Computational correction of spatially variant optical aberrations in 3D single-molecule localization microscopy.

3D single-molecule localization microscopy relies on fitting the shape of point-spread-functions (PSFs) recorded on a wide-field detector. However, optical aberrations distort those shapes, which compromises the accuracy and precision of single-molecule localization microscopy. Here, we employ a computational phase retrieval based on a vectorial PSF model to quantify the spatial variance of optical aberrations in a two-channel ultrawide-field single-molecule localization microscope. The use of a spatially variant PSF model enables accurate and precise emitter localization in x-, y- and z-directions throughout the entire field of view.

All-optical amplitude-phase transmultiplexing of RZ-OOK and RZ-BPSK to RZ-QPSK by polarization-insensitive XPM using a nonlinear birefringent AlGaAs waveguide.

Polarization-insensitive (PI) phase-transmultiplexing (PTM) of a 10-Gb/s return-to-zero ON-OFF keying (RZ-OOK) pump and a 10-Gb/s RZbinary phase-shift keying (RZ-BPSK) probe to 20-Gb/s RZ-quadrature-PSK (RZ-QPSK) has been successfully demonstrated for the first time in a passive, birefringent AlGaAs waveguide, utilizing PI cross-phase modulation (PI-XPM). For differential QPSK (DQPSK)-detection, a 10 − 9-BER pre-amplified receiver sensitivity penalty of ≈ 2.5 dB for the in-phase component and ≈ 4.9 dB for the quadrature component were found. The penalties were relative to the FPGA-precoded RZ-DQPSK baseline for a pump-probe detuning of ≈ 12 nm, when the probe state of polarization was scrambled and the pump was launched off-axis into the waveguide.

Enhanced electro-optic phase shifts in suspended waveguides.

We demonstrate enhanced electro-optic phase shifts in suspended InGaAs/InGaAsP quantum well waveguides compared to attached waveguides. The enhancement stems from an improved overlap between the optical mode and the multiple quantum well layers in thin waveguides when the semiconductor material beneath the waveguide is selectively etched. The measured voltage length product is 0.41 V-cm and the measured propagation loss is 2.3 +/- 0.7 dB/cm for the TE mode in the optical L-band.

A microelectromechanically tunable asymmetric Fabry-Perot quantum well modulator at 1.55 microm.

Placing a quantum well modulator in an asymmetric Fabry- Perot cavity enables significantly higher contrast ratios than are possible in a conventional surface-normal quantum well modulator. However, fixed-cavity asymmetric Fabry-Perot quantum well modulators require extremely precise and uniform crystal growth and are sensitive to small fluctuations in temperature or angle of incidence. Here, we experimentally demonstrate an InP-based microelectromechanically tunable asymmetric Fabry-Perot quantum well modulator that operates in the optical C-band. By actuating a suspended InGaAlAs reflector, the cavity mode can be perfectly matched to the appropriate quantum well absorption wavelength. The devices exhibit contrast ratios over 30 (15 dB) at 8 volts quantum well bias and modulation speeds of 1 MHz.

Low-loss suspended quantum well waveguides.

We have used surface micromachining to fabricate suspended InGaAs/InGaAsP quantum well waveguides that are supported by lateral tethers. The average measured TE propagation loss in our samples is 4.1 dB/cm, and the average measured TE loss per tether pair is 0.21 dB. These measurements are performed at wavelengths in the optical L-band, just 125 nm below the quantum well band gap.

Anal condyloma: a comparison between HIV positive and negative patients.

HIV positive and negative patients with anal condylomata were compared to determine an association with squamous cell neoplasia, its disease progression, and response to treatment. From 1992 to 2003, 61 patients were diagnosed with anal condylomata by anal biopsy. Thirty-four patients were HIV+ and 27 patients were considered HIV-. Upon retrospective chart review, details on disease progression, development of malignancy, and subsequent treatment were collected. Sixty-one per cent of HIV+ patients had a neoplastic process in contrast to 25 per cent of HIV- patients (P = 0.005). Five patients demonstrated disease progression, of which four were HIV+. Three HIV+ patients were treated for invasive carcinoma with excision and standard chemoradiation therapy. Two patients with T3 lesions developed recurrence and died. Eighteen HIV+ patients had noninvasive carcinoma and were treated with local excision without recurrence at mean follow-up of 28 months. HIV+ patients were shown to have more condylomata harboring squamous cell neoplasia than HIV- patients. Noninvasive carcinoma can be treated effectively with local excision, independent of HIV status; however, long-term follow-up is needed. Chemoradiation therapy in patients who are relatively healthy and have stage I disease may be successful. The role for chemoradiation in AIDS patients with stage III disease remains unclear.

