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All free-living microorganisms homeostatically maintain the fluidity of their membranes by adapting lipid composition to environmental temperatures. Here, we quantify enzymes and metabolic intermediates of the Escherichia coli fatty acid and phospholipid synthesis pathways, to describe how this organism measures temperature and restores optimal membrane fluidity within a single generation after a temperature shock. A first element of this regulatory system is a temperature-sensitive metabolic valve that allocates flux between the saturated and unsaturated fatty acid synthesis pathways via the branchpoint enzymes FabI and FabB. A second element is a transcription-based negative feedback loop that counteracts the temperature-sensitive valve. The combination of these elements accelerates membrane adaptation by causing a transient overshoot in the synthesis of saturated or unsaturated fatty acids following temperature shocks. This strategy is comparable to increasing the temperature of a water bath by adding water that is excessively hot rather than adding water at the desired temperature. These properties are captured in a mathematical model, which we use to show how hard-wired parameters calibrate the system to generate membrane compositions that maintain constant fluidity across temperatures. We hypothesize that core features of the E. coli system will prove to be ubiquitous features of homeoviscous adaptation systems., (© 2024. The Author(s).)

The unique asymmetry of the Gram-negative outer membrane, with glycerophospholipids (GPLs) in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet, works to resist external stressors and prevent the entry of toxic compounds. Thus, GPL and LPS synthesis must be tightly controlled to maintain the integrity of this essential structure. We sought to decipher why organisms like Escherichia coli possess two redundant pathways-PlsB and PlsX/Y-for synthesis of the GPL precursor lysophosphatidic acid (LPA). LPA is then converted by PlsC to the universal precursor for GPL synthesis, phosphatidic acid (PA). PlsB and PlsC are essential in E. coli , indicating they serve as the major pathway for PA synthesis. While loss of PlsX or PlsY individually has little consequence on the cell, the absence of both was lethal. To understand the synthetic lethality of this seemingly redundant PlsX/Y pathway, we performed a suppressor screen. Suppressor analysis indicated that ∆ plsXY requires increased levels of glycerol-3-phosphate (G3P), a GPL precursor. In agreement, ∆ plsXY required supplementation with G3P for survival. Furthermore, loss of PlsX dysregulated fatty acid synthesis, resulting in increased long-chain fatty acids. We show that although PlsX/Y together contribute to PA synthesis, they also contribute to the regulation of overall membrane biogenesis. Thus, synthetic lethality of ∆ plsXY is multifactorial, suggesting that PlsX/Y has been maintained as a redundant system to fine-tune the synthesis of major lipids and promote cell envelope homeostasis.IMPORTANCEGram-negative bacteria must maintain optimal ratios of glycerophospholipids and lipopolysaccharide within the cell envelope for viability. Maintenance of proper outer membrane asymmetry allows for resistance to toxins and antibiotics. Here, we describe additional roles of PlsX and PlsY in Escherichia coli beyond lysophosphatidic acid synthesis, a key precursor of all glycerophospholipids. These findings suggest that PlsX and PlsY also play a larger role in impacting homeostasis of lipid synthesis.

