Procedures for the statistical evaluation of method comparisons and instrument tests often have a requirement for distributional properties of the experimental data, but this requirement is frequently not met. In our paper we propose a new linear regression procedure with no special assumptions regarding the distribution of the samples and the measurement errors. The result does not depend on the assignment of the methods (instruments) to X and Y. After testing a linear relationship between X and Y confidence limits are given for the slope beta and the intercept alpha; they are used to determine whether there is only a chance difference between beta and 1 and between alpha and 0. The mathematical background is amplified separately in an appendix.

The ability of personality traits to predict important life outcomes has traditionally been questioned because of the putative small effects of personality. In this article, we compare the predictive validity of personality traits with that of socioeconomic status (SES) and cognitive ability to test the relative contribution of personality traits to predictions of three critical outcomes: mortality, divorce, and occupational attainment. Only evidence from prospective longitudinal studies was considered. In addition, an attempt was made to limit the review to studies that controlled for important background factors. Results showed that the magnitude of the effects of personality traits on mortality, divorce, and occupational attainment was indistinguishable from the effects of SES and cognitive ability on these outcomes. These results demonstrate the influence of personality traits on important life outcomes, highlight the need to more routinely incorporate measures of personality into quality of life surveys, and encourage further research about the developmental origins of personality traits and the processes by which these traits influence diverse life outcomes.

The rate of protein diffusion in bacterial cytoplasm may constrain a variety of cellular functions and limit the rates of many biochemical reactions in vivo. In this paper, we report noninvasive measurements of the apparent diffusion coefficient of green fluorescent protein (GFP) in the cytoplasm of Escherichia coli. These measurements were made in two ways: by photobleaching of GFP fluorescence and by photoactivation of a red-emitting fluorescent state of GFP (M. B. Elowitz, M. G. Surette, P. E. Wolf, J. Stock, and S. Leibler, Curr. Biol. 7:809-812, 1997). The apparent diffusion coefficient, Da, of GFP in E. coli DH5alpha was found to be 7.7 +/- 2.5 microm2/s. A 72-kDa fusion protein composed of GFP and a cytoplasmically localized maltose binding protein domain moves more slowly, with Da of 2.5 +/- 0.6 microm2/s. In addition, GFP mobility can depend strongly on at least two factors: first, Da is reduced to 3.6 +/- 0.7 microm2/s at high levels of GFP expression; second, the addition to GFP of a small tag consisting of six histidine residues reduces Da to 4.0 +/- 2.0 microm2/s. Thus, a single effective cytoplasmic viscosity cannot explain all values of Da reported here. These measurements have implications for the understanding of intracellular biochemical networks.

The complement system is an important mediator of the acute inflammatory response, and an effective inhibitor would suppress tissue damage in many autoimmune and inflammatory diseases. Such an inhibitor might be found among the endogenous regulatory proteins of complement that block the enzymes that activate C3 and C5. Of these proteins, complement receptor type 1 (CR1; CD35) has the most inhibitory potential, but its restriction to a few cell types limits its function in vivo. This limitation was overcome by the recombinant, soluble human CR1, sCR1, which lacks the transmembrane and cytoplasmic domains. The sCR1 bivalently bound dimeric forms of its ligands, C3b and methylamine-treated C4 (C4-ma), and promoted their inactivation by factor I. In nanomolar concentrations, sCR1 blocked complement activation in human serum by the two pathways. The sCR1 had complement inhibitory and anti-inflammatory activities in a rat model of reperfusion injury of ischemic myocardium, reducing myocardial infarction size by 44 percent. These findings identify sCR1 as a potential agent for the suppression of complement-dependent tissue injury in autoimmune and inflammatory diseases.

