The Trypanosoma cruzi RNA-binding protein RBP42 is expressed in the cytoplasm throughout the life cycle of the parasite.

Trypanosoma cruzi, the protozoan parasite that causes Chagas disease in humans, has a complex life cycle that promotes survival in disparate environments. In each environment, the parasite must fine-tune its metabolic pathways to divide and multiply. In the absence of recognizable transcriptional gene regulation, it is apparent that protein levels are determined by post-transcriptional mechanisms. Post-transcriptional gene control is influenced by RNA-binding proteins that target mRNAs in the cell's cytoplasm. To initiate the study of post-transcriptional activities in T. cruzi, we studied this organism's ortholog of RBP42, a trypanosomal RNA-binding protein. RBP42 was originally detected in Trypanosoma brucei and was shown to target a subset of mRNAs that encode proteins governing central carbon metabolism. T. cruzi RBP42 structurally resembles T. brucei RBP42, sharing an NTF2 domain at its amino terminus and a single RNA-binding domain (specifically, the RNA recognition motif, or RRM), at its carboxy terminus. A phylogenetic analysis reveals that an NTF2 and a single RRM are distinguishing features of all RBP42 orthologs within the broad kinetoplastid grouping. T. cruzi RBP42 is expressed in all life cycle stages of the parasite as determined by immunoblot and immunofluorescence microscopy. In each case, the protein is localized to the cytoplasm, indicating a role for T. cruzi RBP42 in post-transcriptional activities in all stages of the parasite life cycle. We speculate that RBP42 influences the dynamic metabolic pathways responsible for parasite infection and transmission.

An essential domain of an early-diverged RNA polymerase II functions to accurately decode a primitive chromatin landscape.

A unique feature of RNA polymerase II (RNA pol II) is its long C-terminal extension, called the carboxy-terminal domain (CTD). The well-studied eukaryotes possess a tandemly repeated 7-amino-acid sequence, called the canonical CTD, which orchestrates various steps in mRNA synthesis. Many eukaryotes possess a CTD devoid of repeats, appropriately called a non-canonical CTD, which performs completely unknown functions. Trypanosoma brucei, the etiologic agent of African Sleeping Sickness, deploys an RNA pol II that contains a non-canonical CTD to accomplish an unusual transcriptional program; all protein-coding genes are transcribed as part of a polygenic precursor mRNA (pre-mRNA) that is initiated within a several-kilobase-long region, called the transcription start site (TSS), which is upstream of the first protein-coding gene in the polygenic array. In this report, we show that the non-canonical CTD of T. brucei RNA pol II is important for normal protein-coding gene expression, likely directing RNA pol II to the TSSs within the genome. Our work reveals the presence of a primordial CTD code within eukarya and indicates that proper recognition of the chromatin landscape is a central function of this RNA pol II-distinguishing domain.

Enhancing tolerance to short-chain alcohols by engineering the Escherichia coli AcrB efflux pump to secrete the non-native substrate n-butanol.

The microbial conversion of sugars to fuels is a promising technology, but the byproducts of biomass pretreatment processes and the fuels themselves are often toxic at industrially relevant levels. One promising solution to these problems is to engineer efflux pumps to secrete fuels and inhibitory chemicals from the cell, increasing microbial tolerance and enabling higher fuel titer. Toward that end, we used a directed evolution strategy to generate variants of the Escherichia coli AcrB efflux pump that act on the non-native substrate n-butanol, enhancing growth rates of E. coli in the presence of this biofuel by up to 25%. Furthermore, these variants confer improved tolerance to isobutanol and straight-chain alcohols up to n-heptanol. Single amino acid changes in AcrB responsible for this phenotype were identified. We have also shown that both the chemical and genetic inactivation of pump activity eliminate the tolerance conferred by AcrB pump variants, supporting our assertion that the variants secrete the non-native substrates. This strategy can be applied to create an array of efflux pumps that modulate the intracellular concentrations of small molecules of interest to microbial fuel and chemical production.

Change, exchange, and rearrange: protein engineering for the biotechnological production of fuels, pharmaceuticals, and other chemicals.

Enzymes are indispensable in the effort to produce chemicals from fuels to pharmaceuticals in an ecologically friendly manner. They have the potential to catalyze reactions with high specificity and efficiency without the use of hazardous chemicals. Nature provides an extensive collection of enzymes, but often these must be altered to perform desired functions under required conditions. Advances in protein engineering permit the design and/or directed evolution of enzymes specifically tailored for such industrial applications. Recent years have seen the development of improved enzymes to assist in both the conversion of biomass into fuels and chemicals, and the creation of key intermediates in pharmaceutical production.

