Genome-wide screen identifies novel genes required for Borrelia burgdorferi survival in its Ixodes tick vector.

Borrelia burgdorferi, the causative agent of Lyme disease in humans, is maintained in a complex biphasic life cycle, which alternates between tick and vertebrate hosts. To successfully survive and complete its enzootic cycle, B. burgdorferi adapts to diverse hosts by regulating genes required for survival in specific environments. Here we describe the first ever use of transposon insertion sequencing (Tn-seq) to identify genes required for B. burgdorferi survival in its tick host. We found that insertions into 46 genes resulted in a complete loss of recovery of mutants from larval Ixodes ticks. Insertions in an additional 56 genes resulted in a >90% decrease in fitness. The screen identified both previously known and new genes important for larval tick survival. Almost half of the genes required for survival in the tick encode proteins of unknown function, while a significant portion (over 20%) encode membrane-associated proteins or lipoproteins. We validated the results of the screen for five Tn mutants by performing individual competition assays using mutant and complemented strains. To better understand the role of one of these genes in tick survival, we conducted mechanistic studies of bb0017, a gene previously shown to be required for resistance against oxidative stress. In this study we show that BB0017 affects the regulation of key borrelial virulence determinants. The application of Tn-seq to in vivo screening of B. burgdorferi in its natural vector is a powerful tool that can be used to address many different aspects of the host pathogen interaction.

A high-throughput genetic screen identifies previously uncharacterized Borrelia burgdorferi genes important for resistance against reactive oxygen and nitrogen species.

Borrelia burgdorferi, the causative agent of Lyme disease in humans, is exposed to reactive oxygen and nitrogen species (ROS and RNS) in both the tick vector and vertebrate reservoir hosts. B. burgdorferi contains a limited repertoire of canonical oxidative stress response genes, suggesting that novel gene functions may be important for protection of B. burgdorferi against ROS or RNS exposure. Here, we use transposon insertion sequencing (Tn-seq) to conduct an unbiased search for genes involved in resistance to nitric oxide, hydrogen peroxide, and tertiary-butyl hydroperoxide in vitro. The screens identified 66 genes whose disruption resulted in increased susceptibility to at least one of the stressors. These genes include previously characterized mediators of ROS and RNS resistance (including components of the nucleotide excision repair pathway and a subunit of a riboflavin transporter), as well as novel putative resistance candidates. DNA repair mutants were among the most sensitive to RNS in the Tn-seq screen, and survival assays with individual Tn mutants confirmed that the putative ribonuclease BB0839 is involved in resistance to nitric oxide. In contrast, mutants lacking predicted inner membrane proteins or transporters were among the most sensitive to ROS, and the contribution of three such membrane proteins (BB0017, BB0164, and BB0202) to ROS sensitivity was confirmed using individual Tn mutants and complemented strains. Further analysis showed that levels of intracellular manganese are significantly reduced in the Tn::bb0164 mutant, identifying a novel role for BB0164 in B. burgdorferi manganese homeostasis. Infection of C57BL/6 and gp91phox-/- mice with a mini-library of 39 Tn mutants showed that many of the genes identified in the in vitro screens are required for infectivity in mice. Collectively, our data provide insight into how B. burgdorferi responds to ROS and RNS and suggests that this response is relevant to the in vivo success of the organism.

The Association of ICU Acuity With Outcomes of Patients at Low Risk of Dying.

