Guardians of the phloem - forisomes and beyond.

The phloem is a highly specialized vascular tissue that forms a fundamentally important transport and signaling pathway in plants. It is therefore a system worth protecting. The main function of the phloem is to transport the products of photosynthesis throughout the whole plant, but it also transports soluble signaling molecules and propagates electrophysiological signals. The phloem is constantly threatened by mechanical injuries, phloem-sucking pests and parasites, and the spread of pathogens, which has led to the evolution of efficient defense mechanisms. One such mechanism involves structural phloem proteins, which are thought to facilitate sieve element occlusion following injury and to defend the plant against pathogens. In leguminous plants, specialized structural phloem proteins known as forisomes form unique mechanoproteins via sophisticated molecular interaction and assembly mechanisms, thus enabling reversible sieve element occlusion. By understanding the structure and function of forisomes and other structural phloem proteins, we can develop a toolbox for biotechnological applications in material science and medicine. Furthermore, understanding the involvement of structural phloem proteins in plant defense mechanisms will allow phloem engineering as a new strategy for the development of crop varieties that are resistant to pests, pathogens and parasites.

The Ca2+ response of a smart forisome protein is dependent on polymerization.

Forisomes are giant self-assembling mechanoproteins that undergo reversible structural changes in response to Ca2+ and various other stimuli. Artificial forisomes assembled from the monomer MtSEO-F1 can be used as smart biomaterials, but the molecular basis of their functionality is not understood. To determine the role of protein polymerization in forisome activity, we tested the Ca2+ association of MtSEO-F1 dimers (the basic polymerization unit) by circular dichroism spectroscopy and microscale thermophoresis. We found that soluble MtSEO-F1 dimers neither associate with Ca2+ nor undergo structural changes. However, polarization modulation infrared reflection absorption spectroscopy revealed that aggregated MtSEO-F1 dimers and fully-assembled forisomes associate with Ca2+ , allowing the hydration of poorly-hydrated protein areas. A change in the signal profile of complete forisomes indicated that Ca2+ interacts with negatively-charged regions in the protein complexes that only become available during aggregation. We conclude that aggregation is required to establish the Ca2+ response of forisome polymers.

The functionality of plant mechanoproteins (forisomes) is dependent on the dual role of conserved cysteine residues.

Forisomes are giant polyprotein complexes that undergo reversible conformational rearrangements from a spindle-like to a plug-like state in response to Ca2+ or changes in pH. They act as valves in the plant vasculature, and reproduce this function in vitro to regulate flow in microfluidic capillaries controlled by electro-titration. Heterologous expression in yeast or plants allows the large-scale production of tailor-made artificial forisomes for technical applications. Here we investigated the unexpected disintegration of artificial forisomes in response to Ca2+ following the deletion of the M1 motif in the MtSEO-F1 protein or the replacement of all four conserved cysteine residues therein. This phenomenon could be mimicked in wild-type forisomes under reducing conditions by adding a thiol alkylating agent. We propose a model in which reversible changes in forisome structure depend on cysteine residues with ambiguous redox states, allowing the formation of intermolecular disulfide bridges (confirmed by mass spectrometry) as well as noncovalent thiol interactions to connect forisome substructures in the dispersed state. This is facilitated by the projection of the M1 motif from the MtSEO-F1 protein as part of an extended loop. Our findings support the rational engineering of disintegrating forisomes to control the release of peptides or enzymes in microfluidic systems.

Identification and molecular analysis of interaction sites in the MtSEO-F1 protein involved in forisome assembly.

Forisomes are large mechanoprotein complexes found solely in legumes such as Medicago truncatula. They comprise several "sieve element occlusion by forisome" (SEO-F) subunits, with MtSEO-F1 as the major structure-forming component. SEO-F proteins possess three conserved domains -an N-terminal domain (SEO-NTD), a potential thioredoxin fold, and a C-terminal domain (SEO-CTD)- but structural and biochemical data are scarce and little is known about the contribution of these domains to forisome assembly. To identify key amino acids involved in MtSEO-F1 dimerization and complex formation, we investigated protein-protein interactions by bimolecular fluorescence complementation and the analysis of yeast two-hybrid and random mutagenesis libraries. We identified a SEO-NTD core region as the major dimerization site, with abundant hydrophobic residues and rare charged residues suggesting dimerization is driven by the hydrophobic effect. We also found that ~45% of the full-length MtSEO-F1 sequence must be conserved for higher-order protein assembly, indicating that large interaction surfaces facilitate stable interactions, contributing to the high resilience of forisome bodies. Interestingly, the removal of 62 amino acids from the C-terminus did not disrupt forisome assembly. This is the first study unraveling interaction sites and mechanisms within the MtSEO-F1 protein at the level of dimerization and complex formation.