Feeding is influenced by hypothalamic neuropeptides that promote (orexigenic peptides) or inhibit feeding. Of these, neuropeptide Y (NPY) in the arcuate nucleus and melanin-concentrating hormone (MCH) and orexins/hypocretins in the lateral hypothalamus have received attention because their expression is increased during fasting and because they promote feeding when administered centrally. Surprisingly, absence of the orexigenic neuropeptide NPY fails to alter feeding or body weight in normal mice. As deficiency of a single component of the pathway that limits food intake (such as leptin or receptors for melanocortin-4) causes obesity, it has been suggested that orexigenic signals are more redundant than those limiting food intake. To define further the physiological role of MCH and to test the redundancy of orexigenic signals, we generated mice carrying a targeted deletion of the MCH gene. MCH-deficient mice have reduced body weight and leanness due to hypophagia (reduced feeding) and an inappropriately increased metabolic rate, despite their reduced amounts of both leptin and arcuate nucleus pro-opiomelanocortin messenger RNA. Our results show that MCH is a critical regulator of feeding and energy balance which acts downstream of leptin and the melanocortin system, and that deletion of a gene encoding a single orexigenic peptide can result in leanness.

The gastrointestinal epithelium forms the boundary between the body and external environment. It effectively provides a selective permeable barrier that limits the permeation of luminal noxious molecules, such as pathogens, toxins, and antigens, while allowing the appropriate absorption of nutrients and water. This selective permeable barrier is achieved by intercellular tight junction (TJ) structures, which regulate paracellular permeability. Disruption of the intestinal TJ barrier, followed by permeation of luminal noxious molecules, induces a perturbation of the mucosal immune system and inflammation, and can act as a trigger for the development of intestinal and systemic diseases. In this context, much effort has been taken to understand the roles of extracellular factors, including cytokines, pathogens, and food factors, for the regulation of the intestinal TJ barrier. Here, I discuss the regulation of the intestinal TJ barrier together with its implications for the pathogenesis of diseases.

Plants associate with a wide range of microorganisms, with both detrimental and beneficial outcomes. Central to plant survival is the ability to recognize invading microorganisms and either limit their intrusion, in the case of pathogens, or promote the association, in the case of symbionts. To aid in this recognition process, elaborate communication and counter-communication systems have been established that determine the degree of ingress of the microorganism into the host plant. In this Review, I describe the common signalling processes used by plants during mutualistic interactions with microorganisms as diverse as arbuscular mycorrhizal fungi and rhizobial bacteria.

BACKGROUND

Nonnegative Matrix Factorization (NMF) is an unsupervised learning technique that has been applied successfully in several fields, including signal processing, face recognition and text mining. Recent applications of NMF in bioinformatics have demonstrated its ability to extract meaningful information from high-dimensional data such as gene expression microarrays. Developments in NMF theory and applications have resulted in a variety of algorithms and methods. However, most NMF implementations have been on commercial platforms, while those that are freely available typically require programming skills. This limits their use by the wider research community.

RESULTS

Our objective is to provide the bioinformatics community with an open-source, easy-to-use and unified interface to standard NMF algorithms, as well as with a simple framework to help implement and test new NMF methods. For that purpose, we have developed a package for the R/BioConductor platform. The package ports public code to R, and is structured to enable users to easily modify and/or add algorithms. It includes a number of published NMF algorithms and initialization methods and facilitates the combination of these to produce new NMF strategies. Commonly used benchmark data and visualization methods are provided to help in the comparison and interpretation of the results.

CONCLUSIONS

The NMF package helps realize the potential of Nonnegative Matrix Factorization, especially in bioinformatics, providing easy access to methods that have already yielded new insights in many applications. Documentation, source code and sample data are available from CRAN.

Anti-human immunodeficiency virus type 1 (HIV-1) antibodies whose binding to gp120 is enhanced by CD4 binding (CD4i antibodies) are generally considered nonneutralizing for primary HIV-1 isolates. However, a novel CD4i-specific Fab fragment, X5, has recently been found to neutralize a wide range of primary isolates. To investigate the precise nature of the extraordinary neutralizing ability of Fab X5, we evaluated the abilities of different forms (immunoglobulin G [IgG], Fab, and single-chain Fv) of X5 and other CD4i monoclonal antibodies to neutralize a range of primary HIV-1 isolates. Our results show that, for a number of isolates, the size of the neutralizing agent is inversely correlated with its ability to neutralize. Thus, the poor ability of CD4i-specific antibodies to neutralize primary isolates is due, at least in part, to steric factors that limit antibody access to the gp120 epitopes. Studies of temperature-regulated neutralization or fusion-arrested intermediates suggest that the steric effects are important in limiting the binding of IgG to the viral envelope glycoproteins after HIV-1 has engaged CD4 on the target cell membrane. The results identify hurdles in using CD4i epitopes as targets for antibody-mediated neutralization in vaccine design but also indicate that the CD4i regions could be efficiently targeted by small molecule entry inhibitors.

