We present a completely automated algorithm for estimating the parameters of the contrast transfer function (CTF) of a transmission electron microscope. The primary contribution of this paper is the determination of the astigmatism prior to the estimation of the CTF parameters. The CTF parameter estimation is then reduced to a 1D problem using elliptical averaging. We have also implemented an automated method to calculate lower and upper cutoff frequencies to eliminate regions of the power spectrum which perturb the estimation of the CTF parameters. The algorithm comprises three optimization subproblems, two of which are proven to be convex. Results of the CTF estimation method are presented for images of carbon support films as well as for images of single particles embedded in ice and suspended over holes in the support film. A MATLAB implementation of the algorithm, called ACE, is freely available.

BACKGROUND

The objective of this study was to assess the impact of intracardiac echocardiography (ICE) on the long-term success and complications in patients undergoing pulmonary vein isolation (PVI) for treatment of atrial fibrillation (AF).

RESULTS

Three hundred fifteen patients underwent PVI for treatment of AF. Each patient underwent ostial isolation of all PVs using a cooled-tip ablation catheter. PVI was performed using circular mapping (CM) alone (group 1, 56 patients), CM and ICE (group 2, 107 patients), and CM and ICE with titration of radiofrequency energy based on visualization of microbubbles by ICE (group 3, 152 patients). After a mean follow-up time of 417+/-145 days, 19.6% (11 of 56), 16.8% (18 of 107), and 9.8% (15 of 152) of patients in groups 1, 2, and 3 experienced recurrence of AF, respectively. Moreover, whereas no group 3 patient experienced severe (>70%) PV stenosis, severe PV stenosis was documented in 3 (3.5%) of 56 patients in group 1 and in 2 (1.8%) of 107 patients in group 2 (P<0.05). No embolic events were detected in group 3 patients.

CONCLUSIONS

Intracardiac echocardiography improves the outcome of cooled-tip PVI. Power adjustment guided by direct visualization of microbubble formation reduces the risk of PV stenosis and improves long-term cure.

BACKGROUND

Vibrio vulnificus is a gram-negative bacterium that causes septicaemia and wound infection. Cases occur sporadically, and no previous outbreaks due to a common source or a clonal strain have been reported. In the summer and autumn of 1996 and 1997, an outbreak of invasive V. vulnificus infection occurred in Israel in people who had recently handled fresh, whole fish purchased from artificial fish-ponds.

METHODS

We reviewed clinical and epidemiological information, and undertook an environmental investigation to assess disease characteristics, modes of transmission, phenotypic characteristics of the bacterium, and fish-marketing policy. The clonal nature of 19 isolates was studied by biotyping, pulsed-field gel electrophoresis, and restriction-fragment length polymorphism (RFLP) analysis of a PCR fragment.

RESULTS

During 1996-97, 62 cases of wound infection and bacteraemia occurred. 57 patients developed cellulitis, four had necrotising fasciitis, and one developed osteomyelitis. In all cases, the fish were cultivated in inland fish-ponds. In the summer of 1996, fish-pond managers initiated a new marketing policy, in which fish were sold alive instead of being packed in ice. Phenotypically, the isolates had five atypical biochemical test results. The isolates were non-typeable by pulsed-field gel electrophoresis, and all had the same PCR-RFLP pattern which had not been seen previously.

CONCLUSIONS

The cause of the outbreak was a new strain of V. vulnificus, classified as biogroup 3. A new fish-marketing policy that began in 1996 may have exposed susceptible people to the organism.