Reactions catalysed by iron-sulfur (Fe-S) enzymes appear in a variety of biosynthetic pathways that produce valuable natural products. Harnessing these biosynthetic pathways by expression in microbial cell factories grown on an industrial scale would yield enormous economic and environmental benefits. However, Fe-S enzymes often become bottlenecks that limits the productivity of engineered pathways. As a consequence, achieving the production metrics required for industrial application remains a distant goal for Fe-S enzyme-dependent pathways. Here, we identify and review three core challenges in harnessing Fe-S enzyme activity, which all stem from the properties of Fe-S clusters: 1) limited Fe-S cluster supply within the host cell, 2) Fe-S cluster instability, and 3) lack of specialized reducing cofactor proteins often required for Fe-S enzyme activity, such as enzyme-specific flavodoxins and ferredoxins. We highlight successful methods developed for a variety of Fe-S enzymes and electron carriers for overcoming these difficulties. We use heterologous nitrogenase expression as a grand case study demonstrating how each of these challenges can be addressed. We predict that recent breakthroughs in protein structure prediction and design will prove well-suited to addressing each of these challenges. A reliable toolkit for harnessing Fe-S enzymes in engineered metabolic pathways will accelerate the development of industry-ready Fe-S enzyme-dependent biosynthesis pathways., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Iron-sulfur (Fe-S) clusters are ancient and ubiquitous protein cofactors and play irreplaceable roles in many metabolic and regulatory processes. Fe-S clusters are built and distributed to Fe-S enzymes by dedicated protein networks. The core components of these networks are widely conserved and highly versatile. However, Fe-S proteins and enzymes are often inactive outside their native host species. We sought to systematically investigate the compatibility of Fe-S networks with non-native Fe-S enzymes. By using collections of Fe-S enzyme orthologs representative of the entire range of prokaryotic diversity, we uncovered a striking correlation between phylogenetic distance and probability of functional expression. Moreover, coexpression of a heterologous Fe-S biogenesis pathway increases the phylogenetic range of orthologs that can be supported by the foreign host. We also find that Fe-S enzymes that require specific electron carrier proteins are rarely functionally expressed unless their taxon-specific reducing partners are identified and co-expressed. We demonstrate how these principles can be applied to improve the activity of a radical S -adenosyl methionine(rSAM) enzyme from a Streptomyces antibiotic biosynthesis pathway in Escherichia coli . Our results clarify how oxygen sensitivity and incompatibilities with foreign Fe-S and electron transfer networks each impede heterologous activity. In particular, identifying compatible electron transfer proteins and heterologous Fe-S biogenesis pathways may prove essential for engineering functional Fe-S enzyme-dependent pathways., Competing Interests: FD, EF, PG, HS, MP, RM, FB, SL, Nv, ND, Cd, MP, EB, SG, BP, SO, FB, GB No competing interests declared, (© 2022, D'Angelo et al.)

Growth and division are central to cell size. Bacteria achieve size homeostasis by dividing when growth has added a constant size since birth, termed the adder principle, by unknown mechanisms. 1 , 2 Growth is well known to be regulated by guanosine tetraphosphate (ppGpp), which controls diverse processes from ribosome production to metabolic enzyme activity and replication initiation and whose absence or excess can induce stress, filamentation, and small growth-arrested cells. 3-6 These observations raise unresolved questions about the relation between ppGpp and size homeostasis mechanisms during normal exponential growth. Here, to untangle effects of ppGpp and nutrients, we gained control of cellular ppGpp by inducing the synthesis and hydrolysis enzymes RelA and Mesh1. We found that ppGpp not only exerts control over the growth rate but also over cell division and thus the steady state cell size. In response to changes in ppGpp level, the added size already establishes its new constant value while the growth rate still adjusts, aided by accelerated or delayed divisions. Moreover, the magnitude of the added size and resulting steady-state birth size correlate consistently with the ppGpp level, rather than with the growth rate, which results in cells of different size that grow equally fast. Our findings suggest that ppGpp serves as a key regulator that coordinates cell size and growth control., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

