Combining text mining with clinical decision support in clinical practice: a scoping review.

OBJECTIVE: Combining text mining (TM) and clinical decision support (CDS) could improve diagnostic and therapeutic processes in clinical practice. This review summarizes current knowledge of the TM-CDS combination in clinical practice, including their intended purpose, implementation in clinical practice, and barriers to such implementation. MATERIALS AND METHODS: A search was conducted in PubMed, EMBASE, and Cochrane Library databases to identify full-text English language studies published before January 2022 with TM-CDS combination in clinical practice. RESULTS: Of 714 identified and screened unique publications, 39 were included. The majority of the included studies are related to diagnosis (n = 26) or prognosis (n = 11) and used a method that was developed for a specific clinical domain, document type, or application. Most of the studies selected text containing parts of the electronic health record (EHR), such as reports (41%, n = 16) and free-text narratives (36%, n = 14), and 23 studies utilized a tool that had software "developed for the study". In 15 studies, the software source was openly available. In 79% of studies, the tool was not implemented in clinical practice. Barriers to implement these tools included the complexity of natural language, EHR incompleteness, validation and performance of the tool, lack of input from an expert team, and the adoption rate among professionals. DISCUSSION/CONCLUSIONS: The available evidence indicates that the TM-CDS combination may improve diagnostic and therapeutic processes, contributing to increased patient safety. However, further research is needed to identify barriers to implementation and the impact of such tools in clinical practice.

Contextualized Drug-Drug Interaction Management Improves Clinical Utility Compared With Basic Drug-Drug Interaction Management in Hospitalized Patients.

Drug-drug interactions (DDIs) frequently trigger adverse drug events or reduced efficacy. Most DDI alerts, however, are overridden because of irrelevance for the specific patient. Basic DDI clinical decision support (CDS) systems offer limited possibilities for decreasing the number of irrelevant DDI alerts without missing relevant ones. Computerized decision tree rules were designed to context-dependently suppress irrelevant DDI alerts. A crossover study was performed to compare the clinical utility of contextualized and basic DDI management in hospitalized patients. First, a basic DDI-CDS system was used in clinical practice while contextualized DDI alerts were collected in the background. Next, this process was reversed. All medication orders (MOs) from hospitalized patients with at least one DDI alert were included. The following outcome measures were used to assess clinical utility: positive predictive value (PPV), negative predictive value (NPV), number of pharmacy interventions (PIs)/1,000 MOs, and the median time spent on DDI management/1,000 MOs. During the basic DDI management phase 1,919 MOs/day were included, triggering 220 DDI alerts/1,000 MOs; showing 57 basic DDI alerts/1,000 MOs to pharmacy staff; PPV was 2.8% with 1.6 PIs/1,000 MOs costing 37.2 minutes/1,000 MOs. No DDIs were missed by the contextualized CDS system (NPV 100%). During the contextualized DDI management phase 1,853 MOs/day were included, triggering 244 basic DDI alerts/1,000 MOs, showing 9.6 contextualized DDIs/1,000 MOs to pharmacy staff; PPV was 41.4% (P < 0.01), with 4.0 PIs/1,000 MOs (P < 0.01) and 13.7 minutes/1,000 MOs. The clinical utility of contextualized DDI management exceeds that of basic DDI management.

Barriers and facilitators for systematically registering adverse drug reactions in electronic health records: a qualitative study with Dutch healthcare professionals.

BACKGROUND: Systematically registering ADRs in electronic health records (EHRs) likely contribute to patient safety as it enables the exchange of drug safety data. Currently, ADRs registrations by healthcare professionals (HCPs) is suboptimal. This study aimed to identify barriers and facilitators perceived by HCPs to register ADRs systematically in EHRs. RESEARCH DESIGN AND METHODS: A qualitative study with individual interviews was conducted among specialist physicians and hospital pharmacists from 10 different Dutch hospitals. A semi-structured interview guide was used to identify experienced barriers and facilitators for systematically registering ADRs. Data was analyzed following thematic analysis. Themes within barriers and facilitators were aligned with the Capability-Opportunity-Motivation-Behavior (COM-B) framework. RESULTS: In total, 16 HCPs were interviewed. Identified barriers were: lack of knowledge to recognize ADRs, time constraints, inadequate IT system, lack of support, stuck in routine, and not recognizing the importance of registering ADRs. Identified facilitators were: enhanced knowledge and awareness of ADRs, functional IT systems, expanding accountability for registration, and motivation toward registering. CONCLUSIONS: Barriers and facilitators for registering spanned all aspects of the COM-B model and occurred in individual, social and environmental domains. Addressing these aspects could improve the registration of ADRs and may contribute to patient safety.

