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Abstract Background Scavenger receptors are important components of the innate immune system in the lung, allowing alveolar macrophages to bind and phagocytose numerous unopsonized targets. Mice with genetic deletions of scavenger receptors, such as SR-A and MARCO, are susceptible to infection or inflammation from inhaled pathogens or dusts. However, the signaling pathways required for scavenger receptor-mediated phagocytosis of unopsonized particles have not been characterized. Methods We developed a scanning cytometry-based high-throughput assay of macrophage phagocytosis that quantitates bound and internalized unopsonized latex beads. This assay allowed the testing of a panel of signaling inhibitors which have previously been shown to target opsonin-dependent phagocytosis for their effect on unopsonized bead uptake by human in vitro-derived alveolar macrophage-like cells. The non-selective scavenger receptor inhibitor poly(I) and the actin destabilizer cytochalasin D were used to validate the assay and caused near complete abrogation of bead binding and internalization, respectively. Results Microtubule destabilization using nocodazole dramatically inhibited bead internalization. Internalization was also significantly reduced by inhibitors of tyrosine kinases (genistein and herbimycin A), protein kinase C (staurosporine, chelerythrine chloride and Gö 6976), phosphoinositide-3 kinase (LY294002 and wortmannin), and the JNK and ERK pathways. In contrast, inhibition of phospholipase C by U-73122 had no effect. Conclusion These data indicate the utility of scanning cytometry for the analysis of phagocytosis and that phagocytosis of unopsonized particles has both shared and distinct features when compared to opsonin-mediated phagocytosis.

The saddle nose deformity is associated with dorsal collapse and can have both function and cosmetic problems. The saddle nose can cause nasal obstruction by narrowing the nasal cavities, eliciting dynamic internal and external nasal valve narrowing, and abnormally widening the internal and external nasal valves altering airflow dynamics, sinonasal passageways, and olfaction. The saddle nose is challenging to treat due to skin contracture, lack of donor tissue, and difficulties in esthetic integration. A focus on balancing the functional and esthetic concerns during treatment is paramount., Competing Interests: Disclosure The authors have nothing to disclose., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Rhinoplasty is a challenging surgery and results are not always perfect. There are many obstacles to achieving optimal results. Among these are inadequate instrumentation, the unpredictability of healing, imprecise planning, and many more. Furthermore, selecting patients who can most benefit from surgery is equally important. In this article, some of the more pressing areas of rhinoplasty that need innovation are discussed. From proper patient selection, to advances in education, to the standardization of training programs, to the development of sophisticated implants, the future of rhinoplasty surgery lies in continued creativity and innovation., Competing Interests: None declared., (Thieme. All rights reserved.)

Rhinoplasty surgeons must continually strive to refine and improve their results. Improvement may be borne out of new surgical techniques, alternative perioperative medicines, or through the application of new technologies. One technology that has recently gained popularity are piezoelectric osteotomes (POs). These instruments offer a powered alternative to the traditional chisel osteotome. This article provides a history of these two modalities, reviews the most recent data surrounding piezoelectric osteotomy, and provides a framework to compare the pros and cons of each osteotomy option., Competing Interests: None declared., (Thieme. All rights reserved.)

Many dilemmas in rhinoplasty tempt surgeons to use exogenous materials. We have long looked toward implants to decrease operative time, to achieve a more reliable result, or when there is a paucity of autologous material. More than ever, the innovative and highly lucrative field of nasal implantology is developing technologically advanced products. This article looks at some popular nasal implant choices with a look toward what might be on the horizon., Competing Interests: None declared., (Thieme. All rights reserved.)