OBJECTIVE: Many ICU patients do not require critical care interventions. Whether aggressive care environments increase risks to low-acuity patients is unknown. We evaluated whether ICU acuity was associated with outcomes of low mortality-risk patients. We hypothesized that admission to high-acuity ICUs would be associated with worse outcomes. This hypothesis was based on two possibilities: 1) high-acuity ICUs may have a culture of aggressive therapy that could lead to potentially avoidable complications and 2) high-acuity ICUs may focus attention toward the many sicker patients and away from the fewer low-risk patients. DESIGN: Retrospective cohort study. SETTING: Three hundred twenty-two ICUs in 199 hospitals in the Philips eICU database between 2010 and 2015. PATIENTS: Adult ICU patients at low risk of dying, defined as an Acute Physiology and Chronic Health Evaluation-IVa-predicted mortality of 3% or less. EXPOSURE: ICU acuity, defined as the mean Acute Physiology and Chronic Health Evaluation IVa score of all admitted patients in a calendar year, stratified into quartiles. MEASUREMENTS AND MAIN RESULTS: We used generalized estimating equations to test whether ICU acuity is independently associated with a primary outcome of ICU length of stay and secondary outcomes of hospital length of stay, hospital mortality, and discharge destination. The study included 381,997 low-risk patients. Mean ICU and hospital length of stay were 1.8 ± 2.1 and 5.2 ± 5.0 days, respectively. Mean Acute Physiology and Chronic Health Evaluation IVa-predicted hospital mortality was 1.6% ± 0.8%; actual hospital mortality was 0.7%. In adjusted analyses, admission to low-acuity ICUs was associated with worse outcomes compared with higher-acuity ICUs. Specifically, compared with the highest-acuity quartile, ICU length of stay in low-acuity ICUs was increased by 0.24 days; in medium-acuity ICUs by 0.16 days; and in high-acuity ICUs by 0.09 days (all p < 0.001). Similar patterns existed for hospital length of stay. Patients in lower-acuity ICUs had significantly higher hospital mortality (odds ratio, 1.28 [95% CI, 1.10-1.49] for low-; 1.24 [95% CI, 1.07-1.42] for medium-, and 1.14 [95% CI, 0.99-1.31] for high-acuity ICUs) and lower likelihood of discharge home (odds ratio, 0.86 [95% CI, 0.82-0.90] for low-, 0.88 [95% CI, 0.85-0.92] for medium-, and 0.95 [95% CI, 0.92-0.99] for high-acuity ICUs). CONCLUSIONS: Admission to high-acuity ICUs is associated with better outcomes among low mortality-risk patients. Future research should aim to understand factors that confer benefit to patients with different risk profiles.

Identifying Distinct Subgroups of ICU Patients: A Machine Learning Approach.

OBJECTIVES: Identifying subgroups of ICU patients with similar clinical needs and trajectories may provide a framework for more efficient ICU care through the design of care platforms tailored around patients' shared needs. However, objective methods for identifying these ICU patient subgroups are lacking. We used a machine learning approach to empirically identify ICU patient subgroups through clustering analysis and evaluate whether these groups might represent appropriate targets for care redesign efforts. DESIGN: We performed clustering analysis using data from patients' hospital stays to retrospectively identify patient subgroups from a large, heterogeneous ICU population. SETTING: Kaiser Permanente Northern California, a healthcare delivery system serving 3.9 million members. PATIENTS: ICU patients 18 years old or older with an ICU admission between January 1, 2012, and December 31, 2012, at one of 21 Kaiser Permanente Northern California hospitals. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We used clustering analysis to identify putative clusters among 5,000 patients randomly selected from 24,884 ICU patients. To assess cluster validity, we evaluated the distribution and frequency of patient characteristics and the need for invasive therapies. We then applied a classifier built from the sample cohort to the remaining 19,884 patients to compare the derivation and validation clusters. Clustering analysis successfully identified six clinically recognizable subgroups that differed significantly in all baseline characteristics and clinical trajectories, despite sharing common diagnoses. In the validation cohort, the proportion of patients assigned to each cluster was similar and demonstrated significant differences across clusters for all variables. CONCLUSIONS: A machine learning approach revealed important differences between empirically derived subgroups of ICU patients that are not typically revealed by admitting diagnosis or severity of illness alone. Similar data-driven approaches may provide a framework for future organizational innovations in ICU care tailored around patients' shared needs.

Targeted mutagenesis of intergenic regions in the Neisseria gonorrhoeae gonococcal genetic island reveals multiple regulatory mechanisms controlling type IV secretion.