Clinical Decision Support System-Assisted Pharmacy Intervention Reduces Feeding Tube-Related Medication Errors in Hospitalized Patients: A Focus on Medication Suitable for Feeding-Tube Administration.

BACKGROUND: Administering medication through an enteral feeding tube (FT) is a frequent cause of errors resulting in increased morbidity and cost. Studies on interventions to prevent these errors in hospitalized patients, however, are limited. OBJECTIVE: The objective was to study the effect of a clinical decision support system (CDSS)-assisted pharmacy intervention on the incidence of FT-related medication errors (FTRMEs) in hospitalized patients. METHODS: A pre-post intervention study was conducted between October 2014 and May 2015 in Catharina Hospital, the Netherlands. Patients who were admitted to the wards of bowel and liver disease, oncology, or neurology; using oral medication; and had an enteral FT were included. Preintervention patients were given care as usual. The intervention consisted of implementing a CDSS-assisted pharmacy check while also implementing standard operating procedures and educating personnel. An FTRME was defined as the administration of inappropriate medication through an enteral FT. The incidence was expressed as the number of FTRMEs per medication administration. Multivariate Poisson regression was used to calculate the incidence ratio (IR) comparing both phases. RESULTS: Eighty-one patients were included, 38 during preintervention and 43 during the intervention phase. Incidence of FTRMEs in the preintervention phase was 0.15 (95% CI, 0.07-0.23) vs 0.02 (95% CI, 0.00-0.04) in the intervention phase, resulting in an adjusted IR of 0.13 (95% CI, 0.10-0.18). DISCUSSION: Incidence of FTRMEs, as well as the IR, is comparable to previous studies. CONCLUSION: The intervention resulted in a substantial reduction in the incidence of FTRMEs.

Citalopram intoxication in four week old infant.

BACKGROUND: In contrast to intoxications in toddlers which can be due to accidental ingestions, many intoxications in infants are due to medication errors. To our knowledge, this is the first case report of a citalopram intoxication in an infant, and may offer new insight on possible screening methods for intoxication as well as pharmacokinetics of citalopram in small infants. CASE PRESENTATION: This case report describes an unintentional citalopram intoxication in a 4 week old infant due to a vitamin D drops 'look alike' error. The infant showed extreme jitteriness and opisthotonus at presentation, as well as prolonged signs of gastro-oesophageal reflux. No cardiac rhythm disturbances or convulsions were seen. The clinical course combined with Finnegan scores was correlated to and supported by pharmacokinetic and pharmacokinetic data of citalopram in the patient. CONCLUSIONS: Using Finnegan scores in general pediatric practice could help objectify follow-up of acute intoxications in young infants with neurological symptoms.

Clinical rule-guided pharmacists' intervention in hospitalized patients with hypokalaemia: A time series analysis.