Cytokines and the pathogenesis of nosocomial pneumonia.

BACKGROUND: Nosocomial pneumonia (NP) in injured patients is a significant clinical problem. We hypothesize that the pathogenesis of NP in injured patients involves an imbalanced cytokine response within the alveolar airspace that may inhibit effector cell function. METHODS: Proinflammatory (IL-8) and anti-inflammatory (IL-10) levels were measured in bronchoalveolar lavage (BAL) fluid from multitrauma patients on admission, 24, 48, and 72 hours post-injury and following lipopolysaccharide (LPS) induction of alveolar cells. Patients were compared based on IL-8 levels and the development of NP. RESULTS: A high level of IL-8 on admission was associated with the development of NP. In addition, levels of IL-8 were significantly greater in NP-positive patients at all time points. The IL-10 levels decreased from admission values in NP-negative patients but increased in NP-positive patients. Furthermore, a high level of IL-10 ( > 120 pg/mL) at 72 hours post-injury was associated with the development of NP. Alveolar cells from NP-positive patients produced significantly more IL-10 in response to LPS than cells from NP-negative patients. CONCLUSIONS: The pathogenesis of NP in injured patients involves an early and severe IL-8 process within the lung followed by an exaggerated IL-10 response that may inhibit effector cell function.

Surveillance of antibiotic resistance in invasive isolates of Neisseria meningitidis in Australia 1994-1999.

A total of 1434 strains of Neisseria meningitidis isolated from cases of invasive meningococcal disease (IMD) in Australia between 1994 and 1999 were examined by standard methods for susceptibility to antibiotics used for treatment and prophylaxis. The proportion of isolates fully susceptible to penicillin decreased from 45% in 1994 to 26% in 1999 (P<0.001). All the other isolates were less sensitive to penicillin except for two meningococci with a penicillin MIC of 1 mg/l. The geometric mean penicillin MIC increased from 0.045 to 0.065 mg/l from 1994 to 1999. There was no significant difference in the geometric mean penicillin MICs of serogroup B and serogroup C meningococci. Penicillin susceptibility was significantly associated with a poorer outcome. Isolates from survivors of IMD had a higher geometric mean penicillin MIC (0.06 mg/l) than those from fatal cases (0.048 mg/l) (P< 0.001). This suggests that factors other than the decrease in susceptibility to penicillin observed were more relevant to outcome in IMD. All isolates were fully susceptible to ceftriaxone. Rifampicin resistance was infrequent (eight isolates in 6 years) and sporadic. A single isolate had decreased quinolone susceptibility. Despite the significant shift in susceptibility to penicillin recorded, this group of antibiotics remains a suitable treatment for IMD in Australia.

Direct detection of ultrafast thermal transients by use of a chirped, supercontinuum white-light pulse.

An experimental technique is demonstrated that permits direct optical measurement of ultrafast material transients during a single excitation-relaxation cycle. Reflection of a linearly chirped, supercontinuum optical pulse from a gold film with changing surface temperature induced by an ultrafast pump pulse allows the thermal transients to be encoded onto the spectrum of the probe pulse. Calibrating the chirp of the probe pulse and the wavelength sensitivity of the sample permits mapping of the measured transient into the time domain. Measurements are completed over the course of 100 ps with subpicosecond time resolution. Results obtained with this technique are compared with similar measurements obtained with conventional pump-probe correlation techniques.

Optical pattern recognition by use of a segmented semiconductor optical amplifier.

A technique for high-speed, all-optical pattern recognition based on cross correlation in a segmented semiconductor optical amplifier (SSOA) is presented. A counterpropagating pump-probe setup is used to perform cross correlation of the spatial gain-loss pattern in the SSOA with the optical data pattern (pump), and the result is read out with a counterpropagating probe. Cross correlation of 4-bit patterns at 85 Gbits/s is experimentally demonstrated. Simulations show reasonable agreement with experimental measurements and are used to address scalability to higher bit rates and longer data patterns.

Contribution of dietary protein to sulfide production in the large intestine: an in vitro and a controlled feeding study in humans.