The role of hydrogen bonding and amphiphilic packing in the self-assembly of peptide-amphiphiles (PAs) was investigated using a series of 26 PA derivatives, including 19 N-methylated variants and 7 alanine mutants. These were studied by circular dichroism spectroscopy, a variety of Fourier transform infrared spectroscopies, rheology, and vitreous ice cryo-transmission electron microscopy. From these studies, we have been able to determine which amino acids are critical for the self-assembly of PAs into nanofibers, why the nanofiber is favored over other possible nanostructures, the orientation of hydrogen bonding with respect to the nanofiber axis, and the constraints placed upon the portion of the peptide most intimately associated with the biological environment. Furthermore, by selectively eliminating key hydrogen bonds, we are able to completely change the nanostructure resulting from self-assembly in addition to modifying the macroscopic mechanical properties associated with the assembled gel. This study helps to clarify the mechanism of self-assembly for peptide amphiphiles and will thereby help in the design of future generations of PAs.

We used comparative genomics to investigate the distribution of conserved DNA-binding motifs in the regulatory regions of genes involved in iron and manganese homeostasis in alpha-proteobacteria. Combined with other computational approaches, this allowed us to reconstruct the metal regulatory network in more than three dozen species with available genome sequences. We identified several classes of cis-acting regulatory DNA motifs (Irr-boxes or ICEs, RirA-boxes, Iron-Rhodo-boxes, Fur-alpha-boxes, Mur-box or MRS, MntR-box, and IscR-boxes) in regulatory regions of various genes involved in iron and manganese uptake, Fe-S and heme biosynthesis, iron storage, and usage. Despite the different nature of the iron regulons in selected lineages of alpha-proteobacteria, the overall regulatory network is consistent with, and confirmed by, many experimental observations. This study expands the range of genes involved in iron homeostasis and demonstrates considerable interconnection between iron-responsive regulatory systems. The detailed comparative and phylogenetic analyses of the regulatory systems allowed us to propose a theory about the possible evolution of Fe and Mn regulons in alpha-proteobacteria. The main evolutionary event likely occurred in the common ancestor of the Rhizobiales and Rhodobacterales, where the Fur protein switched to regulating manganese transporters (and hence Fur had become Mur). In these lineages, the role of global iron homeostasis was taken by RirA and Irr, two transcriptional regulators that act by sensing the physiological consequence of the metal availability rather than its concentration per se, and thus provide for more flexible regulation.

The benefits of regular exercise, physical fitness and sports participation on cardiovascular and brain health are undeniable. Physical activity reduces the risk for cardiovascular disease, type 2 diabetes, hypertension, obesity, and stroke, and produces beneficial effects on cholesterol levels, antioxidant systems, inflammation, and vascular function. Exercise also enhances psychological health, reduces age-related loss of brain volume, improves cognition, reduces the risk of developing dementia, and impedes neurodegeneration. Nonetheless, the play of sports is associated with risks, including a risk for mild TBI (mTBI) and, rarely, catastrophic traumatic injury and death. There is also growing awareness that repetitive mTBIs, such as concussion and subconcussion, can occasionally produce persistent cognitive, behavioral, and psychiatric problems as well as lead to the development of a neurodegeneration, chronic traumatic encephalopathy (CTE). In this review, we summarize the beneficial aspects of sports participation on psychological, emotional, physical and cognitive health, and specifically analyze some of the less common adverse neuropathological outcomes, including concussion, second-impact syndrome, juvenile head trauma syndrome, catastrophic sudden death, and CTE. CTE is a latent neurodegeneration clinically associated with behavioral changes, executive dysfunction and cognitive impairments, and pathologically characterized by frontal and temporal lobe atrophy, neuronal and axonal loss, and abnormal deposits of paired helical filament (PHF)-tau and 43 kDa TAR deoxyribonucleic acid (DNA)-binding protein (TDP-43). CTE often occurs as a sole diagnosis, but may be associated with other neurodegenerative disorders, including motor neuron disease (CTE-MND). Although the incidence and prevalence of CTE are not known, CTE has been reported most frequently in American football players and boxers. Other sports associated with CTE include ice hockey, professional wrestling, soccer, rugby, and baseball.