WHAT IS KNOWN AND OBJECTIVE: Physicians' response to moderate and severe hypokalaemia in hospitalized patients is frequently suboptimal, leading to increased risk of cardiac arrhythmias and sudden death. While actively alerting physicians on all critical care values using telephone or electronic pop-ups can improve response, it can also lead to alert fatigue and frustration due to non-specific and overdue alerts. Therefore, a new method was tested. A clinical rule built into a clinical decision support system (CDSS) generated alerts for patients with a serum potassium level (SPL) <2.9 mmol/L without a prescription for potassium supplementation. If the alert was deemed clinically relevant, a pharmacist contacted the physician. The aim of this study was to evaluate the impact of the clinical rule-guided pharmacists' intervention compared to showing passive alerts in the electronic health records on outcome in patients who developed hypokalaemia (<2.9 mmol/L) during hospitalization. METHODS: A before (2007-2009) and after (2010-2017) study with time series design was performed. Pre-intervention, physicians were shown passive alerts for hypokalaemia in the electronic health records. During the intervention period, in addition to these passive alerts, a pharmacist provided the physician with a specific advice on patients with untreated hypokalaemia, guided by the generated alerts. Unique patients >18 years with SPL <2.9 mmol/L measured at least 24 hours after hospitalization in whom no potassium supplementation was initiated within 4 hours after measurement and normalization of SPL was not achieved within these 4 hours were included. Haemodialysis patients were excluded. The percentage of hypokalaemic patients with a subsequent prescription for potassium supplementation, time to subsequent potassium supplementation prescription, the percentage of patients who achieved normokalaemia (SPL ≥ 3.0 mmol/L), time to achieve normokalaemia and total duration of hospitalization were compared. RESULTS AND DISCUSSION: A total of 693 patients were included, of whom 278 participated in the intervention phase. The percentage of patients prescribed supplementation as well as time to prescription improved from 76.0% in 31.1 hours to 92.0% in 11.3 hours (P < .01). Time to achieve SPL ≥3.0 mmol/L improved, P < .009. No changes, however, were observed in the percentage of patients who achieved normokalaemia or time to reach normokalaemia, 87.5% in 65.2 hours pre-intervention compared to 90.2% (P = .69) in 64.0 hours (P = .71) in the intervention group. A non-significant decrease of 8.2 days was observed in the duration of hospitalization: 25.4 compared to 17.2 days (P = .29). WHAT IS NEW AND CONCLUSION: Combining CDSS alerting with a pharmacist evaluation is an effective method to improve response rate, time to supplementation and time to initial improvement, defined as SPL ≥3.0 mmol/L. However, it showed no significant effect on the percentage of patients achieving normokalaemia, time to normokalaemia or hospitalization. The discrepancy between rapid supplementation and improvement on the one hand and failure to improve time to normokalaemia on the other warrants further study.

Psychotropic Drug Prescription and the Risk of Falls in Nursing Home Residents.

BACKGROUND: Falling is a common and serious problem in the elderly. Previous studies suggest that the use of psychotropic drugs increases the risk of falling. However, the contribution of these drugs on fall risk has not been quantified on a daily basis among the general population of nursing homes until now. OBJECTIVE: To assess the association between fall incidence and the prescription of psychotropic drugs and different categories of psychotropic drugs (antipsychotics, antidepressants, and benzodiazepines) among a general nursing home population. DESIGN: Retrospective observational study, data collection per person-day. SETTING: 9 nursing homes in Eindhoven, the Netherlands. PARTICIPANTS: 2368 nursing home residents, resulting in 538,575 person-days. MAIN OUTCOME MEASURE: Association between the prescription of psychotropic drugs and falls. RESULTS: A total of 2368 nursing home residents were included, which resulted in a data set of 538,575 person-days. Prescription of at least 1 psychotropic drug per day occurred during a total of 318,128 person-days (59.1%). Scheduled prescriptions with or without an as-needed prescription were involved in a total of 270,781 person-days (50.3%). The prescription of psychotropic drugs on a scheduled basis was found to be associated with almost a 3-fold increase in fall incidence (OR 2.88; 95% CI 1.52-5.44). An increase in fall incidence was found following the prescription of antipsychotics (OR 1.97; 95% CI 1.51-2.59) and antidepressants (OR 2.26; 95% CI 1.73-2.95). This increased fall risk was found for prescriptions on a scheduled basis as well as for prescriptions on an as-needed basis. CONCLUSION: The prescription of psychotropic drugs is associated with a strongly increased risk of falling among nursing home residents. To our knowledge, this is the first study among the general nursing home population in which the association between daily falls and daily prescriptions of psychotropic drugs and groups of psychotropic drugs was specified.

A Novel Catastrophic Presentation of X-Linked Adrenoleukodystrophy.