Forizymes - functionalised artificial forisomes as a platform for the production and immobilisation of single enzymes and multi-enzyme complexes.

The immobilisation of enzymes plays an important role in many applications, including biosensors that require enzyme activity, stability and recyclability in order to function efficiently. Here we show that forisomes (plant-derived mechanoproteins) can be functionalised with enzymes by translational fusion, leading to the assembly of structures designated as forizymes. When forizymes are expressed in the yeast Saccharomyces cerevisiae, the enzymes are immobilised by the self-assembly of forisome subunits to form well-structured protein bodies. We used glucose-6-phosphate dehydrogenase (G6PDH) and hexokinase 2 (HXK2) as model enzymes for the one-step production and purification of catalytically active forizymes. These structures retain the typical stimulus-response reaction of the forisome and the enzyme remains active even after multiple assay cycles, which we demonstrated using G6PDH forizymes as an example. We also achieved the co-incorporation of both HXK2 and G6PDH in a single forizyme, facilitating a two-step reaction cascade that was 30% faster than the coupled reaction using the corresponding enzymes on different forizymes or in solution. Our novel forizyme immobilisation technique therefore not only combines the sensory properties of forisome proteins with the catalytic properties of enzymes but also allows the development of multi-enzyme complexes for incorporation into technical devices.

Identification of 3-sulfinopropionyl coenzyme A (CoA) desulfinases within the Acyl-CoA dehydrogenase superfamily.

In a previous study, the essential role of 3-sulfinopropionyl coenzyme A (3SP-CoA) desulfinase acyl-CoA dehydrogenase (Acd) in Advenella mimigardefordensis strain DPN7(T) (AcdDPN7) during degradation of 3,3'-dithiodipropionic acid (DTDP) was elucidated. DTDP is a sulfur-containing precursor substrate for biosynthesis of polythioesters (PTEs). AcdDPN7 showed high amino acid sequence similarity to acyl-CoA dehydrogenases but was unable to catalyze a dehydrogenation reaction. Hence, it was investigated in the present study whether 3SP-CoA desulfinase activity is an uncommon or a widespread property within the acyl-CoA dehydrogenase superfamily. Therefore, proteins of the acyl-CoA dehydrogenase superfamily from Advenella kashmirensis WT001, Bacillus cereus DSM31, Cupriavidus necator N-1, Escherichia coli BL21, Pseudomonas putida KT2440, Burkholderia xenovorans LB400, Ralstonia eutropha H16, Variovorax paradoxus B4, Variovorax paradoxus S110, and Variovorax paradoxus TBEA6 were expressed in E. coli strains. All purified acyl-CoA dehydrogenases appeared as homotetramers, as revealed by size exclusion chromatography. AcdS110, AcdB4, AcdH16, and AcdKT2440 were able to dehydrogenate isobutyryl-CoA. AcdKT2440 additionally dehydrogenated butyryl-CoA and valeryl-CoA, whereas AcdDSM31 dehydrogenated only butyryl-CoA and valeryl-CoA. No dehydrogenation reactions were observed with propionyl-CoA, isovaleryl-CoA, succinyl-CoA, and glutaryl-CoA for any of the investigated acyl-CoA dehydrogenases. Only AcdTBEA6, AcdN-1, and AcdLB400 desulfinated 3SP-CoA and were thus identified as 3SP-CoA desulfinases within the acyl-CoA dehydrogenase family, although none of these three Acds dehydrogenated any of the tested acyl-CoA thioesters. No appropriate substrates were identified for AcdBL21 and AcdWT001. Spectrophotometric assays provided apparent Km and Vmax values for active substrates and indicated the applicability of phylogenetic analyses to predict the substrate range of uncharacterized acyl-CoA dehydrogenases. Furthermore, C. necator N-1 was found to utilize 3SP as the sole source of carbon and energy.