1. A rapid colorimetric and apparently specific micromethod for the determination of total glutathione in small amounts of tissue is described. Generally, less than 30mg. of tissue is sufficient and this is homogenized in ice-cold 3% metaphosphoric acid. The product is filtered through sintered glass and neutralized or diluted before being added to a cuvette containing phosphate buffer, pH7.1, 5,5'-dithiobis-(2-nitrobenzoic acid), EDTA and glutathione reductase. Addition of NADPH(2) to the system initiates a progressive reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) by catalytic amounts of GSH, and this causes a colour increase at 412mmu. The rate of this change, calculated over 5min., is proportional to the total amount of glutathione present, and consequently unknown concentrations may be determined by reference to standards. 2. A preparation (based on that of Racker, 1955) of a suitable sample of glutathione reductase from yeast is described. 3. A less specific and less sensitive determination of extracted thiol groups with 5,5'-dithiobis-(2-nitrobenzoic acid) at pH8.0, based on observations of Ellman (1959) and Jocelyn (1962), is also described. 4. Although the precise nature of the reaction is not known, evidence is put forward to support a process of cyclo-reduction. GSSG is reduced enzymically to GSH, which reacts with 5,5'-dithiobis-(2-nitrobenzoic acid) to produce a coloured ion: [Formula: see text] (E(max.) 412mmu) and a mixed disulphide. This disulphide reacts with further quantities of GSH to liberate another ion and GSSG, which then re-enters the cycle.

The evolution of oxygenic photosynthesis had a profound impact on the Earth's surface chemistry, leading to a sharp rise in atmospheric oxygen between 2.45 and 2.32 billion years (Gyr) ago and the onset of extreme ice ages. The oldest widely accepted evidence for oxygenic photosynthesis has come from hydrocarbons extracted from approximately 2.7-Gyr-old shales in the Pilbara Craton, Australia, which contain traces of biomarkers (molecular fossils) indicative of eukaryotes and suggestive of oxygen-producing cyanobacteria. The soluble hydrocarbons were interpreted to be indigenous and syngenetic despite metamorphic alteration and extreme enrichment (10-20 per thousand) of (13)C relative to bulk sedimentary organic matter. Here we present micrometre-scale, in situ (13)C/(12)C measurements of pyrobitumen (thermally altered petroleum) and kerogen from these metamorphosed shales, including samples that originally yielded biomarkers. Our results show that both kerogen and pyrobitumen are strongly depleted in (13)C, indicating that indigenous petroleum is 10-20 per thousand lighter than the extracted hydrocarbons. These results are inconsistent with an indigenous origin for the biomarkers. Whatever their origin, the biomarkers must have entered the rock after peak metamorphism approximately 2.2 Gyr ago and thus do not provide evidence for the existence of eukaryotes and cyanobacteria in the Archaean eon. The oldest fossil evidence for eukaryotes and cyanobacteria therefore reverts to 1.78-1.68 Gyr ago and approximately 2.15 Gyr ago, respectively. Our results eliminate the evidence for oxygenic photosynthesis approximately 2.7 Gyr ago and exclude previous biomarker evidence for a long delay (approximately 300 million years) between the appearance of oxygen-producing cyanobacteria and the rise in atmospheric oxygen 2.45-2.32 Gyr ago.

In the accompanying paper by Weil et al. (1996) we show that staurosporine (STS), in the presence of cycloheximide (CHX) to inhibit protein synthesis, induces apoptotic cell death in a large variety of nucleated mammalian cell types, suggesting that all nucleated mammalian cells constitutively express all of the proteins required to undergo programmed cell death (PCD). The reliability of that conclusion depends on the evidence that STS-induced, and (STS + CHS)-induced, cell deaths are bona fide examples of PCD. There is rapidly accumulating evidence that some members of the Ced-3/Interleukin-1 beta converting enzyme (ICE) family of cysteine proteases are part of the basic machinery of PCD. Here we show that Z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a cell-permeable, irreversible, tripeptide inhibitor of some of these proteases, suppresses STS-induced and (STS + CHX)-induced cell death in a wide variety of mammalian cell types, including anucleate cytoplasts, providing strong evidence that these are all bona fide examples of PCD. We show that the Ced-3/ICE family member CPP32 becomes activated in STS-induced PCD, and that Bcl-2 inhibits this activation. Most important, we show that, in some cells at least, one or more CPP32-family members, but not ICE itself, is required for STS-induced PCD. Finally, we show that zVAD-fmk suppresses PCD in the interdigital webs in developing mouse paws and blocks the removal of web tissue during digit development, suggesting that this inhibition will be a useful tool for investigating the roles of PCD in various developmental processes.