Microbes are routinely engineered to synthesize high-value chemicals from renewable materials through synthetic biology and metabolic engineering. Microbial biosynthesis often relies on expression of heterologous biosynthetic pathways, i.e., enzymes transplanted from foreign organisms. Metallocluster enzymes are one of the most ubiquitous family of enzymes involved in natural product biosynthesis and are of great biotechnological importance. However, the functional expression of recombinant metallocluster enzymes in live cells is often challenging and represents a major bottleneck. The activity of metallocluster enzymes requires essential supporting pathways, involved in protein maturation, electron supply, and/or enzyme stability. Proper function of these supporting pathways involves specific protein-protein interactions that remain poorly characterized and are often overlooked by traditional synthetic biology approaches. Consequently, engineering approaches that focus on enzymatic expression and carbon flux alone often overlook the particular needs of metallocluster enzymes. This review highlights the biotechnological relevance of metallocluster enzymes and discusses novel synthetic biology strategies to advance their industrial application, with a particular focus on iron-sulfur cluster enzymes. Strategies to enable functional heterologous expression and enhance recombinant metallocluster enzyme activity in industrial hosts include: (1) optimizing specific maturation pathways; (2) improving catalytic stability; and (3) enhancing electron transfer. In addition, we suggest future directions for developing microbial cell factories that rely on metallocluster enzyme catalysis.

The molecule guanosine tetraphophosphate (ppGpp) is most commonly considered an alarmone produced during acute stress. However, ppGpp is also present at low concentrations during steady-state growth. Whether ppGpp controls the same cellular targets at both low and high concentrations remains an open question and is vital for understanding growth rate regulation. It is widely assumed that basal ppGpp concentrations vary inversely with growth rate, and that the main function of basal ppGpp is to regulate transcription of ribosomal RNA in response to environmental conditions. Unfortunately, studies to confirm this relationship and to define regulatory targets of basal ppGpp are limited by difficulties in quantifying basal ppGpp. In this Perspective we compare reported concentrations of basal ppGpp in E. coli and quantify ppGpp within several strains using a recently developed analytical method. We find that although the inverse correlation between ppGpp and growth rate is robust across strains and analytical methods, absolute ppGpp concentrations do not absolutely determine RNA synthesis rates. In addition, we investigated the consequences of two separate RNA polymerase mutations that each individually reduce (but do not abolish) sensitivity to ppGpp and find that the relationship between ppGpp, growth rate, and RNA content of single-site mutants remains unaffected. Both literature and our new data suggest that environmental conditions may be communicated to RNA polymerase via an additional regulator. We conclude that basal ppGpp is one of potentially several agents controlling ribosome abundance and DNA replication initiation, but that evidence for additional roles in controlling macromolecular synthesis requires further study., (Copyright © 2020 Imholz, Noga, van den Broek and Bokinsky.)

Every cell must produce enough membrane to contain itself. However, the mechanisms by which the rate of membrane synthesis is coupled with the rate of cell growth remain unresolved. By comparing substrate and enzyme concentrations of the fatty acid and phospholipid synthesis pathways of Escherichia coli across a 3-fold range of carbon-limited growth rates, we show that the rate of membrane phospholipid synthesis during steady-state growth is determined principally through allosteric control of a single enzyme, PlsB. Due to feedback regulation of the fatty acid pathway, PlsB activity also indirectly controls synthesis of lipopolysaccharide, a major component of the outer membrane synthesized from a fatty acid synthesis intermediate. Surprisingly, concentrations of the enzyme that catalyzes the committed step of lipopolysaccharide synthesis (LpxC) do not differ across steady-state growth conditions, suggesting that steady-state lipopolysaccharide synthesis is modulated primarily via indirect control by PlsB. In contrast to steady-state regulation, we found that responses to environmental perturbations are triggered directly via changes in acetyl coenzyme A (acetyl-CoA) concentrations, which enable rapid adaptation. Adaptations are further modulated by ppGpp, which regulates PlsB activity during slow growth and growth arrest. The strong reliance of the membrane synthesis pathway upon posttranslational regulation ensures both the reliability and the responsiveness of membrane synthesis. IMPORTANCE How do bacterial cells grow without breaking their membranes? Although the biochemistry of fatty acid and membrane synthesis is well known, how membrane synthesis is balanced with growth and metabolism has remained unclear. This is partly due to the many control points that have been discovered within the membrane synthesis pathways. By precisely establishing the contributions of individual pathway enzymes, our results simplify the model of membrane biogenesis in the model bacterial species Escherichia coli Specifically, we found that allosteric control of a single enzyme, PlsB, is sufficient to balance growth with membrane synthesis and to ensure that growing E. coli cells produce sufficient membrane. Identifying the signals that activate and deactivate PlsB will resolve the issue of how membrane synthesis is synchronized with growth., (Copyright © 2020 Noga et al.)

