecto-apyrase in nodulation.

Root nodulation is regulated by a variety of mechanisms. Ecto-apyrase is an enzyme proposed to control the concentration of extracellular nucleotides. Transgenic expression of the soybean GS52 ecto-apyrase was shown to stimulate nodulation. However, mutation of the enzyme to disrupt enzymatic activity prevented this effect. Therefore, the data suggest that the enzymatic activity of the ecto-apyrase is critical for nodulation enhancement, suggesting a direct effect on extracellular nucleotide hydrolysis. In this article, we propose a hypothetical mechanism for plant ecto-apyrase function during nodulation.

Intracellular visualization of BrdU-labeled plasmid DNA/cationic liposome complexes.

Difficulties in specific detection of transfected DNA in cells represent an important limitation in the study of the gene transfer process. We studied the cellular entry and fate of a plasmid DNA complexed with a cationic lipid, Vectamidine (3-tetradecylamino-N-tert-butyl-N'-tetradecylpropionamidine) in BHK21 cells. To facilitate its detection inside the cells, bromodeoxyuridine (BrdU) was incorporated into plasmid DNA under conditions that minimize plasmid alteration. BrdU was localized in cells incubated with Vectamidine/BrdU-labeled plasmid DNA complexes by immunogold labeling and electron microscopy (EM). Labeling was predominantly associated with aggregated liposome structures at the surface of and inside the cells. EM observations of cells transfected with Vectamidine/DNA complexes showed that the liposome/DNA aggregates accumulate in large vesicles in the cell cytosol. On the other hand, using rhodamine-labeled Vectamidine and revealing BrdU with FITC-conjugated antibodies permitted simultaneous detection in the cells of both components of the complexes with confocal laser scanning microscopy. The DNA and lipids co-localized at the surface of and inside the cells, indicating that the complex is internalized as a whole. Our results show that the BrdU-labeled plasmid DNA detection system can be a useful tool to visualize exogenous DNA entry into cells by a combination of electron and confocal microscopy.