RNA polymerase sliding on DNA can couple the transcription of nearby bacterial operons.

DNA transcription initiates after an RNA polymerase (RNAP) molecule binds to the promoter of a gene. In bacteria, the canonical picture is that RNAP comes from the cytoplasmic pool of freely diffusing RNAP molecules. Recent experiments suggest the possible existence of a separate pool of polymerases, competent for initiation, which freely slide on the DNA after having terminated one round of transcription. Promoter-dependent transcription reinitiation from this pool of posttermination RNAP may lead to coupled initiation at nearby operons, but it is unclear whether this can occur over the distance and timescales needed for it to function widely on a bacterial genome in vivo. Here, we mathematically model the hypothesized reinitiation mechanism as a diffusion-to-capture process and compute the distances over which significant interoperon coupling can occur and the time required. These quantities depend on molecular association and dissociation rate constants between DNA, RNAP, and the transcription initiation factor σ70; we measure these rate constants using single-molecule experiments in vitro. Our combined theory/experimental results demonstrate that efficient coupling can occur at physiologically relevant σ70 concentrations and on timescales appropriate for transcript synthesis. Coupling is efficient over terminator-promoter distances up to ∼1,000 bp, which includes the majority of terminator-promoter nearest neighbor pairs in the Escherichia coli genome. The results suggest a generalized mechanism that couples the transcription of nearby operons and breaks the paradigm that each binding of RNAP to DNA can produce at most one messenger RNA.

RNA polymerase sliding on DNA can couple the transcription of nearby bacterial operons.

DNA transcription initiates after an RNA polymerase (RNAP) molecule binds to the promoter of a gene. In bacteria, the canonical picture is that RNAP comes from the cytoplasmic pool of freely diffusing RNAP molecules. Recent experiments suggest the possible existence of a separate pool of polymerases, competent for initiation, which freely slide on the DNA after having terminated one round of transcription. Promoter-dependent transcription reinitiation from this pool of post-termination RNAP may lead to coupled initiation at nearby operons, but it is unclear whether this can occur over the distance- and time-scales needed for it to function widely on a bacterial genome in vivo. Here, we mathematically model the hypothesized reinitiation mechanism as a diffusion-to-capture process and compute the distances over which significant inter-operon coupling can occur and the time required. These quantities depend on previously uncharacterized molecular association and dissociation rate constants between DNA, RNAP and the transcription initiation factor σ 70 ; we measure these rate constants using single-molecule experiments in vitro. Our combined theory/experimental results demonstrate that efficient coupling can occur at physiologically relevant σ 70 concentrations and on timescales appropriate for transcript synthesis. Coupling is efficient over terminator-promoter distances up to ∼ 1, 000 bp, which includes the majority of terminator-promoter nearest neighbor pairs in the E. coli genome. The results suggest a generalized mechanism that couples the transcription of nearby operons and breaks the paradigm that each binding of RNAP to DNA can produce at most one messenger RNA. SIGNIFICANCE STATEMENT: After transcribing an operon, a bacterial RNA polymerase can stay bound to DNA, slide along it, and reini-tiate transcription of the same or a different operon. Quantitative single-molecule biophysics experiments combined with mathematical theory demonstrate that this reinitiation process can be quick and efficient over gene spacings typical of a bacterial genome. Reinitiation may provide a mechanism to orchestrate the transcriptional activities of groups of nearby operons.

Clinical and Radiographic Predictors of Progression and Survival in Relapsed/Refractory Lymphoma Patients Receiving Anti-CD19 CAR T-cell Therapy.

INTRODUCTION: Chimeric antigen receptor (CAR) T-cell therapy has revolutionized treatment of relapsed/refractory (R/R) B-cell lymphomas, though certain patients do not respond to treatment or relapse afterwards. The purpose of this study is to determine patient variables that are predictive of response to CAR-T therapy. METHODS: We conducted a retrospective review of 59 R/R B-cell non-Hodgkin lymphoma patients who received anti-CD19 CAR T-cell therapy. Risk factors for progression free survival (PFS) and overall survival (OS) were identified and multivariate logistic regression models for PFS and OS at 1 year were created using stepwise selection. The final multivariate logistic regression models were used to estimate the area under the receiver operating curve (AUROC). RESULTS: At median follow up of 25.6 months, median overall survival was not reached, and median progression free survival was 5.7 months. Stage IV disease (odds ratio (OR) 9.335, P = .025) was identified as a predictive variable for progression at day 365 with an AUC of 0.7922 (P < .001). IPI (OR 2.828, P = .014), ALC ≥ 0.50 at collection (OR 0.183, P = .043), CRP ≥ 11 (OR 6.177, P = .019), and tocilizumab administration (OR 0.062, P = .005) as predictors for death at day 365 with an AUC 0.8626 (P < .001). CONCLUSION: Clinical variables identify R/R lymphoma patients who are at risk for progression and poor overall survival after CAR T-cell therapy. IPI, CRP, ALC, and tocilizumab administration may be predictors of survival.