We completed a systematic search of the epidemiologic literature on built environment and obesity and identified 63 relevant papers, which were then evaluated for the quality of between-study evidence. We were able to classify studies into one of two primary approaches for defining place and corresponding geographic areas of influence: those based on contextual effects derived from shared pre-determined administrative units and those based on individually unique geographic buffers. The 22 contextual papers evaluated 80 relations, 38 of which did not achieve statistical significance. The 15 buffer papers evaluated 40 relations, 24 of which did not achieve statistical significance. There was very little between-study similarity in methods in both types of approaches, which prevented estimation of pooled effects. The great heterogeneity across studies limits what can be learned from this body of evidence.

Neutralizing antibodies elicited by prior infection or vaccination are likely to be key for future protection of individuals and populations against SARS-CoV-2. Moreover, passively administered antibodies are among the most promising therapeutic and prophylactic anti-SARS-CoV-2 agents. However, the degree to which SARS-CoV-2 will adapt to evade neutralizing antibodies is unclear. Using a recombinant chimeric VSV/SARS-CoV-2 reporter virus, we show that functional SARS-CoV-2 S protein variants with mutations in the receptor binding domain (RBD) and N-terminal domain that confer resistance to monoclonal antibodies or convalescent plasma can be readily selected. Notably, SARS-CoV-2 S variants that resist commonly elicited neutralizing antibodies are now present at low frequencies in circulating SARS-CoV-2 populations. Finally, the emergence of antibody-resistant SARS-CoV-2 variants that might limit the therapeutic usefulness of monoclonal antibodies can be mitigated by the use of antibody combinations that target distinct neutralizing epitopes.

Keywords: immunology; infectious disease; inflammation; microbiology; viruses.

OBJECTIVE

To examine trends and effects of weight loss treatment on health-related quality of life (HRQL) in the severely obese over 10 years.

METHODS

Swedish obese subjects (SOS) intervention study is a controlled, longitudinal trial of the health effects of weight loss in the severely obese.

METHODS

A total of 655 of 851 surgically treated and 621 of 852 conventionally treated obese men (body mass index, BMI>or=34) and women (BMI>or=38) who completed 10 years of the study.

METHODS

HRQL was assessed before treatment and after 0.5, 1, 2, 3, 4, 6, 8 and 10 years.

RESULTS

HRQL change during the 10-year observation period largely followed phases of weight loss, weight regain and weight stability. Improvements and deteriorations in HRQL were associated with the magnitude of weight loss or regain, except regarding anxiety. Peak improvements in the surgical group were observed during the first year of weight loss, whereas the weight regain phase (mainly between 1- and 6-year follow-up) was accompanied by a gradual decline in HRQL. The period from 6- to 10-year follow-up was characterized by relatively stable observations in both weight and HRQL. At 10 years, net gains were noted in all HRQL domains compared to baseline. Comparisons of treatment effects on HRQL in the surgical vs conventional group after 10 years showed significantly better outcome in the surgical group on current health perceptions, social interaction, psychosocial functioning and depression, whereas no significant differences were found for overall mood and anxiety. Long-term results of the study suggest that a maintained weight loss of about 10% is sufficient for positive long-term effects on HRQL, a limit that was reached in about two-thirds of the surgically treated patients who completed 10 years of the study.

CONCLUSIONS

Long-lasting weight reduction in the severely obese has a general long-standing positive outcome on HRQL. Bariatric surgery is a favorable option for the treatment of severe obesity, resulting in long-term weight loss and HRQL improvements in a majority of patients. However, difficulties among some surgical patients to control and maintain weight loss over time should not be ignored. Future research should study if the long-term efficacy of bariatric surgery may be further enhanced by implementing lifestyle modification techniques in the postoperative management of patients.