Septorhinoplasty is among the most common facial plastic and reconstructive surgeries and its complexities are reflected in a relatively high revision rate. The patient with a postoperative twisted nose presents an additional challenge and requires that the surgeon display empathy and possess a deep knowledge of the aesthetic and functional intricacies of the nose. Correction of the twisted nose should be approached in a systematic fashion with unique considerations for each "third" of the nose. While there are many options in the overall surgical armamentarium, each surgeon will find specific techniques that are most efficacious and reproducible for their individual practice. This article discusses select surgical "pearls" and techniques that can aid the surgeon in their own surgical decision-making., Competing Interests: None., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Objective: To evaluate whether a diagnosis of allergic rhinitis affects surgical outcomes of open septorhinoplasty (OSR) and to examine whether OSR provides the same level of improvement in quality of life to patients with and without allergic rhinitis., Study Design, Setting, Subjects and Methods: We performed a retrospective evaluation of 646 patients who underwent open septorhinoplasty in a tertiary otolaryngology practice between 2008 and 2015. Preoperative and postoperative quality of life (QoL) measurement using the validated Nasal Obstruction Symptom Evaluation (NOSE) Scale was performed on 307 patients meeting inclusion criteria. These patients were then divided into two groups based on a diagnosis of allergic rhinitis (non-AR vs AR). Comparisons were then made based on quality of life improvements by the NOSE score., Results: There were 213 patients in the non-AR group vs. 94 patients in the AR group. After OSR, patients in both groups experienced significant improvement in nasal airway obstruction. Pre-op NOSE score averages were similar for the non-AR and AR groups (69.9 vs 73.4 p = 0.087). Average improvement in NOSE score for the non-AR and AR groups at 30 days was 48.6 vs 45.9 (p = 0.41); and at 90 day of 48.1 vs 51.5 (p = 0.402)., Conclusion: Patients with and without allergic rhinitis experience similar OSR outcomes as measured by the NOSE score. Open septorhinoplasty addresses multiple components contributing to nasal airway obstruction, and may offset the effects of allergic rhinitis. When indicated, it should be offered to patients with allergic rhinitis after complete medical management., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Introduction: The effect of marijuana on human health has been studied extensively. Marijuana intoxication has been shown to affect performance, attention span, and reaction time. The public health relationship between trauma and cannabis use has also been studied, with mixed conclusions. In this report, the effect of marijuana legalization on many aspects of facial trauma at two hospitals in Denver, Colorado is examined., Methods: A retrospective review of the electronic medical records was undertaken. Mann-Whitney U tests were used to compare age of patients before and after legalization, and chi squared analyses were used to compare mechanism of injury, and fracture types before and after recreational marijuana legalization in Denver, Colorado. Geographical location of patients was also considered., Results: No significant increase was found in race before and after marijuana legalization (p=0.19). A significant increase in age was found before (M=39.54,SD=16.37), and after (M=41.38,SD=16.66) legalization (p<0.01). Maxillary and skull base fracture proportions significantly increased following legalization (p<0.001 and p<0.001respectively). No significant differences were seen in the proportion of patients who lived in urban and rural counties before and after legalization (p>0.05)., Conclusion: Public health efforts should be directed towards educating residents and visitors of Colorado on the effects and toxicology of marijuana. More epidemiologic studies are needed for further assessment of the long-term effects of the legalization of marijuana on the population., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Silver nanoparticles (AgNPs) have attracted great attention due to its optical, electrical and thermal properties. Cellulosic supports for these nanoparticles are of particular interest because of its availability, flexibility and biocompatibility. In this work, AgNPs were synthesized using two cellulosic materials, cellophane (CP) and filter paper (FP), as matrix support. Cellulosic materials were immersed in an aqueous solution of silver nitrate containing polyvinylpyrrolidone (PVP) and then reduced with hydroxylamine. The obtained nanocomposites (CP-AgNPs and FP-AgNPs) were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (DRX) and scanning electron microscopy (SEM). AgNPs of near 15nm anchored onto cellulosic surfaces were detected. The thermal properties of these materials were investigated through thermogravimetry (TG). Their kinetic of thermal decomposition was studied by the Vyasovkin method of dynamic isoconvertion, which indicated a catalytic effect of AgNPs in the cellulose thermal decomposition reaction., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

We report effects of age, age(2), sex and additive genetic factors on variability in gray matter thickness, surface area and white matter integrity in 1,010 subjects from the Genetics of Brain Structure and Function Study. Age was more strongly associated with gray matter thickness and fractional anisotropy of water diffusion in white matter tracts, while sex was more strongly associated with gray matter surface area. Widespread heritability of neuroanatomic traits was observed, suggesting that brain structure is under strong genetic control. Furthermore, our findings indicate that neuroimaging-based measurements of cerebral variability are sensitive to genetic mediation. Fundamental studies of genetic influence on the brain will help inform gene discovery initiatives in both clinical and normative samples.

It is becoming progressively more understandable that different phytochemicals isolated from edible plants interfere with specific stages of carcinogenesis. Cancer cells have evolved hallmark mechanisms to escape from death. Concordant with this approach, there is a disruption of spatiotemproal behaviour of signaling cascades in cancer cells, which can escape from apoptosis because of downregulation of tumor suppressor genes and over- expression of oncogenes. Genomic instability, intra-tumor heterogeneity, cellular plasticity and metastasizing potential of cancer cells all are related to molecular alterations. Data obtained through in vitro studies has convincingly revealed that curcumin, EGCG, resveratrol and quercetin are promising anticancer agents. Their efficacy has been tested in tumor xenografted mice and considerable experimental findings have stimulated researchers to further improve the bioavailability of these nutraceuticals. We partition this review into different sections with emphasis on how bioavailability of curcumin, EGCG, resveratrol and quercetin has improved using different nanotechnology approaches.