OBJECTIVE: We report a novel presentation of childhood cerebral X-linked adrenoleukodystrophy: status epilepticus followed by abrupt and catastrophic neurologic deterioration. METHODS: A description of the clinical presentation, laboratory evaluation, and imaging findings leading to a diagnosis of X-linked adrenoleukodystrophy. RESULTS: A 3-year-old male with prior history of autism presented with fever, diarrhea, and status epilepticus requiring a pentobarbital coma. Admission labs were notable only for a glucose level of 22 mg/dL, which stabilized after correction. The child never returned to his prior neurologic baseline, with complete loss of gross motor, fine motor, and speech skills. Serial brain magnetic resonance imaging (MRI)/magnetic resonance spectroscopy (MRS) was notable for progressive diffuse cortical signal changes with swelling, diffusion restriction, and ultimately laminar necrosis. Nine months after presentation, CSF (cerebrospinal fluid) protein and MRS lactate were persistently elevated, concerning for a neurodegenerative disorder. This led to testing for mitochondrial disease, followed by lysosomal and peroxisomal disorders. Very long-chain fatty acids were elevated. Identification of a pathogenic ABCD1 mutation confirmed the diagnosis of X-linked adrenoleukodystrophy. CONCLUSIONS: Boys with childhood cerebral X-linked adrenoleukodystrophy typically present with gradual behavioral changes. Rare reports of boys presenting with transient altered mental status or status epilepticus describe a recovery to their pre-presentation baseline. To our knowledge this is the first X-ALD patient to present with status epilepticus with abrupt and catastrophic loss of neurologic function. X-linked adrenoleukodystrophy should be suspected in young males presenting with seizures, acute decline in neurologic function, with persistently elevated CSF protein and MRS lactate.

Prevalence and level of Escherichia coli O157:H7 in feces and on hides of feedlot steers fed diets with or without wet distillers grains with solubles.

The objective of this study was to determine if wet distillers grains with solubles (WDGS) from corn in diets affected Escherichia coli O157:H7 in growing and finishing cattle; steers (n = 603) were randomly assigned to diets with or without WDGS. Hide and fecal samples were collected monthly (October through June) from each animal for enumeration and enrichment of E. coli O157:H7. In the growing phase (0 or 13.9% WDGS diets), fecal prevalence for E. coli O157:H7 in steers fed a diet with WDGS was twice that of the prevalence in control steers (P < 0.001). In the finishing phase (0 or 40% WDGS diets), the average prevalence in feces (P < 0.001) and on hides (P < 0.001) was higher for cattle fed WDGS. The average percentage of fecal E. coli O157:H7 enumerable samples during the finishing phase for cattle fed WDGS was 2.7% compared with 0.1% for control steers (P < 0.001). The average percentage of E. coli O157:H7 enumerable hide samples was not different between diets, but the cattle fed WDGS had higher levels (P < 0.05) of the pathogen. Animals fed WDGS had higher levels of E. coli (P < 0.001), higher pH values (P < 0.001), and lower concentrations of L-lactate (P < 0.001) in feces than those values of the control steers. These results indicate that feeding 40% WDGS could increase the level and prevalence of E. coli O157:H7 in and on feedlot cattle when E. coli O157:H7 is seasonally low.

Prevalence of Mycobacterium avium subsp. paratuberculosis in ileocecal lymph nodes and on hides and carcasses from cull cows and fed cattle at commercial beef processing plants in the United States.