Regional muscle changes in adult dysfunctional hip conditions of femoroacetabular impingement and hip dysplasia.

OBJECTIVE: To analyze regional muscle CT density and bulk in femoroacetabular impingement (FAI) and hip dysplasia (HD) versus controls. MATERIALS AND METHODS: Patients who obtained perioperative CT imaging for FAI and HD before surgery were retrospectively studied. Asymptomatic controls included for comparison. Two readers independently evaluated regional hip muscle [iliopsoas (IP), rectus femoris (RF), gluteus minimus (Gm), and medius (GM)] density, muscle area, and muscle circumference. Inter-observer reliability calculated using intra-class correlation coefficient (ICC). RESULTS: A consecutive series of 25 FAI patients, 16 HD patients, and 38 controls were recruited in the study. FAI patients had significantly greater Gm and GM circumferences as well as greater RF and IP areas on the normal side compared to the asymptomatic control group (p values 0.004, 0.032, 0.033, and 0.028, respectively). In addition, Gm and RF circumferences and RF area were significantly larger (p values 0.029, 0.036, and 0.014, respectively) in FAI patients on the affected side compared to the control group. HD patients had significantly smaller Gm and GM circumferences on the affected side than normal side measurements in FAI group (p values 0.043 and 0.003, respectively). Normal side GM circumference was also smaller in HD patients than normal side FAI hips (p value 0.02). There was no significant difference between the measurements on normal and abnormal sides in each disease group. No significant difference was found between measurements of HD compared to controls (p > 0.05). No muscle density differences were seen among different groups. There was moderate to excellent inter-reader reliability for all measurements except Gm muscle density. CONCLUSIONS: Muscle analysis was able to quantify differences among patients with FAI, HD, and asymptomatic controls. These changes could indicate either a muscle imbalance contributing to the pathology or disuse atrophy, which may have implications for specific muscle-strengthening therapies and rehabilitation procedures in such patients.

CT bone density analysis of low-impact proximal femur fractures using Hounsfield units.

AIM: To quantify and compare changes in bone mineral density (BMD) via CT analysis in patients with and without spontaneous femoral fractures. MATERIALS AND METHODS: Consecutive series of patients with CT imaging for spontaneous femoral fractures were compared to the age and gender matched controls. Bone density fixed region of interest measurements were obtained at the site of the fracture, proximally at the femoral head, and distally at the lesser trochanter in fracture patients and controls. Inter- and intrapatient comparisons were performed, including Chi-square and t-test analyses. RESULTS: 24 spontaneous fractures and 25 controls were analyzed with no significant differences in mean age, gender, or body mass index. There were differences in the bone density between the fracture and contralateral non-fracture sides at (p = 0.0001) and distal (p < 0.0001) to the fracture. Proximal and distal bone density differences existed between case fracture and control non-fracture sites (p < 0.0001, p = 0.0001), and between the case non-fracture and control non-fracture sites (p < 0.0001, p < 0.0001). The reliability for measurements was good to excellent proximally (ICC = 0.63-0.87), moderate to excellent at the fracture site (ICC = 0.43-0.78), and fair to good distal (ICC = 0.24-0.68) to the fracture site. CONCLUSION: Patients with spontaneous femoral fractures exhibit lower bone density than the asymptomatic controls. Bone insufficiency is best demonstrated proximal or distal to, rather than at the fracture site.

The International Society of Arthroscopy, Knee Surgery and Orthopaedic Sports Medicine classification of knee meniscus tears: three-dimensional MRI and arthroscopy correlation.