Carbapenems, a family of β-lactam antibiotics, are among the most powerful bactericidal compounds in clinical use. However, as rational engineering of native carbapenem-producing microbes is not currently possible, the present carbapenem supply relies upon total chemical synthesis of artificial carbapenem derivatives. To enable access to the full diversity of natural carbapenems, we have engineered production of a simple carbapenem antibiotic within Escherichia coli. By increasing concentrations of precursor metabolites and identifying a reducing cofactor of a bottleneck enzyme, we improved productivity by 60-fold over the minimal pathway and surpassed reported titers obtained from carbapenem-producing Streptomyces species. We stabilized E. coli metabolism against antibacterial effects of the carbapenem product by artificially inhibiting membrane synthesis, which further increased antibiotic productivity. As all known naturally occurring carbapenems are derived from a common intermediate, our engineered strain provides a platform for biosynthesis of tailored carbapenem derivatives in a genetically tractable and fast-growing species.

The production of fatty acids from simple nutrients occurs via a complex biosynthetic pathway with dozens of intermediate compounds and multiple branch points. Despite its importance for microbial physiology and biotechnology, critical aspects of fatty acid biosynthesis, especially dynamics of in vivo regulation, remain poorly characterized. We have developed a liquid chromatography/mass spectroscopy (LC-MS) method for relative quantification of fatty acid synthesis intermediates in Escherichia coli, a model organism for studies of fatty acid metabolism. The acyl carrier protein, a vehicle for the substrates and intermediates of fatty acid synthesis, is extracted from E. coli, proteolytically digested, resolved using reverse-phase LC, and detected using electrospray ionization coupled with a tandem MS. Our method reliably resolves 21 intermediates of fatty acid synthesis, with an average relative standard deviation in ratios of individual acyl-ACP species to total ACP concentrations of 20%. We demonstrate that fast sampling and quenching of cells is essential to accurately characterize intracellular concentrations of ACP species. We apply our method to examine the rapid response of fatty acid metabolism to the antibiotic cerulenin. We anticipate that our method will enable the characterization of in vivo regulation and kinetics of microbial fatty acid synthesis at unprecedented detail and will improve integration of fatty acid synthesis into models of microbial metabolism.

Persistence is a phenomenon whereby a subpopulation of bacterial cells enters a transient growth-arrested state that confers antibiotic tolerance. While entrance into persistence has been linked to the activities of toxin proteins, the molecular mechanisms by which toxins induce growth arrest and the persistent state remain unclear. Here, we show that overexpression of the protein kinase HipA in Escherichia coli triggers growth arrest by activating synthesis of the alarmone guanosine tetraphosphate (ppGpp) by the enzyme RelA, a signal typically associated with amino acid starvation. We further demonstrate that chemically suppressing ppGpp synthesis with chloramphenicol relieves inhibition of DNA replication initiation and RNA synthesis in HipA-arrested cells and restores vulnerability to β-lactam antibiotics. HipA-arrested cells maintain glucose uptake and oxygen consumption and accumulate amino acids as a consequence of translational inhibition. We harness the active metabolism of HipA-arrested cells to provide a bacteriophage-resistant platform for the production of biotechnologically relevant compounds, which may represent an innovative solution to the costly problem of phage contamination in industrial fermentations.