Gonococci secrete chromosomal DNA into the extracellular environment using a type IV secretion system (T4SS). The secreted DNA acts in natural transformation and initiates biofilm development. Although the DNA and its effects are detectable, structural components of the T4SS are present at very low levels, suggestive of uncharacterized regulatory control. We sought to better characterize the expression and regulation of T4SS genes and found that the four operons containing T4SS genes are transcribed at very different levels. Increasing transcription of two of the operons through targeted promoter mutagenesis did not increase DNA secretion. The stability and steady-state levels of two T4SS structural proteins were affected by a homolog of tail-specific protease. An RNA switch was also identified that regulates translation of a third T4SS operon. The switch mechanism relies on two putative stem-loop structures contained within the 5' untranslated region of the transcript, one of which occludes the ribosome binding site and start codon. Mutational analysis of these stem loops supports a model in which induction of an alternative structure relieves repression. Taken together, these results identify multiple layers of regulation, including transcriptional, translational and post-translational mechanisms controlling T4SS gene expression and DNA secretion.

TraK and TraB are conserved outer membrane proteins of the Neisseria gonorrhoeae Type IV secretion system and are expressed at low levels in wild-type cells.

Neisseria gonorrhoeae uses a type IV secretion system (T4SS) to secrete chromosomal DNA into the medium, and this DNA is effective in transforming other gonococci via natural transformation. In addition, the T4SS is important in the initial stages of biofilm development and mediates intracellular iron uptake in the absence of TonB. To better understand the mechanism of type IV secretion in N. gonorrhoeae, we examined the expression levels and localization of two predicted T4SS outer membrane proteins, TraK and TraB, in the wild-type strain as well as in overexpression strains and in a strain lacking all of the T4SS proteins. Despite very low sequence similarity to known homologues, TraB (VirB10 homolog) and TraK (VirB9 homolog) localized similarly to related proteins in other systems. Additionally, we found that TraV (a VirB7 homolog) interacts with TraK, as in other T4SSs. However, unlike in other systems, neither TraK nor TraB required the presence of other T4SS components for proper localization. Unlike other gonococcal T4SS proteins we have investigated, protein levels of the outer membrane proteins TraK and TraB were extremely low in wild-type cells and were undetectable by Western blotting unless overexpressed or tagged with a FLAG3 triple-epitope tag. Localization of TraK-FLAG3 in otherwise wild-type cells using immunogold electron microscopy of thin sections revealed a single gold particle on some cells. These results suggest that the gonococcal T4SS may be present in single copy per cell and that small amounts of T4SS proteins TraK and TraB are sufficient for DNA secretion.

Characterization of SARS-CoV-2 Aerosols Dispersed During Noninvasive Respiratory Support of Patients With COVID-19.

BACKGROUND: In the midst of the COVID-19 pandemic, noninvasive respiratory support (NRS) therapies such as high-flow nasal cannula (HFNC) and noninvasive ventilation (NIV) were central to respiratory care. The extent to which these treatments increase the generation and dispersion of infectious respiratory aerosols is not fully understood. The objective of this study was to characterize SARS-CoV-2 aerosol dispersion from subjects with COVID-19 undergoing NRS therapy. METHODS: Several different aerosol sampling devices were used to collect air samples in the vicinity of 31 subjects with COVID-19, most of whom were receiving NRS therapy, primarily HFNC. Aerosols were collected onto filters and analyzed for the presence of SARS-CoV-2 RNA. Additional measurements were collected in an aerosol chamber with healthy adult subjects using respiratory therapy devices under controlled and reproducible conditions. RESULTS: Fifty aerosol samples were collected from subjects receiving HFNC or NIV therapy, whereas 6 samples were collected from subjects not receiving NRS. Only 4 of the 56 aerosol samples were positive for SARS-CoV-2 RNA, and all positive samples were collected using a high air flow scavenger mask collection device placed in close proximity to the subject. The chamber measurements with healthy subjects did not show any significant increase in aerosol dispersion caused by the respiratory therapy devices compared to baseline. CONCLUSIONS: Our findings demonstrate very limited detection of SARS-CoV-2-containing aerosols in the vicinity of subjects with COVID-19 receiving NRS therapies in the clinical setting. These results, combined with controlled chamber measurements showing that HFNC and NIV device usage was not associated with increased aerosol dispersion, suggest that NRS therapies do not result in increased dispersal of aerosols in the clinical setting.