A sensitivie and simple procedure for the detection and quantitation of soluble complement (C)- fixing immune complexes in sera of patients with various disease states has been developed by utilizing C receptors on Raji cells. These cells lack membrane-bound immunoglobulin but have receptors for IgG Fc, C3b, C3d, and possibly with other C proteins. Uptake experiments showed that both aggregated human gamma globulin (AHG) and 7S IgG bound to receptors for IgG Fc; however, AHG reacted with C bound to cells only via receptors for C and this binding was much more efficient than via IgG Fc receptors. AHG was used as an in vitro model of human immune complexes and its uptake by Raji cells was quantitated by 125I-radiolabeled antihuman IgG. The limit of sensitivity of this test was 6 mug AHG/ml serum. The ability of Raji cells to detect AHG in serum depended on the amount of radioactive antibody used and the size of aggregates. The presence of an excess of C somewhat inhibited binding of AHG containing C to Raji cells. The efficient binding of AHG by receptors for C on Raji cells was used for the detection and quantitation of immune complexes in human sera. Raji cells were incubated with sera to be tested and then reacted with excess radiolabeled antihuman IgG; the amount of radioactivity bound to the washed cells was determined and referred to a standard curve of radioactive antibody uptake by cells previously incubated with increasing amounts of AHG in serum. Thereby immune complexes were detected and quantitated in serum hepatitis, systemic lupus erythematosus, vasculitis, subacute sclerosing panencephalitis, dengue hemorrhagic fever, and malignancies.

BACKGROUND

Elderly patients may have limited ability to read and comprehend medical information pertinent to their health.

OBJECTIVE

To determine the prevalence of low functional health literacy among community-dwelling Medicare enrollees in a national managed care organization.

METHODS

Cross-sectional survey.

METHODS

Four Prudential HealthCare plans (Cleveland, Ohio; Houston, Tex; south Florida; Tampa, Fla).

METHODS

A total of 3260 new Medicare enrollees aged 65 years or older were interviewed in person between June and December 1997 (853 in Cleveland, 498 in Houston, 975 in south Florida, 934 in Tampa); 2956 spoke English and 304 spoke Spanish as their native language. MAIN OUTCOME MEASURE; Functional health literacy as measured by the Short Test of Functional Health Literacy in Adults.

RESULTS

Overall, 33.9% of English-speaking and 53.9% of Spanish-speaking respondents had inadequate or marginal health literacy. The prevalence of inadequate or marginal functional health literacy among English speakers ranged from 26.8% to 44.0%. In multivariate analysis, study location, race/language, age, years of school completed, occupation, and cognitive impairment were significantly associated with inadequate or marginal literacy. Reading ability declined dramatically with age, even after adjusting for years of school completed and cognitive impairment. The adjusted odds ratio for having inadequate or marginal health literacy was 8.62 (95% confidence interval, 5.55-13.38) for enrollees aged 85 years or older compared with individuals aged 65 to 69 years.

CONCLUSIONS

Elderly managed care enrollees may not have the literacy skills necessary to function adequately in the health care environment. Low health literacy may impair elderly patients' understanding of health messages and limit their ability to care for their medical problems.