Escherichia coli has the potential to be a powerful biocatalyst for the conversion of lignocellulosic biomass into useful materials such as biofuels and polymers. One important challenge in using E. coli for the transformation of biomass sugars is diauxie, or sequential utilization of different types of sugars. We demonstrate that, by increasing the intracellular levels of the transcription factor XylR, the preferential consumption of arabinose before xylose can be eliminated. In addition, XylR augmentation must be finely tuned for robust coutilization of these two hemicellulosic sugars. Using a novel technique for scarless gene insertion, an additional copy of xylR was inserted into the araBAD operon. The resulting strain was superior at cometabolizing mixtures of arabinose and xylose and was able to produce at least 36% more ethanol than wild-type strains. This strain is a useful starting point for the development of an E. coli biocatalyst that can simultaneously convert all biomass sugars.

One approach to reducing the costs of advanced biofuel production from cellulosic biomass is to engineer a single microorganism to both digest plant biomass and produce hydrocarbons that have the properties of petrochemical fuels. Such an organism would require pathways for hydrocarbon production and the capacity to secrete sufficient enzymes to efficiently hydrolyze cellulose and hemicellulose. To demonstrate how one might engineer and coordinate all of the necessary components for a biomass-degrading, hydrocarbon-producing microorganism, we engineered a microorganism naïve to both processes, Escherichia coli, to grow using both the cellulose and hemicellulose fractions of several types of plant biomass pretreated with ionic liquids. Our engineered strains express cellulase, xylanase, beta-glucosidase, and xylobiosidase enzymes under control of native E. coli promoters selected to optimize growth on model cellulosic and hemicellulosic substrates. Furthermore, our strains grow using either the cellulose or hemicellulose components of ionic liquid-pretreated biomass or on both components when combined as a coculture. Both cellulolytic and hemicellulolytic strains were further engineered with three biofuel synthesis pathways to demonstrate the production of fuel substitutes or precursors suitable for gasoline, diesel, and jet engines directly from ionic liquid-treated switchgrass without externally supplied hydrolase enzymes. This demonstration represents a major advance toward realizing a consolidated bioprocess. With improvements in both biofuel synthesis pathways and biomass digestion capabilities, our approach could provide an economical route to production of advanced biofuels.

Increasing energy costs and environmental concerns have emphasized the need to produce sustainable renewable fuels and chemicals. Major efforts to this end are focused on the microbial production of high-energy fuels by cost-effective 'consolidated bioprocesses'. Fatty acids are composed of long alkyl chains and represent nature's 'petroleum', being a primary metabolite used by cells for both chemical and energy storage functions. These energy-rich molecules are today isolated from plant and animal oils for a diverse set of products ranging from fuels to oleochemicals. A more scalable, controllable and economic route to this important class of chemicals would be through the microbial conversion of renewable feedstocks, such as biomass-derived carbohydrates. Here we demonstrate the engineering of Escherichia coli to produce structurally tailored fatty esters (biodiesel), fatty alcohols, and waxes directly from simple sugars. Furthermore, we show engineering of the biodiesel-producing cells to express hemicellulases, a step towards producing these compounds directly from hemicellulose, a major component of plant-derived biomass.

The reverse transcriptase of human immunodeficiency virus (HIV) catalyses a series of reactions to convert the single-stranded RNA genome of HIV into double-stranded DNA for host-cell integration. This task requires the reverse transcriptase to discriminate a variety of nucleic-acid substrates such that active sites of the enzyme are correctly positioned to support one of three catalytic functions: RNA-directed DNA synthesis, DNA-directed DNA synthesis and DNA-directed RNA hydrolysis. However, the mechanism by which substrates regulate reverse transcriptase activities remains unclear. Here we report distinct orientational dynamics of reverse transcriptase observed on different substrates with a single-molecule assay. The enzyme adopted opposite binding orientations on duplexes containing DNA or RNA primers, directing its DNA synthesis or RNA hydrolysis activity, respectively. On duplexes containing the unique polypurine RNA primers for plus-strand DNA synthesis, the enzyme can rapidly switch between the two orientations. The switching kinetics were regulated by cognate nucleotides and non-nucleoside reverse transcriptase inhibitors, a major class of anti-HIV drugs. These results indicate that the activities of reverse transcriptase are determined by its binding orientation on substrates.