Purpose of Review: Alloplasts have long been used in rhinoplasty, but their use remains controversial. Many complications are associated with their implementation in rhinoplasty. This article elucidates these complications and provides recommendations for management., Recent Findings: Several recent articles have been published presenting experience and outcomes regarding alloplast use in rhinoplasty. In many of these studies, a specific section has been dedicated to outlining the complications encountered by the authors. Oftentimes, a short summary of the complications and their management is provided. By examining the data from these studies, one can conclude several things about the management of complications involving alloplastic implants in rhinoplasty: each case must be approached on an individual basis; clinical decision-making is dictated by physical exam findings and severity of the complication; removal of the implant must be strongly considered; and revision rhinoplasty after an alloplastic complication usually necessitates an autologous graft., Summary: Alloplasts continue to be a controversial option in rhinoplasty. The surgeon must be cognizant of the risks and benefits of their use. A frank preoperative discussion of possible complications with the patient is important. Additionally, prompt recognition and appropriate management of complications is essential to minimize permanent sequelae.

We investigate the density decay in the pair-annihilation process A+A→∅ in the case when the particles perform anomalous diffusion on a cubic lattice. The anomalous diffusion is realized via Lévy flights, which are characterized by long-range jumps and lead to superdiffusive behavior. As a consequence, the critical dimension depends continuously on the control parameter of the Lévy flight distribution. This instance is used to study the system close to the critical dimension by means of the nonperturbative renormalization group theory. Close to the critical dimension, the assumption of well-stirred reactants is violated by anticorrelations between the particles, and the law of mass action breaks down. The breakdown of the law of mass action is known to be caused by long-range fluctuations. We identify three interrelated consequences of these fluctuations. First, despite being a nonuniversal quantity and thus depending on the microscopic details, the renormalized reaction rate λ(0) can be approximated by a universal law close to the critical dimension. The emergence of universality relies on the fact that long-range fluctuations suppress the influence of the underlying microscopic details. Second, as criticality is approached, the macroscopic reaction rate decreases such that the law of mass action loses its significance. And third, additional nonanalytic power law corrections complement the analytic law of mass action term. An increasing number of those corrections accumulate and give an essential contribution as the critical dimension is approached. We test our findings for two implementations of Lévy flights that differ in the way they cross over to the normal diffusion in the limit σ→2.

We study the problem of diffusing particles which coalesce upon contact. With the aid of a nonperturbative renormalization group, we first analyze the dynamics emerging below the critical dimension two, where strong fluctuations imply anomalously slow decay. Above two dimensions, the long-time, low-density behavior is known to conform with the law of mass action. For this case, we establish an exact mapping between the physics at the microscopic scale (lattice structure, particle shape and size) and the macroscopic decay rate in the law of mass action. In addition, we identify a term violating this classical law. It originates in long-range and many-particle fluctuations and is a simple, universal function of the macroscopic decay rate.

Objective: To evaluate the incidence of infection and extrusion of porous high-density polyethylene (pHDPE) and expanded polytetrafluoroethylene (ePTFE) implants used in rhinoplasty at a high-volume, academic facial plastic surgery practice., Methods: A total of 662 rhinoplasty procedures performed by 3 faculty surgeons from 1999 to 2008 were retrospectively reviewed. Patient demographics, medical comorbidities, operative details, and postoperative course findings were collected from patient records., Results: The incidence of postoperative infection was 2.8% (19 of 662 patients). In each case of infection, alloplastic material had been used. Infections occurred in 1 in 5 rhinoplasty procedures in which pHDPE implants were used. In patients in whom ePTFE was used alone, the infection rate was 5.3%. Exposure developed in 12% of patients in whom an alloplast was used during surgery. Factors notably not associated with infection on bivariate analysis included sex, surgeon, purpose of procedure (functional vs cosmetic), current tobacco use, or history of cocaine use (P > .05 for all)., Conclusions: To our knowledge, this study represents the largest evaluation of the use of pHDPE implants in rhinoplasty to date. Our findings are in contrast to those of previous studies regarding the use of pHDPE in rhinoplasty and parallel to those regarding the use of ePTFE. Caution is strongly recommended when considering the use of pHDPE in rhinoplasty.

Diffusion-limited reactions are studied in detail on the classical coalescing process. We demonstrate how, with the aid of a recent renormalization group approach, fluctuations can be integrated systematically. We thereby obtain an exact relation between the microscopic physics (lattice structure and particle shape and size) and the macroscopic decay rate in the law of mass action. Moreover, we find a strong violation of the law of mass action. The corresponding term in the kinetic equations originates in long-wavelength fluctuations and is a universal function of the macroscopic decay rate.