Cell-free human immunodeficiency virus type 1 (HIV-1) can initiate infections, but contact between infected and uninfected T cells can enhance viral spread through intercellular structures called virological synapses (VS). The relative contribution of VS to cell-free viral transfer has not been carefully measured. Using an ultrasensitive, fluorescent virus transfer assay, we estimate that when VS between HIV-expressing Jurkat T cells and primary CD4(+) T cells are formed, cell-associated transfer of virus is 18,000-fold more efficient than uptake of cell-free virus. Furthermore, in contrast to cell-free virus uptake, the VS deposits virus rapidly into focal, trypsin-resistant compartments in target T cells. This massive virus internalization requires Env-CD4 receptor interactions but is resistant to inhibition by patient-derived neutralizing antisera that inhibit homologous cell-free virus. Deleting the Env cytoplasmic tail does not abrogate VS-mediated transfer, but it renders the VS sensitive to neutralizing antibodies, suggesting that the tail limits exposure of VS-neutralizing epitopes on the surface of infected cells. Dynamic live imaging of the VS reveals that HIV-expressing cells are polarized and make sustained, Env-dependent contacts with target cells through uropod-like structures. The polarized T-cell morphology, Env-CD4 coordinated adhesion, and viral transfer from HIV-infected to uninfected cells suggest that VS allows HIV-1 to evade antibody neutralization and to disseminate efficiently. Future studies will discern to what extent this massive viral transfer contributes to productive infection or viral dissemination through the migration of virus-carrying T cells.

Experiments using intracellular recording techniques were performed on rat hippocampal neurones in vitro, to study the discharge properties of these cells. When CA 1 pyramidal cells were excited by injecting long depolarizing current pulses (approximately 600-800 ms), they responded with an initial rapid action potential discharge which slowed, or accommodated, and then stopped after 200-300 ms. The train of action potentials was followed by a hyperpolarization which was due primarily to calcium-activated potassium conductance (GK(Ca]. The amplitude of this hyperpolarization increased with an increasing number of action potentials in the initial discharge. Blocking the calcium-activated potassium conductance, by injecting EGTA into the cell, by bathing the cell in cadmium, a calcium channel blocker, or by bathing the cell in calcium-free medium, reduced the after-hyperpolarization (a.h.p.) and accommodation such that the frequency of action potential discharge increased and the duration of this discharge was prolonged. Blocking the calcium-activated potassium conductance had a greater effect on discharge frequency later in the action potential train, as late interspike intervals were shortened more than early ones by the application of cadmium or of calcium-free medium. This was presumably because the calcium-activated potassium conductance was more developed later in the train. Accommodation was not completely abolished in the absence of calcium and presence of cadmium, suggesting that other factors, in addition to calcium-activated potassium conductance, contributed to this process. This remaining accommodation was reduced by low doses of carbachol, suggesting that the M-current also plays a role in accommodation. We conclude that accommodation of the action potential discharge of hippocampal pyramidal cells may be regulated by at least two potassium currents: the calcium-activated potassium current and the M-current. Both of these currents are turned on during excitation of the neurone and act in an inhibitory manner on that neurone to limit further action potential discharge.

Vascular calcification contributes to the high risk of cardiovascular mortality in chronic kidney disease (CKD) patients. Dysregulation of calcium (Ca) and phosphate (P) metabolism is common in CKD patients and drives vascular calcification. In this article, we review the physiological regulatory mechanisms for Ca and P homeostasis and the basis for their dysregulation in CKD. In addition, we highlight recent findings indicating that elevated Ca and P have direct effects on vascular smooth muscle cells (VSMCs) that promote vascular calcification, including stimulation of osteogenic/chondrogenic differentiation, vesicle release, apoptosis, loss of inhibitors, and extracellular matrix degradation. These studies suggest a major role for elevated P in promoting osteogenic/chondrogenic differentiation of VSMC, whereas elevated Ca has a predominant role in promoting VSMC apoptosis and vesicle release. Furthermore, the effects of elevated Ca and P are synergistic, providing a major stimulus for vascular calcification in CKD. Unraveling the complex regulatory pathways that mediate the effects of both Ca and P on VSMCs will ultimately provide novel targets and therapies to limit the destructive effects of vascular calcification in CKD patients.