Single-molecule FRET is a powerful tool for probing the kinetic mechanism of a complex enzymatic reaction. However, not every reaction intermediate can be identified via a distinct FRET value, making it difficult to fully dissect a multistep reaction pathway. Here, we demonstrate a method using sequential kinetic experiments to differentiate each reaction intermediate by a distinct time sequence of FRET signal (a kinetic "fingerprint"). Our model system, the two-way junction hairpin ribozyme, catalyzes a multistep reversible RNA cleavage reaction, which comprises two structural transition steps (docking/undocking) and one chemical reaction step (cleavage/ligation). Whereas the docked and undocked forms of the enzyme display distinct FRET values, the cleaved and ligated forms do not. To overcome this difficulty, we used Mg(2+) pulse-chase experiments to differentiate each reaction intermediate by a distinct kinetic fingerprint at the single-molecule level. This method allowed us to unambiguously determine the rate constant of each reaction step and fully characterize the reaction pathway by using the chemically competent enzyme-substrate complex. We found that the ligated form of the enzyme highly favors the docked state, whereas undocking becomes accelerated upon cleavage by two orders of magnitude, a result different from that obtained with chemically blocked substrate and product analogs. The overall cleavage reaction is rate-limited by the docking/undocking kinetics and the internal cleavage/ligation equilibrium, contrasting the rate-limiting mechanism of the four-way junction ribozyme. These results underscore the kinetic interdependence of reversible steps on an enzymatic reaction pathway and demonstrate a potentially general route to dissect them.

Like most cellular RNA enzymes, the bI5 group I intron requires binding by a protein cofactor to fold correctly. Here, we use single-molecule approaches to monitor the structural dynamics of the bI5 RNA in real time as it assembles with its CBP2 protein cofactor. These experiments show that CBP2 binds to the target RNA in two distinct modes with apparently opposite effects: a "non-specific" mode that forms rapidly and induces large conformational fluctuations in the RNA, and a "specific" mode that forms slowly and stabilizes the native RNA structure. The bI5 RNA folds though multiple pathways toward the native state, typically traversing dynamic intermediate states induced by non-specific binding of CBP2. These results suggest that the protein cofactor-assisted RNA folding involves sequential non-specific and specific protein-RNA interactions. The non-specific interaction potentially increases the local concentration of CBP2 and the number of conformational states accessible to the RNA, which may promote the formation of specific RNA-protein interactions.

Single-molecule experiments significantly expand our capability to characterize complex dynamics of biological processes. This relatively new approach has contributed significantly to our understanding of the RNA folding problem. Recent single-molecule experiments, together with structural and biochemical characterizations of RNA at the ensemble level, show that RNA molecules typically fold across a highly rugged energy landscape. As a result, long-lived folding intermediates, multiple folding pathways, and heterogeneous conformational dynamics are commonly found for RNA enzymes. While initial results have suggested that stable secondary structures are partly responsible for the rugged energy landscape of RNA, a complete mechanistic understanding of the complex folding behavior has not yet been obtained. A combination of single-molecule experiments, which are well suited to analyze transient and heterogeneous dynamic behaviors, with ensemble characterizations that can provide structural information at a superior resolution will likely provide more answers.