BACKGROUND: Hydrogen sulfide is a luminally acting, bacterially derived cell poison that has been implicated in ulcerative colitis. Sulfide generation in the colon is probably driven by dietary components such as sulfur-containing amino acids (SAAs) and inorganic sulfur (eg, sulfite). OBJECTIVE: We assessed the contribution of SAAs from meat to sulfide production by intestinal bacteria with use of both a model culture system in vitro and an in vivo human feeding study. DESIGN: Five healthy men were housed in a metabolic suite and fed a sequence of 5 diets for 10 d each. Meat intake ranged from 0 g/d with a vegetarian diet to 600 g/d with a high-meat diet. Fecal sulfide and urinary sulfate were measured in samples collected on days 9 and 10 of each diet period. Additionally, 5 or 10 g bovine serum albumin or casein/L was added to batch cultures inoculated with feces from 4 healthy volunteers. Concentrations of sulfide, ammonia, and Lowry-reactive substances were measured over 48 h. RESULTS: Mean (+/-SEM) fecal sulfide concentrations ranged from 0.22 +/- 0.02 mmol/kg with the 0-g/d diet to 3.38 +/- 0.31 mmol/kg with the 600-g/d diet and were significantly related to meat intake (P: < 0.001). Sulfide formation in fecal batch cultures supplemented with both bovine serum albumin and casein correlated with protein digestion, as measured by the disappearance of Lowry-reactive substances and the appearance of ammonia. CONCLUSION: Dietary protein from meat is an important substrate for sulfide generation by bacteria in the human large intestine.

Identification of patients at highest risk for ventilator-associated pneumonia in the surgical intensive care unit.

Ventilator-acquired pneumonia (VAP) is a major problem for patients admitted to the surgical intensive care unit mechanically ventilated. Recently, we have identified both clinical and immunologic factors associated with the development of VAP. The clinical risk factors are associated with the severity of the injury and the length of mechanical ventilation. The immunologic risk factors are associated with the local lung inflammatory response that is unchecked and affects local cell function. The combination of the severity of injury, the length of mechanical ventilation, and the failure to "auto-regulate" the lung response places the host at risk of VAP. In the next millennium, if we are to make significant advances in the management of VAP, we will need to understand the pathophysiology of the disease process. Then we can develop preventive strategies that will reduce the morbidity and the associated cost of VAP.

A new method for the determination of sulphide in gastrointestinal contents and whole blood by microdistillation and ion chromatography.

Hydrogen sulphide is produced in the human large intestine by the bacterial reduction of dietary inorganic sulphate and sulphite and by fermentation of sulphur amino acids. Sulphide may damage the colonic epithelium and has been implicated in the pathogenesis of ulcerative colitis. The accurate measurement of sulphide in biological samples, particularly in gut contents is difficult due to the volatile nature of the compound, and the viscosity and turbidity of the samples. Here we describe a method for the determination of sulphide in gut contents and whole blood which overcomes these problems. Initially, samples are treated with zinc acetate to trap sulphide. A microdistillation pretreatment is then used, which releases sulphide from its stable, stored state, coupled to ion chromatography with electrochemical detection. The limit of detection of the method was determined as 2.5 micromol/l, which enabled sulphide levels in gut contents and whole blood samples obtained from humans to be accurately determined. A preliminary investigation in healthy human subjects showed blood sulphide ranged from 10 to 100 micromol/l. Whole blood sulphide did not change significantly when increasing amounts of protein from meat were fed. However, faecal sulphide did show a significant increase from 164 to 754 nmol/g in four subjects fed diets which contained 60 and 420 g meat.

Oral transmission of human immunodeficiency virus by infected seminal fluid and milk: a novel mechanism.

Salivary transmission by the 30 million human immunodeficiency virus (HIV) carriers is rare, despite kissing, aerosolization, and dental treatment. The main protective mechanism of saliva is reported to be inactivation of HIV-transmitting leukocytes by its unique hypotonicity; however, the successful oral transmission of HIV by seminal fluid and milk is unexplained. Whether seminal fluid and milk successfully transmit HIV orally by overcoming the recipient's salivary hypotonic inactivation of HIV-transmitting leukocytes was tested. Isotonic salt solution and normal donor samples of milk, colostrum, seminal fluid, and blood were studied for their ability to overcome the salivary hypotonic inactivation. All samples, in physiologic volumes, prevented the hypotonic saliva from inactivating HIV-transmitting leukocytes by providing solutes and retarding diffusion. This indicates that successful oral transmission of HIV by seminal fluid, milk, and colostrum may be due to their isotonicity, which overcomes hypotonic salivary inactivation of HIV-transmitting leukocytes.