PURPOSE In studies of diffuse large B-cell lymphoma, positron emission tomography with [(18)F]fluorodeoxyglucose (FDG-PET) performed after two to four cycles of chemotherapy has demonstrated prognostic significance. However, some patients treated with immunochemotherapy experience a favorable long-term outcome despite a positive interim FDG-PET scan. To clarify the significance of interim FDG-PET scans, we prospectively studied interim FDG-positive disease within a risk-adapted sequential immunochemotherapy program. PATIENTS AND METHODS From March 2002 to November 2006, 98 patients at Memorial Sloan-Kettering Cancer Center received induction therapy with four cycles of accelerated R-CHOP (rituximab + cyclophosphamide, doxorubicin, vincristine, and prednisone) followed by an interim FDG-PET scan. If the FDG-PET scan was negative, patients received three cycles of ICE (ifosfamide, carboplatin, and etoposide) consolidation therapy. If residual FDG-positive disease was seen, patients underwent biopsy; if the biopsy was negative, they also received three cycles of ICE. Patients with a positive biopsy received ICE followed by autologous stem-cell transplantation. RESULTS At a median follow-up of 44 months, overall and progression-free survival were 90% and 79%, respectively. Ninety-seven patients underwent interim FDG-PET scans; 59 had a negative scan, 51 of whom are progression free. Thirty-eight patients with FDG-PET-positive disease underwent repeat biopsy; 33 were negative, and 26 remain progression free after ICE consolidation therapy. Progression-free survival of interim FDG-PET-positive/biopsy-negative patients was identical to that in patients with a negative interim FDG-PET scan (P = .27). CONCLUSION Interim or post-treatment FDG-PET evaluation did not predict outcome with this dose-dense, sequential immunochemotherapy program. Outside of a clinical trial, we recommend biopsy confirmation of an abnormal interim FDG-PET scan before changing therapy.

Interleukin-1 beta-converting enzyme (ICE) proteolytically cleaves pro-IL-1 beta to its mature, active form. The crystal structure at 2.5 A resolution of a recombinant human ICE-tetrapeptide chloromethylketone complex reveals that the holoenzyme is a homodimer of catalytic domains, each of which contains a p20 and a p10 subunit. The spatial separation of the C-terminus of p20 and the N-terminus of p10 in each domain suggests two alternative pathways of assembly and activation in vivo. ICE is homologous to the C. elegans cell death gene product, CED-3, and these may represent a novel class of cytoplasmic cysteine proteases that are important in programmed cell death (apoptosis). Conservation among members of the ICE/CED-3 family of the amino acids that form the active site region of ICE supports the hypothesis that they share functional similarities.

We have identified a human Bcl-2-interacting protein, p28 Bap31. It is a 28-kD (p28) polytopic integral protein of the endoplasmic reticulum whose COOH-terminal cytosolic region contains overlapping predicted leucine zipper and weak death effector homology domains, flanked on either side by identical caspase recognition sites. In cotransfected 293T cells, p28 is part of a complex that includes Bcl-2/Bcl-XL and procaspase-8 (pro-FLICE). Bax, a pro-apoptotic member of the Bcl-2 family, does not associate with the complex; however, it prevents Bcl-2 from doing so. In the absence (but not presence) of elevated Bcl-2 levels, apoptotic signaling by adenovirus E1A oncoproteins promote cleavage of p28 at the two caspase recognition sites. Purified caspase-8 (FLICE/MACH/Mch5) and caspase-1(ICE), but not caspase-3 (CPP32/apopain/ Yama), efficiently catalyze this reaction in vitro. The resulting NH2-terminal p20 fragment induces apoptosis when expressed ectopically in otherwise normal cells. Taken together, the results suggest that p28 Bap31 is part of a complex in the endoplasmic reticulum that mechanically bridges an apoptosis-initiating caspase, like procaspase-8, with the anti-apoptotic regulator Bcl-2 or Bcl-XL. This raises the possibility that the p28 complex contributes to the regulation of procaspase-8 or a related caspase in response to E1A, dependent on the status of the Bcl-2 setpoint within the complex.