New complementation constructs for inducible and constitutive gene expression in Neisseria gonorrhoeae and Neisseria meningitidis.

We have created new complementation constructs for use in Neisseria gonorrhoeae and Neisseria meningitidis. The constructs contain regions of homology with the chromosome and direct the insertion of a gene of interest into the intergenic region between the genes iga and trpB. In order to increase the available options for gene expression in Neisseria, we designed the constructs to contain one of three different promoters. One of the constructs contains the isopropyl-ß-d-thiogalactopyranoside-inducible lac promoter, which has been widely used in Neisseria. We also designed a construct that contains the strong, constitutive promoter from the gonococcal opaB gene. The third construct contains a tetracycline-inducible promoter, a novel use of this promoter in Neisseria. We demonstrate that anhydrotetracycline can be used to induce gene expression in the pathogenic Neisseria at very low concentrations and without negatively affecting the growth of the organisms. We use these constructs to complement an arginine auxotrophy in N. gonorrhoeae as well as to express a translational fusion of alkaline phosphatase with TraW. TraW is a component of the gonococcal type IV secretion system, and we demonstrate that TraW localizes to the periplasm.

Improving lung cancer screening rates among patients with head and neck cancer in a radiation oncology clinic.

Background: The United States Preventive Services Task Force (USPSTF) recommends lung cancer screening via annual low dose computed tomography (LDCT) for high risk patients. Despite the strong evidence of a mortality benefit from several randomized clinical trials, rates of lung cancer screening remain low. We plan to assess how screening guidelines are implemented in a radiation oncology clinic for patients with head and neck cancer. Methods: A single institution, retrospective chart review was used to identify patients with head and neck cancer seen in a radiation oncology clinic who were potentially eligible for lung cancer screening under the current USPSTF guidelines. Patients who were potentially screening-eligible were enrolled in a phone survey to assess their knowledge about lung cancer screening and willingness to be screened. Results: Of the 184 patients with head and neck cancer seen in the clinic, 8 (4%) patients were eligible for lung cancer screening under the previous USPSTF recommendations, including 1 (0.5%) patient already being screened. One patient (0.5%) became eligible under the expanded guidelines. All 184 patients had smoking history documented. Of the 87 current or former smokers, there were 24 (28%) who did not have pack-years documented; of the 82 former smokers, there were 8 (10%) who did not have quit date documented. Among the 16 phone survey participants (response rate: 70%) only 6 (38%) were aware there is a way to screen for lung cancer and 12 (75%) patients would be interested in screening if they are found to be eligible. Conclusions: These findings highlight a potential opportunity to increase rates of lung cancer screening among patients with head and neck cancer by both enhancing provider awareness as well as patient education at the community level.

Genomic Profiling of Bronchoalveolar Lavage Fluid in Lung Cancer.

Genomic profiling of bronchoalveolar lavage (BAL) samples may be useful for tumor profiling and diagnosis in the clinic. Here, we compared tumor-derived mutations detected in BAL samples from subjects with non-small cell lung cancer (NSCLC) to those detected in matched plasma samples. Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq) was used to genotype DNA purified from BAL, plasma, and tumor samples from patients with NSCLC. The characteristics of cell-free DNA (cfDNA) isolated from BAL fluid were first characterized to optimize the technical approach. Somatic mutations identified in tumor were then compared with those identified in BAL and plasma, and the potential of BAL cfDNA analysis to distinguish lung cancer patients from risk-matched controls was explored. In total, 200 biofluid and tumor samples from 38 cases and 21 controls undergoing BAL for lung cancer evaluation were profiled. More tumor variants were identified in BAL cfDNA than plasma cfDNA in all stages (P < 0.001) and in stage I to II disease only. Four of 21 controls harbored low levels of cancer-associated driver mutations in BAL cfDNA [mean variant allele frequency (VAF) = 0.5%], suggesting the presence of somatic mutations in nonmalignant airway cells. Finally, using a Random Forest model with leave-one-out cross-validation, an exploratory BAL genomic classifier identified lung cancer with 69% sensitivity and 100% specificity in this cohort and detected more cancers than BAL cytology. Detecting tumor-derived mutations by targeted sequencing of BAL cfDNA is technically feasible and appears to be more sensitive than plasma profiling. Further studies are required to define optimal diagnostic applications and clinical utility. SIGNIFICANCE: Hybrid-capture, targeted deep sequencing of lung cancer mutational burden in cell-free BAL fluid identifies more tumor-derived mutations with increased allele frequencies compared with plasma cell-free DNA. See related commentary by Rolfo et al., p. 2826.