The bottom line for prediction of residue solvent accessibility.

A simple method of predicting residue solvent accessibilities in proteins is described, with the intention that it should be used as a baseline by which more sophisticated approaches to prediction can be judged. Comparison with existing methods of predicting residue burial reveals that their performance is often little better than that of the baseline method. The problem of comparing different prediction methods is shown to be complicated by the proliferation of different schemes for classifying residue burial.

Identification of regulators for Ypt1 GTPase nucleotide cycling.

Small GTPases of the Ypt/Rab family are involved in the regulation of vesicular transport. Cycling between the GDP- and GTP-bound forms and the accessory proteins that regulate this cycling are thought to be crucial for Ypt/Rab function. Guanine nucleotide exchange factors (GEFs) stimulate both GDP loss and GTP uptake, and GTPase-activating proteins (GAPs) stimulate GTP hydrolysis. Little is known about GEFs and GAPs for Ypt/Rab proteins. In this article we report the identification and initial characterization of two factors that regulate nucleotide cycling by Ypt1p, which is essential for the first two steps of the yeast secretory pathway. The Ypt1p-GEF stimulates GDP release and GTP uptake at least 10-fold and is specific for Ypt1p. Partially purified Ypt1p-GEF can rescue the inhibition caused by the dominant-negative Ypt1p-D124N mutant of in vitro endoplasmic reticulum-to-Golgi transport. This mutant probably blocks transport by inhibiting the GEF, suggesting that we have identified the physiological GEF for Ypt1p. The Ypt1p-GAP stimulates GTP hydrolysis by Ypt1p up to 54-fold, has a higher affinity for the GTP-bound form of Ypt1p than for the GDP-bound form, and is specific to a subgroup of exocytic Ypt proteins. The Ypt1p-GAP activity is not affected by deletion of two genes that encode known Ypt GAPs, GYP7 and GYP1, nor is it influenced by mutations in SEC18, SEC17, or SEC22, genes whose products are involved in vesicle fusion. The GEF and GAP activities for Ypt1p localize to particulate cellular fractions. However, contrary to the predictions of current models, the GEF activity localizes to the fraction that functions as the acceptor in an endoplasmic reticulum-to-Golgi transport assay, whereas the GAP activity cofractionates with markers for the donor. On the basis of our current and previous results, we propose a new model for the role of Ypt/Rab nucleotide cycling and the factors that regulate this process.

GTP hydrolysis is not important for Ypt1 GTPase function in vesicular transport.

GTPases of the Ypt/Rab family play a key role in the regulation of vesicular transport. Their ability to cycle between the GTP- and the GDP-bound forms is thought to be crucial for their function. Conversion from the GTP- to the GDP-bound form is achieved by a weak endogenous GTPase activity, which can be stimulated by a GTPase-activating protein (GAP). Current models suggest that GTP hydrolysis and GAP activity are essential for vesicle fusion with the acceptor compartment or for timing membrane fusion. To test this idea, we inactivated the GTPase activity of Ypt1p by using the Q67L mutation, which targets a conserved residue that helps catalyze GTP hydrolysis in Ras. We demonstrate that the mutant Ypt1-Q67L protein is severely impaired in its ability to hydrolyze GTP both in the absence and in the presence of GAP and consequently is restricted mostly to the GTP-bound form. Surprisingly, a strain with ypt1-Q67L as the only YPT1 gene in the cell has no observable growth phenotypes at temperatures ranging from 14 to 37 degrees C. In addition, these mutant cells exhibit normal rates of secretion and normal membrane morphology as determined by electron microscopy. Furthermore, the ypt1-Q67L allele does not exhibit dominant phenotypes in cell growth and secretion when overexpressed. Together, these results lead us to suggest that, contrary to current models for Ypt/Rab function, GTP hydrolysis is not essential either for Ypt1p-mediated vesicular transport or as a timer to turn off Ypt1p-mediated membrane fusion but only for recycling of Ypt1p between compartments. Finally, the ypt1-Q67L allele, like the wild type, is inhibited by dominant nucleotide-free YPT1 mutations. Such mutations are thought to exert their dominant phenotype by sequestration of the guanine nucleotide exchange factor (GNEF). These results suggest that the function of Ypt1p in vesicular transport requires not only the GTP-bound form of the protein but also the interaction of Ypt1p with its GNEF.

Nutrient profiles in the everglades: examination along the eutrophication gradient.