Clinical associations between Crohn's disease in humans and Mycobacterium avium subsp. paratuberculosis (MAP) have been suggested but not confirmed. Cattle could be sources for MAP, but little information on MAP prevalence with beef has been reported. Samples of ileocecal lymph nodes and swabs of hides and carcasses from 343 animals at cull cattle slaughtering facilities and 243 animals at fed cattle slaughtering facilities across the United States were analyzed for the presence of MAP. Amplification of genetic sequences detected MAP DNA predominantly on hides and in lymph nodes of samples taken at both types of processing facilities. More than 34% of the cattle at cull cow slaughtering facilities had ileocecal lymph nodes that tested positive for MAP DNA. From these same cattle, hide prevalence was more than twofold greater than the prevalence in ileocecal lymph nodes, suggesting that cross-contamination could be occurring during transport and lairage. The prevalence of MAP DNA decreased during processing, and less than 11% of the carcasses tested positive after interventions in the cull cow processing facilities. Using standard double-decontamination and culture techniques, less than 1% of the postintervention carcasses tested positive for viable MAP at cull cow facilities. In samples from the facilities processing only fed cattle, MAP prevalence of 1% or less was detected for ileocecal lymph node, hide, and carcass samples, and viable MAP was not detected. Based on this study, fed cattle carcasses are unlikely sources of MAP, and carcasses at cull cow plants have only a slight risk for transmitting viable MAP, due to current interventions.

Enumeration of Salmonella from poultry carcass rinses via direct plating methods.

AIM: To evaluate direct plating methods for the estimation of Salmonella load in poultry carcass rinses. METHODS AND RESULTS: Two direct plating tools, the spiral plate count method (SPCM) and the hydrophobic grid membrane filtration (HGMF) method, were adapted to support quantification of Salmonella during poultry processing. Test samples consisted of 180 broiler carcasses from a commercial abattoir, 60 from each of three points in the processing line [pre-inside-outside bird wash (pre-IOBW), prechill and postchill]. The SPCM was used to estimate Salmonella load in pre-IOBW rinses, while HGMF was used to estimate Salmonella levels in prechill and postchill rinses. Carcass rinses were also evaluated for Salmonella prevalence by enrichment methods. Mean prevalences of Salmonella were 95%, 100% and 41.7%, and the geometric mean loads were 3.7 x 10(1), 5.6 x 10(0) and 5.0 x 10(-2) CFU ml(-1) for pre-IOBW, prechill and postchill rinses, respectively. CONCLUSIONS: The methods described are useful for estimating the concentration of viable and typical Salmonella in poultry carcass rinses. SIGNIFICANCE AND IMPACT OF THE STUDY: Direct plating enumeration methods can facilitate the monitoring of Salmonella load on poultry carcasses throughout the production process, and the evaluation of new processing intervention strategies.

Enumeration of Salmonella and Escherichia coli O157:H7 in ground beef, cattle carcass, hide and faecal samples using direct plating methods.

AIM: To develop and validate high throughput methods for the direct enumeration of viable and culturable Salmonella and Escherichia coli O157:H7 in ground beef, carcass, hide and faecal (GCHF) samples from cattle. METHODS AND RESULTS: The hydrophobic grid membrane filtration (HGMF) method and the spiral plate count method (SPCM) were evaluated as rapid tools for the estimation of pathogen load using GCHF samples spiked with known levels of Salmonella serotype Typhimurium. Validation studies showed that for a single determination of each sample type the low end of the detection limits were approx. 2.0 x 10(0) CFU g(-1) for ground beef, 5.0 x 10(-1) CFU (100 cm(2))(-1) for Salmonella and 8.0 x 10(-1) CFU (100 cm(2))(-1) for E. coli O157:H7 on carcasses, 4.0 x 10(1) CFU (100 cm(2))(-1) for hide and 2.0 x 10(2) CFU g(-1) for faecal samples. In addition, ground beef (n = 609), carcass (n = 1520) and hide (n = 3038) samples were collected from beef-processing plants and faecal samples (n = 3190) were collected from feed-lot cattle, and these samples were tested for the presence of Salmonella and E. coli O157:H7 by enrichment and enumeration methods. CONCLUSIONS: The direct enumeration methods described here are amenable to high throughput sample processing and were found to be cost-effective alternatives to other enumeration methods for the estimation of Salmonella and E. coli O157:H7, in samples collected during cattle production and beef processing. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of the methods described here would allow for more routine testing and quantification data collection, providing useful information about the effectiveness of beef processing intervention strategies.

Interventions to reduce/eliminate Escherichia coli O157:H7 in ground beef.