Control of the type 3 secretion system in Vibrio harveyi by quorum sensing through repression of ExsA.

The type 3 secretion system (T3SS) genes of Vibrio harveyi are activated at low cell density and repressed at high cell density by quorum sensing (QS). Repression requires LuxR, the master transcriptional regulator of QS-controlled genes. Here, we determine the mechanism underlying the LuxR repression of the T3SS system. Using a fluorescence-based cell sorting approach, we isolated V. harveyi mutants that are unable to express T3SS genes at low cell density and identified two mutations in the V. harveyi exsBA operon. While LuxR directly represses the expression of exsBA, complementation and epistasis analyses reveal that it is the repression of exsA expression, but not exsB expression, that is responsible for the QS-mediated repression of T3SS genes at high cell density. The present work further defines the genes in the V. harveyi QS regulon and elucidates a mechanism demonstrating how multiple regulators can be linked in series to direct the expression of QS target genes specifically at low or high cell density.

Minimal genomes, maximal productivity: comparative genomics of the photosystem and light-harvesting complexes in the marine cyanobacterium, Prochlorococcus.

Although Prochlorococcus isolates possess the smallest genomes of any extant photosynthetic organism, this genus numerically dominates vast regions of the world's subtropical and tropical open oceans and has evolved to become an important contributor to global biogeochemical cycles. The sequencing of 12 Prochlorococcus genomes provides a glimpse of the extensive genetic heterogeneity and, thus, physiological potential of the lineage. In this study, we present an up-to-date comparative analysis of major proteins of the photosynthetic apparatus in 12 Prochlorococcus genomes. Our analyses reveal a striking diversity within the Prochlorococcus lineage in the major protein complexes of the photosynthetic apparatus. The heterogeneity that has evolved in the photosynthetic apparatus suggests versatility in strategies for optimizing photosynthesis under conditions of environmental variability and stress. This diversity could be particularly important in ensuring the survival of a lineage whose individuals have evolved minimal genomes and, thus, relatively limited repertoires for responding to environmental challenges.

A phase II study of gefitinib, 5-fluorouracil, leucovorin, and oxaliplatin in previously untreated patients with metastatic colorectal cancer.

PURPOSE: We investigated the gefitinib, 5-fluorouracil (5-FU), leucovorin and oxaliplatin (IFOX) regimen as first-line therapy in patients with metastatic colorectal cancer. EXPERIMENTAL DESIGN: Eligible patients had stage IV colorectal adenocarcinoma, and had not received prior chemotherapy for metastatic disease. Each cycle consisted of 14 days. Cycle 1 consisted of oxaliplatin, leucovorin, and 5-FU (FOLFOX-4). All subsequent cycles consisted of FOLFOX-4 with gefitinib at 500 mg orally daily throughout the 14-day cycle. RESULTS: Forty-five patients were enrolled and were assessable for toxicity. Forty-three patients were assessable for response. Thirty-one of the 43 patients (72%) had either a complete or partial response by the Response Evaluation Criteria in Solid Tumors. Median overall survival was 20.5 months. Median time to progression was 9.3 months. Commonly encountered grade 3 or 4 toxicities included diarrhea in 67% of patients and neutropenia in 60%. Grade 2 acneiform skin rash typical of gefitinib occurred in 60% of patients. CONCLUSIONS: IFOX is an active first-line regimen in patients with metastatic colorectal adenocarcinoma, showing higher response rates but also increased toxicities compared with FOLFOX-4 alone in a similar patient population.