Cyclic (rock-paper-scissors-type) population models serve to mimic complex species interactions. Focusing on a paradigmatic three-species model with mutations in one dimension, we observe an interplay between equilibrium and nonequilibrium processes in the stationary state. We exploit these insights to obtain asymptotically exact descriptions of the emerging reactive steady state in the regimes of high and low mutation rates. The results are compared to stochastic lattice simulations. Our methods and findings are potentially relevant for the spatiotemporal evolution of other nonequilibrium stochastic processes.

Arteriovenous malformations (AVMs) of the scalp are rare congenital lesions that can be extremely disfiguring and even life-threatening. Another congenital lesion is the preauricular sinus, which forms when the auricular hillocks fail to fuse. We present a case of an AVM of the scalp that ruptured into a preauricular sinus. The occurrence of these two lesions on the same side of a patient is a notable and previously unreported event. The patient was successfully treated with intravascular embolization and surgical ligation.

Americans over the age of 65 represent one of the fastest growing segments of our population. Rhinoplasty in the elderly man presents a unique set of obstacles. In addition to the typical forces of healing and scar contracture, the aging nose features the altered anatomy and physiology of aging added to an already complicated three-dimensional structure. The heavier, thicker, and more sebaceous skin of men can exacerbate the loss of support associated with aging, creating a scenario of worse functional issues in men as compared with their female counterparts. Approaching the aging nose requires an appreciation of the nuances of the preoperative, intraoperative, and postoperative phases of the patient interaction. Although functional issues predominate in this population, aesthetic concerns also need to be addressed.

Malic acid is a potential biomass-derivable "building block" for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production from glucose proceeds via carboxylation of pyruvate, followed by reduction of oxaloacetate to malate. This redox- and ATP-neutral, CO(2)-fixing pathway has a theoretical maximum yield of 2 mol malate (mol glucose)(-1). A previously engineered glucose-tolerant, C(2)-independent pyruvate decarboxylase-negative S. cerevisiae strain was used as the platform to evaluate the impact of individual and combined introduction of three genetic modifications: (i) overexpression of the native pyruvate carboxylase encoded by PYC2, (ii) high-level expression of an allele of the MDH3 gene, of which the encoded malate dehydrogenase was retargeted to the cytosol by deletion of the C-terminal peroxisomal targeting sequence, and (iii) functional expression of the Schizosaccharomyces pombe malate transporter gene SpMAE1. While single or double modifications improved malate production, the highest malate yields and titers were obtained with the simultaneous introduction of all three modifications. In glucose-grown batch cultures, the resulting engineered strain produced malate at titers of up to 59 g liter(-1) at a malate yield of 0.42 mol (mol glucose)(-1). Metabolic flux analysis showed that metabolite labeling patterns observed upon nuclear magnetic resonance analyses of cultures grown on (13)C-labeled glucose were consistent with the envisaged nonoxidative, fermentative pathway for malate production. The engineered strains still produced substantial amounts of pyruvate, indicating that the pathway efficiency can be further improved.

Background: Repair of ventral hernias represents a challenging problem for surgeons. AlloDerm (LifeCell, Branchburg, NJ, USA), an acellular dermal matrix (ADM) product derived from cadaveric human skin, has gained in popularity in the management of abdominal hernias because of its ability to support neovascularization and therefore resist infection. Surgeons have traditionally used nonabsorbable suture when using ADM in this setting, perhaps because of concerns regarding wound strength. This study was undertaken to examine the influence of suture material on wound breaking strength and complication rates in abdominal wall defects closed with ADM., Methods: Full-thickness abdominal defects were created in athymic rats and immediately repaired with an ADM interposition graft using either interrupted Prolene or Maxon suture. Complications were recorded over time and the animals were sacrificed at 1 month intervals. The abdominal repair complex was harvested and wound strength was measured using a tensiometer., Results: There were no hernias in any of the groups. There were also no cases of major adhesions to the AlloDerm. Two rats in the Prolene group developed a stitch extrusion through the ventral skin. Wound breaking strength increased significantly from the second to third month after surgery in both suture groups (p=0.0000, LSD test). The breaking strength of the abdominal fascia-ADM complex increased over the course of the experiment in both test groups, but no significant difference was found between the groups at any time point (p=0.3157). At 3 months, the breaking strength of the Prolene group and Maxon group was nearly identical (27.1 N +/- SD 7.4 and 29.7 N +/- SD 9.6, respectively)., Conclusions: We were unable to identify a significant difference in breaking strength at the interface between ADM and normal, native fascia when using permanent versus resorbable sutures.