In the future, Arctic warming and the melting of polar glaciers will be considerable, but the magnitude of both is uncertain. We used a global climate model, a dynamic ice sheet model, and paleoclimatic data to evaluate Northern Hemisphere high-latitude warming and its impact on Arctic icefields during the Last Interglaciation. Our simulated climate matches paleoclimatic observations of past warming, and the combination of physically based climate and ice-sheet modeling with ice-core constraints indicate that the Greenland Ice Sheet and other circum-Arctic ice fields likely contributed 2.2 to 3.4 meters of sea-level rise during the Last Interglaciation.

Lymphocyte granule-mediated apoptosis is postulated to entail the formation of membrane pores by perforin. Then soluble granzyme reaches the cytosol either through these pores or by reparative pinocytosis. We demonstrate here that Jurkat cells bind and internalize granzyme B via high affinity binding sites without toxic consequence. Apoptosis occurs, however, if sublytic perforin is added to targets washed free of soluble granzyme B. We suggest that granule-mediated apoptosis mimics viral strategies for cellular entry. Accordingly, co-internalization of granzyme B with adenovirus, a virus that escapes endosomes to reach the cytosol, also induced apoptosis. Poly(ADP-ribose) polymerase cleavage and processing of CPP32, ICE-LAP3, and Mch2 were detected at 30 min, while cytosolic acidification and DNA fragmentation occurred at 60 min. Annexin V binding and membrane permeabilization arose at 4 h. The concurrent activation of the Ced-3 proteases differed from the rate at which each cysteine protease is cleaved in vitro by granzyme B. Thus, granzyme B may not directly process these proteases in whole cells but rather may function by activating a more proximal enzyme. These results indicate that adenovirus-mediated delivery of granzyme B is suitable for elucidating biochemical events that accompany granule-mediated apoptosis.

A real-time alignment and reconstruction scheme for electron microscopic tomography (EMT) has been developed and integrated within our UCSF tomography data collection software. This newly integrated software suite provides full automation from data collection to real-time reconstruction by which the three-dimensional (3D) reconstructed volume is immediately made available at the end of each data collection. Real-time reconstruction is achieved by calculating a weighted back-projection on a small Linux cluster (five dual-processor compute nodes) concurrently with the UCSF tomography data collection running on the microscope's computer, and using the fiducial-marker free alignment data generated during the data collection process. The real-time reconstructed 3D volume provides users with immediate feedback to fully asses all aspects of the experiment ranging from sample choice, ice thickness, experimental parameters to the quality of specimen preparation. This information can be used to guide subsequent data collections. Access to the reconstruction is especially useful in low-dose cryo EMT where such information is very difficult to obtain due to extraordinary low signal to noise ratio in each 2D image. In our environment, we generally collect 2048 x 2048 pixel images which are subsequently computationally binned four-fold for the on-line reconstruction. Based upon experiments performed with thick and cryo specimens at various CCD magnifications (50000x-80000x), alignment accuracy is sufficient to support this reduced resolution but should be refined before calculating a full resolution reconstruction. The reduced resolution has proven to be quite adequate to assess sample quality, or to screen for the best data set for full-resolution reconstruction, significantly improving both productivity and efficiency of system resources. The total time from start of data collection to a final reconstructed volume (512 x 512 x 256 pixels) is about 50 min for a +/-70 degrees 2k x 2k pixel tilt series acquired at every 1 degrees.