Detecting organisational innovations leading to improved ICU outcomes: a protocol for a double-blinded national positive deviance study of critical care delivery.

INTRODUCTION: There is substantial variability in intensive care unit (ICU) utilisation and quality of care. However, the factors that drive this variation are poorly understood. This study uses a novel adaptation of positive deviance approach-a methodology used in public health that assumes solutions to challenges already exist within the system to detect innovations that are likely to improve intensive care. METHODS AND ANALYSIS: We used the Philips eICU Research Institute database, containing 3.3 million patient records from over 50 health systems across the USA. Acute Physiology and Chronic Health Evaluation IVa scores were used to identify the study cohort, which included ICU patients whose outcomes were felt to be most sensitive to organisational innovations. The primary outcomes included mortality and length of stay. Outcome measurements were directly standardised, and bootstrapped CIs were calculated with adjustment for false discovery rate. Using purposive sampling, we then generated a blinded list of five positive outliers and five negative comparators.Using rapid qualitative inquiry (RQI), blinded interdisciplinary site visit teams will conduct interviews and observations using a team ethnography approach. After data collection is completed, the data will be unblinded and analysed using a cross-case method to identify themes, patterns and innovations using a constant comparative grounded theory approach. This process detects the innovations in intensive care and supports an evaluation of how positive deviance and RQI methods can be adapted to healthcare. ETHICS AND DISSEMINATION: The study protocol was approved by the Stanford University Institutional Review Board (reference: 39509). We plan on publishing study findings and methodological guidance in peer-reviewed academic journals, white papers and presentations at conferences.

Inadequacies of Physical Examination as a Cause of Medical Errors and Adverse Events: A Collection of Vignettes.

BACKGROUND: Oversights in the physical examination are a type of medical error not easily studied by chart review. They may be a major contributor to missed or delayed diagnosis, unnecessary exposure to contrast and radiation, incorrect treatment, and other adverse consequences. Our purpose was to collect vignettes of physical examination oversights and to capture the diversity of their characteristics and consequences. METHODS: A cross-sectional study using an 11-question qualitative survey for physicians was distributed electronically, with data collected from February to June of 2011. The participants were all physicians responding to e-mail or social media invitations to complete the survey. There were no limitations on geography, specialty, or practice setting. RESULTS: Of the 208 reported vignettes that met inclusion criteria, the oversight was caused by a failure to perform the physical examination in 63%; 14% reported that the correct physical examination sign was elicited but misinterpreted, whereas 11% reported that the relevant sign was missed or not sought. Consequence of the physical examination inadequacy included missed or delayed diagnosis in 76% of cases, incorrect diagnosis in 27%, unnecessary treatment in 18%, no or delayed treatment in 42%, unnecessary diagnostic cost in 25%, unnecessary exposure to radiation or contrast in 17%, and complications caused by treatments in 4%. The mode of the number of physicians missing the finding was 2, but many oversights were missed by many physicians. Most oversights took up to 5 days to identify, but 66 took longer. Special attention and skill in examining the skin and its appendages, as well as the abdomen, groin, and genitourinary area could reduce the reported oversights by half. CONCLUSIONS: Physical examination inadequacies are a preventable source of medical error, and adverse events are caused mostly by failure to perform the relevant examination.

The Gonococcal Genetic Island and Type IV Secretion in the Pathogenic Neisseria.