For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as l-arabinose, is required. Wild-type strains of Saccharomyces cerevisiae, the organism used in industrial ethanol production, cannot ferment xylose and arabinose. Although metabolic and evolutionary engineering has enabled the efficient alcoholic fermentation of xylose under anaerobic conditions, the conversion of l-arabinose into ethanol by engineered S. cerevisiae strains has previously been demonstrated only under oxygen-limited conditions. This study reports the first case of fast and efficient anaerobic alcoholic fermentation of l-arabinose by an engineered S. cerevisiae strain. This fermentation was achieved by combining the expression of the structural genes for the l-arabinose utilization pathway of Lactobacillus plantarum, the overexpression of the S. cerevisiae genes encoding the enzymes of the nonoxidative pentose phosphate pathway, and extensive evolutionary engineering. The resulting S. cerevisiae strain exhibited high rates of arabinose consumption (0.70 g h(-1) g [dry weight](-1)) and ethanol production (0.29 g h(-1) g [dry weight](-1)) and a high ethanol yield (0.43 g g(-1)) during anaerobic growth on l-arabinose as the sole carbon source. In addition, efficient ethanol production from sugar mixtures containing glucose and arabinose, which is crucial for application in industrial ethanol production, was achieved.

Metabolic engineering of Saccharomyces cerevisiae for ethanol production from D-xylose, an abundant sugar in plant biomass hydrolysates, has been pursued vigorously for the past 15 years. Whereas wild-type S. cerevisiae cannot ferment D-xylose, the keto-isomer D-xylulose can be metabolised slowly. Conversion of D-xylose into D-xylulose is therefore crucial in metabolic engineering of xylose fermentation by S. cerevisiae. Expression of heterologous xylose reductase and xylitol dehydrogenase does enable D-xylose utilisation, but intrinsic redox constraints of this pathway result in undesirable byproduct formation in the absence of oxygen. In contrast, expression of xylose isomerase (XI, EC 5.3.1.5), which directly interconverts D-xylose and D-xylulose, does not have these constraints. However, several problems with the functional expression of various bacterial and Archaeal XI genes have precluded successful use of XI in yeast metabolic engineering. This changed with the discovery of a fungal XI gene in Piromyces sp. E2, expression of which led to high XI activities in S. cerevisiae. When combined with over-expression of the genes of the non-oxidative pentose phosphate pathway of S. cerevisiae, the resulting strain grew anaerobically on D-xylose with a doubling time of ca. 8 h, with the same ethanol yield as on glucose. Additional evolutionary engineering was used to improve the fermentation kinetics of mixed-substrate utilisation, resulting in efficient D-xylose utilisation in synthetic media. Although industrial pilot experiments have already demonstrated high ethanol yields from the D-xylose present in plant biomass hydrolysates, strain robustness, especially with respect to tolerance to inhibitors present in hydrolysates, can still be further improved.

We have recently reported about a Saccharomyces cerevisiae strain that, in addition to the Piromyces XylA xylose isomerase gene, overexpresses the native genes for the conversion of xylulose to glycolytic intermediates. This engineered strain (RWB 217) exhibited unprecedentedly high specific growth rates and ethanol production rates under anaerobic conditions with xylose as the sole carbon source. However, when RWB 217 was grown on glucose-xylose mixtures, a diauxic growth pattern was observed with a relatively slow consumption of xylose in the second growth phase. After prolonged cultivation in an anaerobic, xylose-limited chemostat, a culture with improved xylose uptake kinetics was obtained. This culture also exhibited improved xylose consumption in glucose-xylose mixtures. A further improvement in mixed-sugar utilization was obtained by prolonged anaerobic cultivation in automated sequencing-batch reactors on glucose-xylose mixtures. A final single-strain isolate (RWB 218) rapidly consumed glucose-xylose mixtures anaerobically, in synthetic medium, with a specific rate of xylose consumption exceeding 0.9 gg(-1)h(-1). When the kinetics of zero trans-influx of glucose and xylose of RWB 218 were compared to that of the initial strain, a twofold higher capacity (V(max)) as well as an improved K(m) for xylose was apparent in the selected strain. It is concluded that the kinetics of xylose fermentation are no longer a bottleneck in the industrial production of bioethanol with yeast.