OBJECTIVES: To introduce MRI-based International Society of Arthroscopy, Knee Surgery and Orthopaedic Sports Medicine (ISAKOS) classification system of meniscal tears and correlate it to the surgical findings from arthroscopy. We hypothesized that the ISAKOS classification will provide good inter-modality and inter-rater reliability for use in the routine clinical practice of radiologists and orthopedic surgeons. METHODS: In this HIPAA-compliant cross-sectional study, there were 44 meniscus tears in 39 patients (26 males, 16 females). Consecutive arthroscopy-proven meniscal tears (March 2017 to December 2017) were evaluated by two board-certified musculoskeletal radiologists using isotropic three-dimensional (3D) MRI user-defined reconstructions. The surgically validated ISAKOS classification of meniscal tears was used to describe medial meniscus (MM) and lateral meniscus (LM) tears. Prevalence-adjusted bias-adjusted kappa (PABAK) and conventional kappa, and paired t test and intra-class correlation coefficient (ICC) were calculated for categorical and numerical variables, respectively. RESULTS: For the MM, the PABAK for location, depth, length (ICC), pattern, quality of meniscus tissue, and zone was 0.7-1, 0.65, 0.57, 0.67, 0.78, and 0.39-0.7, respectively. For the LM, the PABAK for location, depth, length (ICC), pattern, quality of meniscus tissue, zone, and central to popliteus hiatus was 0.57-0.95, 0.57, 0.74, 0.93, 0.38, 0.52-0.67, and 0.48, respectively. The mean tear lengths were larger on MRI than on arthroscopy (mean difference MM 9.74 mm (6.66 mm, 12.81 mm; p < 0.001), mean difference LM 4.04 mm (0.31 mm, 7.76 mm; p = 0.034)). CONCLUSIONS: The ISAKOS classification of meniscal tears on 3D MRI provides mostly moderate agreement, which was similar to the agreement at arthroscopy. KEY POINTS: • There is a fair to good inter-method correlation in most categories of ISAKOS meniscus tear classification. • The tear lengths are significantly larger on MRI than on arthroscopy. • The inter-reader correlation on 3D MRI is moderate to excellent, with the exception of lateral meniscus tear patterns.

Exercise Altered the Skeletal Muscle MicroRNAs and Gene Expression Profiles in Burn Rats With Hindlimb Unloading.

This study investigated microRNA and target gene profiles under different conditions of burn, bed rest, and exercise training. Male Sprague-Dawley rats (n = 48) were assigned to sham ambulatory, sham hindlimb unloading, burn ambulatory, or burn plus hindlimb unloading groups. Rats received a 40% TBSA scald burn or sham treatments and were ambulatory or hindlimb unloaded. Rats were further assigned to exercise or no exercise. Plantaris tissues were harvested on day 14 and pooled to analyze for microRNA and gene expression profiles. Compared with the sham ambulatory-no exercise group, 73, 79, and 80 microRNAs were altered 2-fold in the burn ambulatory, sham hindlimb unloading, and burn hindlimb unloading groups, all with no exercise, respectively. More than 70% of microRNAs were upregulated in response to burn and hindlimb unloading, whereas 60% microRNA of the profile decreased in hindlimb unloaded burn rats with exercise training. MiR-182 was the most affected in rat muscle. Gene ontology biological process and pathway analysis showed that the oxidative stress pathway was most stimulated in the hindlimb unloaded burn rats; while in response to exercise training, all genes in related pathways such as hypermetabolic, inflammation, and blood coagulation were alleviated. MicroRNAs and transcript gene profiles were altered in burn and hindlimb unloading groups, with additive effects on hindlimb unloaded burn rats. The altered genes' signal pathways were associated with muscle mass loss and function impairment. Muscle improvement with exercise training was observed in gene levels with microRNA alterations as well.

Evaluating Pre Burn Center Intubation Practices: An Update.

A significant proportion of patients appeared to arrive at our American Burn Association-verified burn center intubated without clear benefit. The current study aims to evaluate regional prehospital intubation practices and their outcomes. All consecutive admissions from November 2012 to June 2014 were reviewed for data points associated with intubation. Demographics and outcomes for patients who were intubated before arrival or within 24 hours of admission were compared using χ, Fisher's exact test, and the Kruskal-Wallis test as appropriate. During this period, 958 patients were admitted. Of these, 120 were intubated before arrival, and 91 survived their injuries. Of these 91 survivors, 45 were extubated within 2 days, suggesting unnecessary intubation rate in 37.5%. Intubation-related complications were roughly three times as common among those intubated before arrival (12.5% vs 4.4%). Patients intubated before arrival to our burn center had a shorter median duration of intubation (1.0 vs 4.0 days), median hospital LOS (5.0 vs 22.0 days), and median intensive care unit length of stay (3.0 vs 10.0 days). Furthermore, we found a significant difference in the pattern of ventilator support duration between those arriving intubated, with a median of 2.0 days, and those intubated at our burn center, with a median of 5.5 days. Patients intubated by pre burn center providers have shorter intubation durations and shorter hospitalizations, suggesting inappropriate use of resources. Impending loss of airway appears unlikely among patients with adequate gas exchange at the time of examination. The current criteria for prehospital intubation should be revised to more accurately identify those who truly benefit from advanced airway maneuvers.