Use of critical pathways to improve efficiency: a cautionary tale.

BACKGROUND: Critical pathways are healthcare management plans that specify patient goals and the sequence and timing of actions necessary to achieve these goals with optimal efficiency. More than 80% of hospitals in the United States use critical pathways for at least some of their patients. Unfortunately, critical pathway effectiveness in improving clinical efficiency is unclear. OBJECTIVES: To assess whether critical pathways have been successful in reducing patient length of stay (LOS) and resource utilization in our tertiary-care academic medical center. STUDY DESIGN: A before-and-after observational study using multivariate linear regression analyses. METHODS: We identified all critical pathways initiated in our medical center between 1993 and 1996 in which at least 50 adult patients would be evaluated in the year preceding and succeeding pathway implementation; 13 pathways satisfied these inclusion criteria. Using a before-and-after design, multivariate linear regression was used to evaluate each pathway's effect on average monthly LOS and resource utilization after adjusting for case mix and secular trends. RESULTS: Three of the 13 pathways were associated with a statistically significant immediate decrease in inpatient LOS: acute myocardial infarction (20.7% decrease; P = .001), cesarean section (14.6% decrease; P = .03), and kidney transplantation (24.5% decrease; P = .003). Only 1 pathway, percutaneous transluminal coronary angioplasty (PTCA), produced a statistically significant decrease in LOS slope (a decrease of 5.2% per month; P = .001). Two pathways were accompanied by a statistically significant immediate reduction in ancillary resource utilization: kidney transplantation (26.4% decrease; P = .001) and community-acquired pneumonia (21.8% decrease; P = .002). Only the PTCA pathway produced a statistically significant decrease in resource utilization slope during the 12-month follow-up period (a decrease of 8.4% per month; P < .001). CONCLUSIONS: Although some pathways did reduce LOS or resource utilization or both, most pathways reduced neither. Because substantial resources must be expended on pathway development, implementation, and maintenance, future efforts should be placed on further evaluating the effectiveness of critical pathways and understanding the reasons behind their success or failure before additional resources are consumed for this management strategy.

Effects of buspirone in withdrawal from opiates.

The purpose of this study is to evaluate the effectiveness of buspirone in attenuating withdrawal symptoms in heroin addicts and methadone-maintained patients following cessation of heroin or methadone use. Subjects were twenty hospitalized male chronic opiate users aged 30-55 who did not present any DSM-IV Axis I disorder with the exception of opioid dependence. For the first five days, patients received doses of methadone that were decreased to 30 mg and were maintained on this dose for the following three days. Methadone was then discontinued, and patients were randomly assigned to buspirone or placebo treatment from day nine to seventeen. The buspirone dose was 15 mg on day nine and 30 mg from day ten to day seventeen. Treatment was double-blind. Withdrawal symptoms were measured with the Objective Opiate Withdrawal Scale (OOWS) and the Subjective Opiate Withdrawal Scale (SOWS). Buspirone-treated patients had significantly lower scores on the OOWS on days thirteen (p=.040), fourteen (p=.025), fifteen (p=.035), and seventeen (p=.035). They also had lower scores on the SOWS on days sixteen (p=.050). It is concluded that buspirone was effective in attenuating the objective and subjective withdrawal symptoms that follow opiate use cessation.

Shifting costs from high-cost to low-cost diagnosis-related groups?

Hospitals are commonly compared with each other within diagnosis-related group (DRG) categories. Administrators infer that hospitals with a higher cost per case within a DRG are less efficient than hospitals with a lower cost per case after case mix and severity adjustment. The authors assess whether hospitals that carry a heavy load of high-cost DRGs potentially distribute the added expenses of treating these patients onto their lower cost DRGs using data gathered from the 47 hospitals in the University Hospital Consortium database between January 1994 and December 1995. The results indicate that given standard hospital allocation practices, some of the costs associated with high-cost patients were likely shifted downward, thereby inflating the cost per case for less expensive patients. As researchers adopt more benchmarking methodologies, it is important to recognize that standard accounting practices in which cost shifting from one class of patient to another may impair the ability to understand the actual cost structure for classes of patients.