After an extensive selection procedure, Saccharomyces cerevisiae strains that express the xylose isomerase gene from the fungus Piromyces sp. E2 can grow anaerobically on xylose with a mu(max) of 0.03 h(-1). In order to investigate whether reactions downstream of the isomerase control the rate of xylose consumption, we overexpressed structural genes for all enzymes involved in the conversion of xylulose to glycolytic intermediates, in a xylose-isomerase-expressing S. cerevisiae strain. The overexpressed enzymes were xylulokinase (EC 2.7.1.17), ribulose 5-phosphate isomerase (EC 5.3.1.6), ribulose 5-phosphate epimerase (EC 5.3.1.1), transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2). In addition, the GRE3 gene encoding aldose reductase was deleted to further minimise xylitol production. Surprisingly the resulting strain grew anaerobically on xylose in synthetic media with a mu(max) as high as 0.09 h(-1) without any non-defined mutagenesis or selection. During growth on xylose, xylulose formation was absent and xylitol production was negligible. The specific xylose consumption rate in anaerobic xylose cultures was 1.1 g xylose (g biomass)(-1) h(-1). Mixtures of glucose and xylose were sequentially but completely consumed by anaerobic batch cultures, with glucose as the preferred substrate.

Due to a growing market for the biodegradable and renewable polymer polylactic acid, the world demand for lactic acid is rapidly increasing. The tolerance of yeasts to low pH can benefit the process economy of lactic acid production by minimizing the need for neutralizing agents. Saccharomyces cerevisiae (CEN.PK background) was engineered to a homofermentative lactate-producing yeast via deletion of the three genes encoding pyruvate decarboxylase and the introduction of a heterologous lactate dehydrogenase (EC 1.1.1.27). Like all pyruvate decarboxylase-negative S. cerevisiae strains, the engineered strain required small amounts of acetate for the synthesis of cytosolic acetyl-coenzyme A. Exposure of aerobic glucose-limited chemostat cultures to excess glucose resulted in the immediate appearance of lactate as the major fermentation product. Ethanol formation was absent. However, the engineered strain could not grow anaerobically, and lactate production was strongly stimulated by oxygen. In addition, under all conditions examined, lactate production by the engineered strain was slower than alcoholic fermentation by the wild type. Despite the equivalence of alcoholic fermentation and lactate fermentation with respect to redox balance and ATP generation, studies on oxygen-limited chemostat cultures showed that lactate production does not contribute to the ATP economy of the engineered yeast. This absence of net ATP production is probably due to a metabolic energy requirement (directly or indirectly in the form of ATP) for lactate export.

When xylose metabolism in yeasts proceeds exclusively via NADPH-specific xylose reductase and NAD-specific xylitol dehydrogenase, anaerobic conversion of the pentose to ethanol is intrinsically impossible. When xylose reductase has a dual specificity for both NADPH and NADH, anaerobic alcoholic fermentation is feasible but requires the formation of large amounts of polyols (e.g., xylitol) to maintain a closed redox balance. As a result, the ethanol yield on xylose will be sub-optimal. This paper demonstrates that anaerobic conversion of xylose to ethanol, without substantial by-product formation, is possible in Saccharomyces cerevisiae when a heterologous xylose isomerase (EC 5.3.1.5) is functionally expressed. Transformants expressing the XylA gene from the anaerobic fungus Piromyces sp. E2 (ATCC 76762) grew in synthetic medium in shake-flask cultures on xylose with a specific growth rate of 0.005 h(-1). After prolonged cultivation on xylose, a mutant strain was obtained that grew aerobically and anaerobically on xylose, at specific growth rates of 0.18 and 0.03 h(-1), respectively. The anaerobic ethanol yield was 0.42 g ethanol x g xylose(-1) and also by-product formation was comparable to that of glucose-grown anaerobic cultures. These results illustrate that only minimal genetic engineering is required to recruit a functional xylose metabolic pathway in Saccharomyces cerevisiae. Activities and/or regulatory properties of native S. cerevisiae gene products can subsequently be optimised via evolutionary engineering. These results provide a gateway towards commercially viable ethanol production from xylose with S. cerevisiae.

The absence of alcoholic fermentation makes pyruvate decarboxylase-negative (Pdc(-)) strains of Saccharomyces cerevisiae an interesting platform for further metabolic engineering of central metabolism. However, Pdc(-) S. cerevisiae strains have two growth defects: (i) growth on synthetic medium in glucose-limited chemostat cultures requires the addition of small amounts of ethanol or acetate and (ii) even in the presence of a C(2) compound, these strains cannot grow in batch cultures on synthetic medium with glucose. We used two subsequent phenotypic selection strategies to obtain a Pdc(-) strain without these growth defects. An acetate-independent Pdc(-) mutant was obtained via (otherwise) glucose-limited chemostat cultivation by progressively lowering the acetate content in the feed. Transcriptome analysis did not reveal the mechanisms behind the C(2) independence. Further selection for glucose tolerance in shake flasks resulted in a Pdc(-) S. cerevisiae mutant (TAM) that could grow in batch cultures ( micro (max) = 0.20 h(-1)) on synthetic medium, with glucose as the sole carbon source. Although the exact molecular mechanisms underlying the glucose-tolerant phenotype were not resolved, transcriptome analysis of the TAM strain revealed increased transcript levels of many glucose-repressible genes relative to the isogenic wild type in nitrogen-limited chemostat cultures with excess glucose. In pH-controlled aerobic batch cultures, the TAM strain produced large amounts of pyruvate. By repeated glucose feeding, a pyruvate concentration of 135 g liter(-1) was obtained, with a specific pyruvate production rate of 6 to 7 mmol g of biomass(-1) h(-1) during the exponential-growth phase and an overall yield of 0.54 g of pyruvate g of glucose(-1).