The hairpin ribozyme is a minimalist paradigm for studying RNA folding and function. In this enzyme, two domains dock by induced fit to form a catalytic core that mediates a specific backbone cleavage reaction. Here, we have fully dissected its reversible reaction pathway, which comprises two structural transitions (docking/undocking) and a chemistry step (cleavage/ligation), by applying a combination of single-molecule fluorescence resonance energy transfer (FRET) assays, ensemble cleavage assays, and kinetic simulations. This has allowed us to quantify the effects that modifications of essential functional groups remote from the site of catalysis have on the individual rate constants. We find that all ribozyme variants show similar fractionations into effectively noninterchanging molecule subpopulations of distinct undocking rate constants. This leads to heterogeneous cleavage activity as commonly observed for RNA enzymes. A modification at the domain junction additionally leads to heterogeneous docking. Surprisingly, most modifications not only affect docking/undocking but also significantly impact the internal chemistry rate constants over a substantial distance from the site of catalysis. We propose that a network of coupled molecular motions connects distant parts of the RNA with its reaction site, which suggests a previously undescribed analogy between RNA and protein enzymes. Our findings also have broad implications for applications such as the action of drugs and ligands distal to the active site or the engineering of allostery into RNA.

How RNA molecules fold into functional structures is a problem of great significance given the expanding list of essential cellular RNA enzymes and the increasing number of applications of RNA in biotechnology and medicine. A critical step toward solving the RNA folding problem is the characterization of the associated transition states. This is a challenging task in part because the rugged energy landscape of RNA often leads to the coexistence of multiple distinct structural transitions. Here, we exploit single-molecule fluorescence spectroscopy to follow in real time the equilibrium transitions between conformational states of a model RNA enzyme, the hairpin ribozyme. We clearly distinguish structural transitions between effectively noninterchanging sets of unfolded and folded states and characterize key factors defining the transition state of an elementary folding reaction where the hairpin ribozyme's two helical domains dock to make several tertiary contacts. Our single-molecule experiments in conjunction with site-specific mutations and metal ion titrations show that the two RNA domains are in a contact or close-to-contact configuration in the transition state even though the native tertiary contacts are at most partially formed. Such a compact transition state without well formed tertiary contacts may be a general property of elementary RNA folding reactions.

Purpose: The UroVysion fluorescence in situ hybridization assay (UroVysion Bladder Cancer Recurrence Kit, Vysis, Inc., Downers Grove, Illinois) is a multi-target assay that detects aneuploidy of chromosomes 3, 7 and 17, and loss of the 9p21 band in exfoliated cells in urine from patients with transitional cell carcinoma. We performed 2 multicenter trials. In 1 trial we compared the sensitivity of the FISH assay to the BTA Stat test (Bion Scientific, Redmond, Washington) and voided cytology in the detection of transitional cell carcinoma. In a separate study of healthy volunteers and patients with other (nontransitional cell carcinoma) conditions we determined the specificity of the FISH assay., Materials and Methods: A total of 176 patients with transitional cell carcinoma in the previous 9 months provided voided urine before cystoscopy. Each specimen was split, preserved and shipped to a central laboratory where all 3 tests were performed. All sites were blinded to results. Sensitivity calculations were based on central pathology review of resected tissue. Specificity was determined by testing 275 volunteers who were healthy and with nontransitional cell carcinoma conditions., Results: The 21 sites enrolled 176 patients with a history of transitional cell carcinoma, with 62 recurrences while undergoing surveillance. Overall sensitivities (with 95% CI) were FISH 71% (95% CI 58 to 82), BTA Stat test 50% (37 to 63) and cytology 26% (16 to 39). FISH was negative in 260 of the 275 healthy volunteers or patients with no history of transitional cell carcinoma (specificity 94.5%)., Conclusions: Sensitivity of the FISH assay is superior to that of cytology and at least equivalent to the BTA Stat test in detecting recurrent transitional cell carcinoma. Its specificity approaches that of cytology. Further testing of its clinical use is warranted.

[formula: see text] A tight-binding, hydrophobic inhibitor of carbonic anhydrase II has been masked with a water-solubilizing, photolabile group derived from o-nitrophenylglycine. This caged inhibitor represents our first effort at the site-specific delivery of prodrugs that can be activated by light. Via this approach, we have begun to address the problems of water insolubility and systemic side effects on administration of tight-binding inhibitors of carbonic anhydrase.