We reported previously that human interleukin-1 beta converting enzyme (ICE) is regulated by the CrmA serpin encoded by cowpox virus. We now report the mechanism and kinetics of this unusual inhibition of a cysteine proteinase by a member of the serpin superfamily previously thought to inhibit serine proteinase only. CrmA possesses several characteristics typical of a number of inhibitory serpins. It is conformationally unstable, unfolding around 3 M urea, and stable to denaturation in 8 M urea upon complex formation with ICE. CrmA rapidly inhibits ICE with an association rate constant (kon) of 1.7 x 10(7) M-1 s-1, forming a tight complex with an equilibrium constant for inhibition (Ki) of less than 4 x 10(-12) M. These data indicate that CrmA is a potent inhibitor of ICE, consistent with the dramatic effects of CrmA on modifying host responses to virus infection. The inhibition of ICE by CrmA is an example of a "cross-class" interaction, in which a serpin inhibits a non-serine proteinase. Since CrmA possesses characteristics shared by inhibitors of serine proteinases, we presume that ICE, though it is a cysteine proteinase, has a substrate binding geometry strikingly close to that of serine proteinases. We reason that it is the substrate binding geometry, not the catalytic mechanism of a proteinase, that dictates its reactivity with protein inhibitors.

Global temperatures are known to have varied over the past 1500 years, but the spatial patterns have remained poorly defined. We used a global climate proxy network to reconstruct surface temperature patterns over this interval. The Medieval period is found to display warmth that matches or exceeds that of the past decade in some regions, but which falls well below recent levels globally. This period is marked by a tendency for La Niña-like conditions in the tropical Pacific. The coldest temperatures of the Little Ice Age are observed over the interval 1400 to 1700 C.E., with greatest cooling over the extratropical Northern Hemisphere continents. The patterns of temperature change imply dynamical responses of climate to natural radiative forcing changes involving El Niño and the North Atlantic Oscillation-Arctic Oscillation.

OBJECTIVE

The purpose of this study was to learn whether preoperative eating habits can be used to predict outcome after vertical banded gastroplasty (VBG) and Roux-en-Y gastric bypass (RYGB). BACKGROUND SUMMARY: Several independent randomized and sequential studies have reported significantly greater weight loss after RYGB in comparison with VBG. Although the mechanism responsible for weight loss after both procedures is restriction of intake rather than malabsorption, the relationships between calorie intake, food preferences, and postoperative weight loss are not well defined.

METHODS

During the past 5 years, 138 patients were prospectively selected for either VBG or RYGB, based on their preoperative eating habits. All patients were screened by a dietitian who determined total calorie intake and diet composition before recommending VBG or RYGB. Thirty patients were selected for VBG; the remaining 108 patients were classified as "sweets eaters" or "snackers" and had RYGB. Detailed recall diet histories also were performed at each postoperative visit.

RESULTS

Early morbidity rate was zero after VBG versus 3% after RYGB. There were no deaths. Mean follow-up was 39 +/- 11 months after VBG and 38 +/- 14 months after RYGB. Mean weight loss peaked at 74 +/- 23 lb at 12 months after VBG and 99 +/- 24 lb at 16 months after RYGB (p < or = 0.001). Twelve of 30 VBG patients lost>> or = 50% of their excess weight versus 100 of 108 RYGB patients (p < or = 0.0001). Milk/ice cream intake was significantly greater postoperatively in patients who underwent VBG versus patients who underwent RYGB after 6 months (p < or = 0.003), whereas solid sweets intake was significantly greater after VBG during the first 18 months postoperatively (p < or = 0.004). Revision of VBG was performed in 6 of 30 patients (20%) for complications or poor weight loss, whereas only 2 of 108 patients who underwent RYGB required surgical revisions (p < or = 0.001).