Evidence is presented that xylose metabolism in the anaerobic cellulolytic fungus Piromyces sp. E2 proceeds via a xylose isomerase rather than via the xylose reductase/xylitol-dehydrogenase pathway found in xylose-metabolising yeasts. The XylA gene encoding the Piromyces xylose isomerase was functionally expressed in Saccharomyces cerevisiae. Heterologous isomerase activities in cell extracts, assayed at 30 degrees C, were 0.3-1.1 micromol min(-1) (mg protein)(-1), with a Km for xylose of 20 mM. The engineered S. cerevisiae strain grew very slowly on xylose. It co-consumed xylose in aerobic and anaerobic glucose-limited chemostat cultures at rates of 0.33 and 0.73 mmol (g biomass)(-1) h(-1), respectively.

Pyruvate decarboxylase-negative (Pdc(-)) mutants of Saccharomyces cerevisiae require small amounts of ethanol or acetate to sustain aerobic, glucose-limited growth. This nutritional requirement has been proposed to originate from (i) a need for cytosolic acetyl coenzyme A (acetyl-CoA) for lipid and lysine biosynthesis and (ii) an inability to export mitochondrial acetyl-CoA to the cytosol. To test this hypothesis and to eliminate the C(2) requirement of Pdc(-) S. cerevisiae, we attempted to introduce an alternative pathway for the synthesis of cytosolic acetyl-CoA. The addition of L-carnitine to growth media did not restore growth of a Pdc(-) strain on glucose, indicating that the C(2) requirement was not solely due to the inability of S. cerevisiae to synthesize this compound. The S. cerevisiae GLY1 gene encodes threonine aldolase (EC 4.1.2.5), which catalyzes the cleavage of threonine to glycine and acetaldehyde. Overexpression of GLY1 enabled a Pdc(-) strain to grow under conditions of carbon limitation in chemostat cultures on glucose as the sole carbon source, indicating that acetaldehyde formed by threonine aldolase served as a precursor for the synthesis of cytosolic acetyl-CoA. Fractionation studies revealed a cytosolic localization of threonine aldolase. The absence of glycine in these cultures indicates that all glycine produced by threonine aldolase was either dissimilated or assimilated. These results confirm the involvement of pyruvate decarboxylase in cytosolic acetyl-CoA synthesis. The Pdc(-) GLY1 overexpressing strain was still glucose sensitive with respect to growth in batch cultivations. Like any other Pdc(-) strain, it failed to grow on excess glucose in batch cultures and excreted pyruvate when transferred from glucose limitation to glucose excess.

The SKP1 gene of Kluyveromyces lactis was isolated as a suppressor of a lethal temperature-sensitive mutation in the Saccharomyces cerevisiae CTF13 gene (Chromosome Transmission Factor 13). KlSKP1 was localized at chromosome V, adjacent to KlPAS3. A similar arrangement of the two genes is present in S. cerevisiae. Disruption of the KISKP1 gene was lethal, whereas overexpression of KlSKP1 lead to a decreased growth rate, to swollen and chain-forming cells with an increased DNA content, and to decreased plasmid stability. In both yeasts, promoter constructs lacking most of the purported binding sequence showed increased transcription levels of KlSKP1 in comparison to constructs with the entire promoter.

The Kluyveromyces lactis UBP2 gene was isolated as a suppressor of a temperature-sensitive mutation in CBF2, a gene coding for a centromere-binding protein of Saccharomyces cerevisiae. The UBP genes are hydrolases than can cleave a ubiquitin moiety from a protein substrate. KlUBP2 is not essential for growth since a disruption of the KlUBP2 gene had little effect, except for a slight decrease in the growth rate. The stability of centromere-containing plasmids was not influenced either. In addition to KlUBP2, five S. cerevisiae genes involved in the ubiquitination pathway could suppress the ts-mutation in the CBF2 gene, namely UBA1, UBA2, UBP1, UBP2 and YUH1, although YUH1 was the only one that could do this like KlUBP2 from a single-copy plasmid. Surprisingly, these genes encode proteins with antagonistic activity as two, UBA1 and UBA2, are ubiquitin-activating enzymes whereas the other three are de-ubiquitinating hydrolases.