The impact of ethnic population dynamics on neonatal ECMO outcomes: a single urban institutional study.

INTRODUCTION: Neonatal extracorporeal membrane oxygenation ECMO has been clinically used for the last 25 y. It has been an effective tool for both cardiac and non cardiac conditions. The impact of ethno-demographic changes on ECMO outcomes however remains unknown. We evaluated a single institution's experience with non cardiac neonatal ECMO over a 28-y period. METHODS: A retrospective review of all neonates undergoing noncardiac ECMO between the y 1984 and 2011 was conducted and stratified into year groups I, II, III (≤1990, 1991-2000, and ≥2001). Demographic, clinical, and outcome data were collected. The patient specifics, ECMO type, ECMO length, blood use, complications, and outcomes were analyzed. Univariate, bivariate, and multivariate analyses were then performed. RESULTS: Data was available for 827 patients. The number of African-American and Hispanic patients increased over the last 27 y (27.5% versus 45.0% and 3.3% versus 21.5%, year group I versus year group III, respectively). The proportion of congenital diaphragmatic hernia (CDH) patients by ethnicity also increased for African-Americans and Hispanics between the two year groups (22.0% to 33.0% and 4.9% to 33.0%, respectively). Similar pattern was noted for non-CDH diagnoses. Low birth weight, low APGAR scores, CDH, primary pulmonary hypertension, central nervous system hemorrhage, and ECMO were independent predictors of mortality. Ethnicity, in itself however, was not associated with mortality on adjusted analysis. CONCLUSION: More African-Americans and Hispanics have required ECMO over the years with a concurrent decrease in the number of Caucasians. While ethnicity was not an independent predictor of mortality, it appears to be a surrogate for fatal but sometime preventable diagnoses among minorities. Further investigations are needed to better delineate the reason behind this disparity.

Open reduction internal fixation versus hemiarthroplasty versus total hip arthroplasty in the elderly: a review of the National Surgical Quality Improvement Program database.

BACKGROUND: Total hip arthroplasty (THA), hemiarthroplasty (HA), and open reduction internal fixation (ORIF) are treatment options for femoral neck fractures. However, the optimal surgical treatment remains unclear. The present study aimed to describe the 30-d postoperative outcomes of THA, HA, and ORIF among patients aged ≥65 y with femoral neck fractures within a national surgical database. MATERIALS AND METHODS: A retrospective analysis of the American College of Surgeons National Surgical Quality Improvement Program for January 2005 through December 2009 was conducted. We included patients aged ≥65 y who had undergone THA, HA, or ORIF for femoral neck fractures. We collected information on patient demographics, comorbidities, risk factors, and complication rates. A logistic regression model was used to assess the variation in overall morbidity and mortality after surgery. RESULTS: Overall, 3423 patients met the inclusion criteria: 674 underwent ORIF, 428 HA, and 2321 THA. Most patients were white (83.6%, n = 2862), female (64.4%, n = 2204), and >70 y old (78.4%, n = 2682). On adjusted multivariate analysis, no differences were found in the 30-d mortality rates among the ORIF, HA, and THA groups. Patients who underwent ORIF (odds ratio 0.51, 95% confidence interval 0.27-0.94) and HA (odds ratio 0.43, 95% confidence interval 0.22-0.84) had a lower likelihood of developing respiratory complications compared with those who underwent THA. CONCLUSIONS: No differences were found in the 30-d mortality rates among the ORIF, HA, and THA groups. ORIF and HA resulted in a lower likelihood of developing respiratory complications than THA.

De novo designed proteins from a library of artificial sequences function in Escherichia coli and enable cell growth.

A central challenge of synthetic biology is to enable the growth of living systems using parts that are not derived from nature, but designed and synthesized in the laboratory. As an initial step toward achieving this goal, we probed the ability of a collection of >10(6) de novo designed proteins to provide biological functions necessary to sustain cell growth. Our collection of proteins was drawn from a combinatorial library of 102-residue sequences, designed by binary patterning of polar and nonpolar residues to fold into stable 4-helix bundles. We probed the capacity of proteins from this library to function in vivo by testing their abilities to rescue 27 different knockout strains of Escherichia coli, each deleted for a conditionally essential gene. Four different strains--ΔserB, ΔgltA, ΔilvA, and Δfes--were rescued by specific sequences from our library. Further experiments demonstrated that a strain simultaneously deleted for all four genes was rescued by co-expression of four novel sequences. Thus, cells deleted for ∼0.1% of the E. coli genome (and ∼1% of the genes required for growth under nutrient-poor conditions) can be sustained by sequences designed de novo.