We examined the concentration profiles of nutrients in the surface water, soil and pore water along the eutrophication gradient of the Water Conservation Area-2A (WCA-2A) in the northern Everglades. Phosphorus levels in the surface waters contributed by the agricultural runoff showed an exponential decrease downstream of the inflow structures attaining background values of 7-12, 7-9 and 5-6 micrograms l-1 of TP, TDP and PO4-P, respectively, at distances of 8-10 km. The pore water PO4-P concentration in the oligotrophic areas ranged between 5 and 10 micrograms l-1. Molar ratios of dissolved inorganic N and P suggest a possible switch in nutrient limitation in the surface water from P in the oligotrophic areas to N in the eutrophic areas (DIN:DIP approximately 5). External nutrient loading has also contributed to a three- to four-fold increase in soil TP concentration and enhanced pore water PO4-P in the northern marshes. Unlike P, C and N concentration in the soils remained fairly uniform along the eutrophication gradient. 210Pb dating of soil cores suggests that the increase in soil P concentration (from < 500 to 1500 micrograms g-1) and P accumulation rate (from 0.06 to 0.46 g P m-2 per year) at the eutrophic site correlates with the installation of inflow structures in 1960-1963 through which agricultural drainage from the Hillsboro canal enters the marshes. Organic P makes up 70-90% of the total P in the soils as uptake by algae and macrophytes is the primary mechanism of P removal in these wetlands. Calcium supply from the underlying bedrock suggested from the surface and pore water chemical profiles has important consequences for P-cycling in the Everglades as Ca-bound P is the major form of inorganic P storage in the soils.

Neural network computer simulation of medical aerosols.

Preliminary investigations have been conducted to assess the potential for using artificial neural networks to simulate aerosol behaviour, with a view to employing this type of methodology in the evaluation and design of pulmonary drug-delivery systems. Details are presented of the general purpose software developed for these tasks; it implements a feed-forward back-propagation algorithm with weight decay and connection pruning, the user having complete run-time control of the network architecture and mode of training. A series of exploratory investigations is then reported in which different network structures and training strategies are assessed in terms of their ability to simulate known patterns of fluid flow in simple model systems. The first of these involves simulations of cellular automata-generated data for fluid flow through a partially obstructed two-dimensional pipe. The artificial neural networks are shown to be highly successful in simulating the behaviour of this simple linear system, but with important provisos relating to the information content of the training data and the criteria used to judge when the network is properly trained. A second set of investigations is then reported in which similar networks are used to simulate patterns of fluid flow through aerosol generation devices, using training data furnished through rigorous computational fluid dynamics modelling. These more complex three-dimensional systems are modelled with equal success. It is concluded that carefully tailored, well trained networks could provide valuable tools not just for predicting but also for analysing the spatial dynamics of pharmaceutical aerosols.

Requirement of nucleotide exchange factor for Ypt1 GTPase mediated protein transport.

Small GTPases of the rab family are involved in the regulation of vesicular transport. It is believed that cycling between the GTP- and GDP-bound forms, and accessory factors regulating this cycling are crucial for rab function. However, an essential role for rab nucleotide exchange factors has not yet been demonstrated. In this report we show the requirement of nucleotide exchange factor activity for Ypt1 GTPase mediated protein transport. The Ypt1 protein, a member of the rab family, plays a role in targeting vesicles to the acceptor compartment and is essential for the first two steps of the yeast secretory pathway. We use two YPT1 dominant mutations that contain alterations in a highly conserved GTP-binding domain, N121I and D124N. YPT1-D124N is a novel mutation that encodes a protein with nucleotide specificity modified from guanine to xanthine. This provides a tool for the study of an individual rab GTPase in crude extracts: a xanthosine triphosphate (XTP)-dependent conditional dominant mutation. Both mutations confer growth inhibition and a block in protein secretion when expressed in vivo. The purified mutant proteins do not bind either GDP or GTP. Moreover, they completely inhibit the ability of the exchange factor to stimulate nucleotide exchange for wild type Ypt1 protein, and are potent inhibitors of ER to Golgi transport in vitro at the vesicle targeting step. The inhibitory effects of the Ypt1-D124N mutant protein on both nucleotide exchange activity and protein transport in vitro can be relieved by XTP, indicating that it is the nucleotide-free form of the mutant protein that is inhibitory. These results suggest that the dominant mutant proteins inhibit protein transport by sequestering the exchange factor from the wild type Ypt1 protein, and that this factor has an essential role in vesicular transport.