The Escherichia coli O157:H7 (E. coli O157:H7) outbreak in the Northwestern United States ushered in an era that has dramatically changed the way beef processors in the United States convert live cattle into meat. Unprecedented cooperation among the beef processors and massive investment in research by the US government and the beef industry have resulted in an acceptable level of control of E. coli O157:H7 in ground beef. The evidence to support the progress in control of E. coli O157:H7 is the CDC data for reduction in human illness as well as the dramatic reduction in the number of E. coli O157:H7-positive samples in USDA-FSIS ground beef monitoring. This manuscript highlights some of the recent findings from our laboratory on the control of E. coli O157:H7 in ground beef. We have also summarized the key events/decisions/milestones that have contributed to the control of E. coli O157:H7 in ground beef in the United States. While there is much to be done to bring E. coli O157:H7 under complete control in the beef sector of the food industry, E. coli O157:H7 also is becoming a major issue in the fresh vegetable sector, as evidenced by the 2006 outbreaks in the United States. We have discussed how the fresh vegetable industry can benefit from the beef industry's experience to expedite the control of E. coli O157:H7 in their products.

Post-harvest interventions to reduce/eliminate pathogens in beef.

In 1999 the foodborne pathogens Salmonella, Listeria, Campylobacter, and Escherichia coli (both O157 and non-O157) were estimated to cause more than 6 million illnesses and approximately 9000 deaths each year. However, the most recent Centers for Disease Control and Prevention report on the sources and incidence of foodborne disease, released in 2004, has shown a dramatic decrease in E. coli O157:H7 infections. Since raw beef products are the most frequently foodborne sources of these pathogens, the results of this report demonstrate that the microbiological quality of raw beef has improved greatly. During the intervening years, post-harvest interventions have continually improved, with new attention to hide decontamination and innovative treatments of carcasses. In addition, a system to hold and test beef trim or ground beef for E. coli O157:H7 before its release into commerce has provided an even greater level of safety. In this paper, we review the latest information on the prevalence of E. coli O157:H7 and other pathogens on beef, the evidence identifying the hide as the primary source of pathogens on beef carcasses, the efficacy of various hide and carcass interventions, and other developments that have led or have the potential to lead to even greater improvements in the microbial quality of beef.

Binding of the initiation factor sigma(70) to core RNA polymerase is a multistep process.

The interaction of RNA polymerase and its initiation factors is central to the process of transcription initiation. To dissect the role of this interface, we undertook the identification of the contact sites between RNA polymerase and sigma(70), the Escherichia coli initiation factor. We identified nine mutationally verified interaction sites between sigma(70) and specific domains of RNA polymerase and provide evidence that sigma(70) and RNA polymerase interact in at least a two-step process. We propose that a cycle of changes in the interface of sigma(70) with core RNA polymerase is associated with progression through the process of transcription initiation.

Genotypic analyses of Escherichia coli O157:H7 and O157 nonmotile isolates recovered from beef cattle and carcasses at processing plants in the Midwestern states of the United States.

Escherichia coli O157:H7 and O157 nonmotile isolates (E. coli O157) previously were recovered from feces, hides, and carcasses at four large Midwestern beef processing plants (R. O. Elder, J. E. Keen, G. R. Siragusa, G. A. Barkocy-Gallagher, M. Koohmaraie, and W. W. Laegreid, Proc. Natl. Acad. Sci. USA 97:2999-3003, 2000). The study implied relationships between cattle infection and carcass contamination within single-source lots as well as between preevisceration and postprocessing carcass contamination, based on prevalence. These relationships now have been verified based on identification of isolates by genomic fingerprinting. E. coli O157 isolates from all positive samples were analyzed by pulsed-field gel electrophoresis of genomic DNA after digestion with XbaI. Seventy-seven individual subtypes (fingerprint patterns) grouping into 47 types were discerned among 343 isolates. Comparison of the fingerprint patterns revealed three clusters of isolates, two of which were closely related to each other. Remarkably, isolates carrying both Shiga toxin genes and nonmotile isolates largely fell into specific clusters. Within lots analyzed, 68.2% of the postharvest (carcass) isolates matched preharvest (animal) isolates. For individual carcasses, 65.3 and 66.7% of the isolates recovered postevisceration and in the cooler, respectively, matched those recovered preevisceration. Multiple isolates were analyzed from some carcass samples and were found to include strains with different genotypes. This study suggests that most E. coli O157 carcass contamination originates from animals within the same lot and not from cross-contamination between lots. In addition, the data demonstrate that most carcass contamination occurs very early during processing.