CONCLUSIONS

These data show that VBG adversely alters postoperative eating behavior toward soft, high-calorie foods, resulting in problematic postoperative weight loss. Conversely, RYGB patients had significantly greater weight loss despite inferior preoperative eating habits. The high rate of surgical revision in conjunction with inconsistent postoperative weight loss has led us to no longer recommend VBG as treatment for morbid obesity.

Fungal spores can account for large proportions of air particulate matter, and they may potentially influence the hydrological cycle and climate as nuclei for water droplets and ice crystals in clouds, fog, and precipitation. Moreover, some fungi are major pathogens and allergens. The diversity of airborne fungi is, however, not well-known. By DNA analysis we found pronounced differences in the relative abundance and seasonal cycles of various groups of fungi in coarse and fine particulate matter, with more plant pathogens in the coarse fraction and more human pathogens and allergens in the respirable fine particle fraction (<3 microm). Moreover, the ratio of Basidiomycota to Ascomycota was found to be much higher than previously assumed, which might also apply to the biosphere.

Apoptosis is now recognized as a normal feature in the development of the nervous system and may also play a role in neurodegenerative diseases and aging. This phenomenon has been investigated intensively during the last 6-7 years, and the progress made in this field is reviewed here. Besides a few in vivo studies, a variety of neuronal preparations from various parts of the brain, the majority of which were primary cultures, and some cell lines have been investigated. Several apoptosis-inducing agents have been identified, and these include lack of neurotrophic support, neurotransmitters, neurotoxicants, modulators of protein phosphorylation and calcium homeostasis, DNA-damaging agents, oxidative stress, nitric oxide, and ceramides. The precise signaling cascade is not well established, and there are lacunae in many suggested pathways. However, it appears certain that the Bcl family of proteins is involved in the apoptotic pathway, and these proteins in turn affect the processing of interleukin-1beta converting enzyme (ICE)/caspases. The available evidence suggests that there may be several apoptotic pathways that may depend on the cell type and the inducing agent, and most of the pathways may converge at the ICE/caspases step.

The noncolligative peptide and glycopeptide antifreezes found in some cold-water fish act by binding to the ice surface and preventing crystal growth, not by altering the equilibrium freezing point of the water. A simple crystal growth and etching technique allows determination of the crystallographic planes where the binding occurs. In the case of elongated molecules, such as the alpha-helical peptides in this report, it also allows a deduction of the molecular alignment on the ice surface. The structurally similar antifreeze peptides from winter flounder (Pseudopleuronectes americanus) and Alaskan plaice (Pleuronectes quadritaberulatus) adsorb onto the (2021) pyramidal planes of ice, whereas the sculpin (Myoxocephalus scorpius) peptide adsorbs on (2110), the secondary prism planes. All three are probably aligned along (0112). These antifreeze peptides have 11-amino acid sequence repeats ending with a polar residue, and each repeat constitutes a distance of 16.5 A along the helix, which nearly matches the 16.7 A repeat spacing along (0112) in ice. This structural match is undoubtedly important, but the mechanism of binding is not yet clear. The suggested mechanism of growth inhibition operates through the influence of local surface curvature upon melting point and results in complete inhibition of the crystal growth even though individual antifreeze molecules bind at only one interface orientation.

Design of freeze-drying processes is often approached with a "trial and error" experimental plan or, worse yet, the protocol used in the first laboratory run is adopted without further attempts at optimization. Consequently, commercial freeze-drying processes are often neither robust nor efficient. It is our thesis that design of an "optimized" freeze-drying process is not particularly difficult for most products, as long as some simple rules based on well-accepted scientific principles are followed. It is the purpose of this review to discuss the scientific foundations of the freeze-drying process design and then to consolidate these principles into a set of guidelines for rational process design and optimization. General advice is given concerning common stability issues with proteins, but unusual and difficult stability issues are beyond the scope of this review. Control of ice nucleation and crystallization during the freezing step is discussed, and the impact of freezing on the rest of the process and final product quality is reviewed. Representative freezing protocols are presented. The significance of the collapse temperature and the thermal transition, denoted Tg', are discussed, and procedures for the selection of the "target product temperature" for primary drying are presented. Furthermore, guidelines are given for selection of the optimal shelf temperature and chamber pressure settings required to achieve the target product temperature without thermal and/or mass transfer overload of the freeze dryer. Finally, guidelines and "rules" for optimization of secondary drying and representative secondary drying protocols are presented.