Study Objective: To determine whether alterations of platelet reactivity occur during obstructive sleep apnea (OSA) and, if so, whether therapy with nasal-continuous positive airway pressure (N-CPAP) alters this reactivity., Design: Patients with suspected moderate to severe OSA had blood drawn for spontaneous platelet aggregation (sAGG) and activation (sACT) measurements at hourly intervals during diagnostic polysomnography (PSG) and, in those with confirmed OSA, on a separate night during which N-CPAP was applied., Setting: Tertiary care center sleep laboratory., Patients: Six patients with OSA had matched blood samples drawn on both diagnostic and N-CPAP treatment nights. Five patients without confirmed OSA served as controls., Interventions: N-CPAP was applied to those patients with OSA and pressures adjusted with goals of eliminating apneas; N-CPAP was then maintained through the night., Measurements and Results: sACT and sAGG were measured using flow cytometric determination of P-selectin expression using a monoclonal antibody. Platelet aggregation was assessed by measuring the proportion of platelets larger than resting platelets by light scatter techniques. Mean values for sACT and sAGG were higher on the diagnostic night compared with treatment night (p = 0.001 and p = 0.003, analysis of variance, respectively). The mean baseline supine sACT compared with completion supine sACT for both diagnostic and N-CPAP nights also revealed significant differences (mean = 16.6 +/- 3.5% vs 36.9 +/- 7.5%, p = 0.04; and 11.9 +/- 3% vs 39.5 +/- 9.1%, p = 0.04). Platelet activation during sleep in five subjects without OSA resembles that found in patients with OSA during N-CPAP., Conclusions: Increased platelet sACT and sAGG occur during sleep in patients with OSA. This effect is greatly reduced by N-CPAP.

Improvement in renal transplantation has been the result of increased clinical experience and laboratory investigation. One of the causes of transplant failure is urinary leakage and slough at the ureteroneocystostomy site. A simplified extravesical ureteral implantation technique is described. This method has been used in more than 200 canine renal transplants producing consistently good results.

An unusual case of asynchronous bilateral renal cell carcinoma presenting as multiple tumors in a solitary kidney is presented. A literature review suggests that the prognosis is improved for those patients with a longer interval between tumors and for those who undergo surgical therapy.

Forty-two patients with chronic obstructive pulmonary disease participated in a comprehensive inpatient rehabilitation program. Criteria for a safe, maximum treadmill exercise regimen were established; all patients pursued this regimen for six weeks, without complications, even though 31 of 39 patients showed a decline in PaO2 with exercise. Postprogram O2 consumption, minute ventilation, heart rate, and respiratory rate were significantly reduced during exercise when compared with preprogram values. Sixteen of 29 patients improved in terms of dyspnea class; an additional 11 of these 29 improved with regard to activities of daily living. Most patients who improved physiologically also improved functionally. Patients able to tolerate higher levels of treadmill exercise initially had the greatest improvement in postcourse exercise performance. The data suggest that precourse functional and exercise classification is useful in selecting candidates who will receive the greatest functional benefits from participation in rehabilitative programs.

Ureteral rejection has been studied in the dog utilizing the isolated transplanted ureter technique. While on adequate immunosuppressive therapy, the allograft ureter remains functionally and histologically normal. When the drugs are discontinued, progressive destructive changes occur within the ureteral wall. As the muscle wall is destroyed the rejection process becomes irreversible. This process helps explain some of the findings seen during clinical renal transplantation.

A case is reported of a spontaneous thrombosis of the left pampiniform plexus. On examination it may be confused for any number of conditions producing spermatic cord enlargements. Surgical intervention is believed to be indicated, not for this specific entity but to rule out some of these other conditions, such as an incarcerated inguinal hernia, torsion of the spermatic cord, or even malignancy.

Benign adenoma of the kidney is rare. A case report of renal oncocytoma, one of the rarest, is presented, and its salient features are discussed.