A coiled-coil from the RNA polymerase beta' subunit allosterically induces selective nontemplate strand binding by sigma(70).

For transcription to initiate, RNA polymerase must recognize and melt promoters. Selective binding to the nontemplate strand of the -10 region of the promoter is central to this process. We show that a 48 amino acid (aa) coiled-coil from the beta' subunit (aa 262--309) induces sigma(70) to perform this function almost as efficiently as core RNA polymerase itself. We provide evidence that interaction between the beta' coiled-coil and region 2.2 of sigma(70) promotes an allosteric transition that allows sigma(70) to selectively recognize the nontemplate strand. As the beta' 262--309 peptide can function with the previously crystallized portion of sigma(70), nontemplate recognition can be reconstituted with only 47 kDa, or 1/10 of holoenzyme.

Mutational analysis of beta '260-309, a sigma 70 binding site located on Escherichia coli core RNA polymerase.

In eubacteria, the final sigma subunit binds to the core RNA polymerase and directs transcription initiation from any of its cognate set of promoters. Previously, our laboratory defined a region of the beta' subunit that interacts with final sigma(70) in vitro. This region of beta' contained heptad repeat motifs indicative of coiled coils. In this work, we used 10 single point mutations of the predicted coiled coils, located within residues 260-309 of beta', to look at disruption of the final sigma(70)-core interaction. Several of the mutants were defective for binding final sigma(70) in vitro. Of these mutants, three (R275Q, E295K, and A302D) caused cells to be inviable in an in vivo assay in which the mutant beta' is the sole source of beta' subunit for the cell. All of the mutants were able to assemble into the core enzyme; however, R275Q, E295K, A302D were defective for Efinal sigma(70) holoenzyme formation. Several of the mutants were also defective for holoenzyme assembly with various minor final sigma factors. In the recently published crystal structure of Thermus aquaticus core RNA polymerase (Zhang, G., Campbell, E. A., Minakhin, L., Richter, C., Severinov, K. , and Darst, S. A. (1999) Cell 98, 811-824), the region homologous to beta'(260-309) of Escherichia coli forms a coiled coil. Modeling of our mutations onto that coiled coil places the most defective mutations on one face of the coiled coil.

Phenotypic stability of chick cardiomyocytes in serum-free media. Preservation of muscarinic receptor expression.

Chick cardiomyocytes cultured in fetal bovine serum (FBS)-supplemented media are phenotypically unstable, becoming noncontractile and unresponsive to stimuli after several days. We report a culturing protocol that preserves the differentiated cardiomyocyte phenotype for at least 9 days in culture. Cardiomyocytes isolated from 11-day chicken embryos, and cultured in either Dulbecco's Modified Earle's Medium (DMEM)/Ham's F12 medium with N-2 supplement or Medium 199 (M199) with 10% FBS continued to beat spontaneously for 4-5 days; only cells cultured in N-2-supplemented medium exhibited spontaneous beating beyond 5 days. Immunostaining for alpha-actinin after 9 days in culture revealed that myofibrils persisted in N-2-supplemented cells, while no myofibrils were observed in the FBS-supplemented cells. For cells in FBS-supplemented media, [3H]thymidine incorporation rates were 7.5 and 3 times greater than that of cells in N-2-supplemented media at Days 4 and 9 in culture, respectively. The effect of growth media on the binding parameters of the muscarinic antagonist, [3H]N-methyl-scopolamine (NMS), was also compared. While B(max) decreased 34% between Days 4 and 9 for cells maintained in N-2-supplemented media, a 77% decrease was observed for cells cultured in FBS-supplemented media. The phenotypic stability of this preparation makes it feasible for the first time to use these cells in experiments that require more than 4 days to complete.