Climate change induced by anthropogenic warming of the earth's atmosphere is a daunting problem. This review examines one of the consequences of climate change that has only recently attracted attention: namely, the effects of climate change on the environmental distribution and toxicity of chemical pollutants. A review was undertaken of the scientific literature (original research articles, reviews, government and intergovernmental reports) focusing on the interactions of toxicants with the environmental parameters, temperature, precipitation, and salinity, as altered by climate change. Three broad classes of chemical toxicants of global significance were the focus: air pollutants, persistent organic pollutants (POPs), including some organochlorine pesticides, and other classes of pesticides. Generally, increases in temperature will enhance the toxicity of contaminants and increase concentrations of tropospheric ozone regionally, but will also likely increase rates of chemical degradation. While further research is needed, climate change coupled with air pollutant exposures may have potentially serious adverse consequences for human health in urban and polluted regions. Climate change producing alterations in: food webs, lipid dynamics, ice and snow melt, and organic carbon cycling could result in increased POP levels in water, soil, and biota. There is also compelling evidence that increasing temperatures could be deleterious to pollutant-exposed wildlife. For example, elevated water temperatures may alter the biotransformation of contaminants to more bioactive metabolites and impair homeostasis. The complex interactions between climate change and pollutants may be particularly problematic for species living at the edge of their physiological tolerance range where acclimation capacity may be limited. In addition to temperature increases, regional precipitation patterns are projected to be altered with climate change. Regions subject to decreases in precipitation may experience enhanced volatilization of POPs and pesticides to the atmosphere. Reduced precipitation will also increase air pollution in urbanized regions resulting in negative health effects, which may be exacerbated by temperature increases. Regions subject to increased precipitation will have lower levels of air pollution, but will likely experience enhanced surface deposition of airborne POPs and increased run-off of pesticides. Moreover, increases in the intensity and frequency of storm events linked to climate change could lead to more severe episodes of chemical contamination of water bodies and surrounding watersheds. Changes in salinity may affect aquatic organisms as an independent stressor as well as by altering the bioavailability and in some instances increasing the toxicity of chemicals. A paramount issue will be to identify species and populations especially vulnerable to climate-pollutant interactions, in the context of the many other physical, chemical, and biological stressors that will be altered with climate change. Moreover, it will be important to predict tipping points that might trigger or accelerate synergistic interactions between climate change and contaminant exposures.

Leucine-Rich Repeat Kinase 2 (LRRK2) is a causative gene for the autosomal dominant form of Parkinson's disease (PD). The gene encodes the approximately 280 kDa LRRK2 protein composed of domains such as leucine-rich repeats, Ras in complex proteins (Roc) followed by C-terminal of Roc (COR), mitogen-activated protein kinase kinase kinase (MAPKKK) and WD40. However, the normal function of the protein as well as its contribution to the pathogenesis of PD remains largely unknown. Here we describe the localization of LRRK2 in Golgi apparatus, plasma membrane and synaptic vesicles in cultured cells including mouse primary neurons. The membrane association of LRRK2 resists solubilization by ice-cold 1% Triton X-100, indicating its association through lipid rafts. To investigate whether mutations found in PD patients affect the localization of LRRK2, we transfected various LRRK2 mutants into cultured cells and performed fractionation experiments. Unexpectedly, the mutants are collected in both membrane and soluble fractions in a manner similar to wild type (WT). I2020T mutant LRRK2 associates with lipid rafts, similar to the WT. The lipid raft association of LRRK2 mutants as well as WT LRRK2 suggests that alteration of LRRK2 function on lipid rafts contributes to the pathogenesis of PD.