Eighty percent of Neisseria gonorrhoeae strains and some Neisseria meningitidis strains encode a 57-kb gonococcal genetic island (GGI). The GGI was horizontally acquired and is inserted in the chromosome at the replication terminus. The GGI is flanked by direct repeats, and site-specific recombination at these sites results in excision of the GGI and may be responsible for its original acquisition. Although the role of the GGI in N. meningitidis is unclear, the GGI in N. gonorrhoeae encodes a type IV secretion system (T4SS). T4SS are versatile multi-protein complexes and include both conjugation systems as well as effector systems that translocate either proteins or DNA-protein complexes. In N. gonorrhoeae, the T4SS secretes single-stranded chromosomal DNA into the extracellular milieu in a contact-independent manner. Importantly, the DNA secreted through the T4SS is effective in natural transformation and therefore contributes to the spread of genetic information through Neisseria populations. Mutagenesis experiments have identified genes for DNA secretion including those encoding putative structural components of the apparatus, peptidoglycanases which may act in assembly, and relaxosome components for processing the DNA and delivering it to the apparatus. The T4SS may also play a role in infection by N. gonorrhoeae. During intracellular infection, N. gonorrhoeae requires the Ton complex for iron acquisition and survival. However, N. gonorrhoeae strains that do not express the Ton complex can survive intracellularly if they express structural components of the T4SS. These data provide evidence that the T4SS is expressed during intracellular infection and suggest that the T4SS may provide an advantage for intracellular survival. Here we review our current understanding of how the GGI and type IV secretion affect natural transformation and pathogenesis in N. gonorrhoeae and N. meningitidis.

Differential gene expression patterns and interaction networks in BCR-ABL-positive and -negative adult acute lymphoblastic leukemias.

PURPOSE: To identify gene expression patterns and interaction networks related to BCR-ABL status and clinical outcome in adults with acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: DNA microarrays were used to profile a set of 54 adult ALL specimens from the Medical Research Council UKALL XII/Eastern Cooperative Oncology Group E2993 trial (21 p185BCR-ABL-positive, 16 p210BCR-ABL-positive and 17 BCR-ABL-negative specimens). RESULTS: Using supervised and unsupervised analysis tools, we detected significant transcriptomic changes in BCR-ABL-positive versus -negative specimens, and assessed their validity in an independent cohort of 128 adult ALL specimens. This set of 271 differentially expressed genes (including GAB1, CIITA, XBP1, CD83, SERPINB9, PTP4A3, NOV, LOX, CTNND1, BAALC, and RAB21) is enriched for genes involved in cell death, cellular growth and proliferation, and hematologic system development and function. Network analysis demonstrated complex interaction patterns of these genes, and identified FYN and IL15 as the hubs of the top-scoring network. Within the BCR-ABL-positive subgroups, we identified genes overexpressed (PILRB, STS-1, SPRY1) or underexpressed (TSPAN16, ADAMTSL4) in p185BCR-ABL-positive ALL relative to p210BCR-ABL-positive ALL. Finally, we constructed a gene expression- and interaction-based outcome predictor consisting of 27 genes (including GRB2, GAB1, GLI1, IRS1, RUNX2, and SPP1), which correlated with overall survival in BCR-ABL-positive adult ALL (P = .0001), independent of age (P = .25) and WBC count at presentation (P = .003). CONCLUSION: We identified prominent molecular features of BCR-ABL-positive adult ALL, which may be useful for developing novel therapeutic targets and prognostic markers in this disease.

Substrate electric dipole moment exerts a pH-dependent effect on electron transfer in Escherichia coli photolyase.

Transient absorption spectroscopy is used to demonstrate that the electric dipole moment of the substrate cyclobutane thymine dimer affects the charge recombination reaction between fully reduced flavin adenine dinucleotide (FADH-) and the neutral radical tryptophan 306 (Trp306*) in Escherichia coli DNA photolyase. At pH 7.4, the charge recombination is slowed by a factor of 1.75 in the presence of substrate, but not at pH 5.4. Photolyase does bind substrate at pH 5.4, and it seems that this pH effect originates from the conversion of FADH- to FADH2 at lower pH. The free-energy changes calculated from the electric field parameters and from the change in electron transfer rate are in good agreement and support the idea that the substrate electric dipole is responsible for the observed change in electron transfer rate. It is expected that the substrate electric field will also modify the physiologically important from excited 1FADH- to the substrate in the DNA repair reaction.