The soil bacterium Agrobacterium tumefaciens can transfer a part of its tumour-inducing (Ti) plasmid, the T-DNA, to plant cells. The virulence (vir) genes, also located on the Ti plasmid, encode proteins involved in the transport of T-DNA into the plant cell. Once in the plant nucleus, T-DNA is able to integrate into the plant genome by an illegitimate recombination mechanism. The host range of A. tumefaciens is not restricted to plant species. A. tumefaciens is also able to transfer T-DNA to the yeast Saccharomyces cerevisiae. In this paper we demonstrate transfer of T-DNA from A. tumefaciens to the yeast Kluyveromyces lactis. Furthermore, we found that T-DNA serves as an ideal substrate for gene targeting in K. lactis. We have studied the efficiency of gene targeting at the K. lactis TRP1 locus using either direct DNA transfer (electroporation) or T-DNA transfer from Agrobacterium. We found that gene targeting using T-DNA was at least ten times more efficient than using linear double-stranded DNA introduced by electroporation. Therefore, the outcome of gene targeting experiments in some organisms may depend strongly upon the DNA substrate used.

The gene coding for the centromere-binding factor 5 (CBF5) of Kluyveromyces lactis has been isolated by hybridization of a Saccharomyces cerevisiae CBF5 DNA probe to a K. lactis library. The amino acid sequence of KlCbf5 is highly homologous, 88% identity, to ScCbf5, but also to the rat protein Nap57 (64% identity). The main difference between both yeast proteins and the rat protein is the presence of a lysine-rich domain with KKE/D repeats in the C-terminal part of the protein. These repeats are thought to be involved in binding of the protein to microtubules. Deletion of the KKE/D domain in KlCbf5 however, has no discernible effect on growth on rich medium, sensitivity to the microtubule-destabilizing drug benomyl or segregation of a reporter plasmid. On the other hand, insertion of two leucine residues adjacent to the KKE domain increases the loss rate of a reporter plasmid. In both yeasts complementation of a lethal CBF5 disruption with the heterologous gene results in a slight increase in benomyl sensitivity. A possible role of CBF5 in chromosome segregation will be discussed.

The KlCPF1 gene, coding for the centromere and promoter factor CPF1 from Kluyveromyces lactis, has been cloned by functional complementation of the methionine auxotrophic phenotype of a Saccharomyces cerevisiae mutant lacking ScCPF1. The amino-acid sequences of both CPF1 proteins show a relatively-low overall identity (31%), but a highly-homologous C-terminal domain (86%). This region constitutes the DNA-binding domain with basic-helix-loop-helix and leucine-zipper motifs, features common to the myc-related transcription factor family. The N-terminal two-thirds of the CPF1 proteins show no significant similarity, although the presence of acidic regions is a shared feature. In KlCPF1, the acidic region is a prominent stretch of approximately 40 consecutive aspartate and glutamate residues, suggesting that this part might be involved in transcriptional activation. In-vitro mobility-shift experiments were used to establish that both CPF1 proteins bind to the consensus binding site RTCACRTG (CDEI element). In contrast to S. cerevisiae, CPF1 gene-disruption is lethal in K. lactis. The homologous CPF1 genes were transformed to both S. cerevisiae and K. lactis cpf1-null strains. Indistinguishable phenotypes were observed, indicating that, not withstanding the long nonconserved N-terminal region, the proteins are sufficiently homologous to overcome the phenotypes associated with cpf1 gene-disruption.

Transcriptional and translational fusions between the reading frame of the beta-D-glucuronidase gene (gusA) and the 2' as well as the 1' promoter of mannopine synthase (mas), a TR locus of Agrobacterium tumefaciens, were made. The expression of these constructs was studied in the transgenic F1 offspring of independent tobacco transformants at the protein level by assaying for GUS activity and western blot analysis of the GUS protein and at the steady-state mRNA level. In leaves, stems and roots no correlation was found between steady-state levels of GUS mRNA and enzyme activity. In older tissues significantly higher GUS activities were found. This is explained by the stable character of the GUS protein together with an accumulation of protein upon ageing. Three to ten times higher GUS activities were found for in vitro grown plants than for greenhouse-grown plants of the same offspring, despite similar levels of GUS mRNA. Roots from in vitro grown plants display three to ten times higher GUS activities than stems and leaves. In transgenic plants grown in vitro, containing a translational fusion with two AUGs in phase, the initiation of translation in leaf material occurred at both AUGs. Initiation of translation at the first AUG, however, was ten times more frequent. In contrast, initiation in roots from in vitro grown plants occurred exclusively at the second AUG.