Cisplatin is a chemotherapeutic drug used to treat a variety of cancers. Both intrinsic and acquired resistance to cisplatin, as well as toxicity, limit its effectiveness. Molecular mechanisms that underlie cisplatin resistance are poorly understood. Here we demonstrate that deletion of the yeast CTR1 gene, which encodes a high-affinity copper transporter, results in increased cisplatin resistance and reduced intracellular accumulation of cisplatin. Copper, which causes degradation and internalization of Ctr1 protein (Ctr1p), enhances survival of wild-type yeast cells exposed to cisplatin and reduces cellular accumulation of the drug. Cisplatin also causes degradation and delocalization of Ctr1p and interferes with copper uptake in wild-type yeast cells. Mouse cell lines lacking one or both mouse Ctr1 (mCtr1) alleles exhibit increased cisplatin resistance and decreased cisplatin accumulation in parallel with mCtr1 gene dosage. We propose that cisplatin uptake is mediated by the copper transporter Ctr1p in yeast and mammals. The link between Ctr1p and cisplatin transport may explain some cases of cisplatin resistance in humans and suggests ways of modulating sensitivity and toxicity to this important anticancer drug.

The ecology of hydrocarbon degradation by microbial populations in the natural environment is reviewed, emphasizing the physical, chemical, and biological factors that contribute to the biodegradation of petroleum and individual hydrocarbons. Rates of biodegradation depend greatly on the composition, state, and concentration of the oil or hydrocarbons, with dispersion and emulsification enhancing rates in aquatic systems and absorption by soil particulates being the key feature of terrestrial ecosystems. Temperature and oxygen and nutrient concentrations are important variables in both types of environments. Salinity and pressure may also affect biodegradation rates in some aquatic environments, and moisture and pH may limit biodegradation in soils. Hydrocarbons are degraded primarily by bacteria and fungi. Adaptation by prior exposure of microbial communities to hydrocarbons increases hydrocarbon degradation rates. Adaptation is brought about by selective enrichment of hydrocarbon-utilizing microorganisms and amplification of the pool of hydrocarbon-catabolizing genes. The latter phenomenon can now be monitored through the use of DNA probes. Increases in plasmid frequency may also be associated with genetic adaptation. Seeding to accelerate rates of biodegradation has been shown to be effective in some cases, particularly when used under controlled conditions, such as in fermentors or chemostats.

The integration of complex information in working memory, and its effect on capacity, shape the limits of conscious cognition. The literature conflicts on whether short-term visual memory represents information as integrated objects. A change-detection paradigm using objects defined by color with location or shape was used to investigate binding in short-term visual memory. Results showed that features from the same dimension compete for capacity, whereas features from different dimensions can be stored in parallel. Binding between these features can occur, but focused attention is required to create and maintain the binding over time, and this integrated format is vulnerable to interference. In the proposed model, working memory capacity is limited both by the independent capacity of simple feature stores and by demands on attention networks that integrate this distributed information into complex but unified thought objects.

The wealth of new data on membrane protein structures and functions is changing our general view of membrane architecture. Some of the key themes that are emerging are that membranes are patchy, with segregated regions of structure and function, that lipid regions vary in thickness and composition, and that crowding and ectodomains limit exposure of lipid to the adjacent aqueous regions.

BACKGROUND

Dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy and cause of cardiac transplantation in children. However, the epidemiology and clinical course of DCM in children are not well established.

OBJECTIVE

To provide a detailed description of the incidence, causes, outcomes, and related risk factors for DCM in children.

METHODS

Longitudinal study based on a population-based, prospective cohort of children diagnosed as having DCM since January 1, 1996, at 89 pediatric cardiac centers and a retrospectively collected cohort of patients seen primarily at large tertiary care centers in North America and who had diagnoses between January 1, 1990, and December 31, 1995, and were enrolled through February 2003.

METHODS

A total of 1426 children from the United States and Canada diagnosed as having DCM at younger than 18 years. Primary DCM was determined by strict echocardiographic and/or pathologic criteria. Patients with disease due to endocrine, immunologic, drug toxicity, and other causes were excluded.

METHODS

Annual incidence per 100,000 children; mortality; cardiac transplantation.

RESULTS

The annual incidence of DCM in children younger than 18 years was 0.57 cases per 100,000 per year overall. The annual incidence was higher in boys than in girls (0.66 vs 0.47 cases per 100,000; P<.001), in blacks than in whites (0.98 vs 0.46 cases per 100,000; P<.001), and in infants (<1 year) than in children (4.40 vs 0.34 cases per 100,000; P<.001). The majority of children (66%) had idiopathic disease. The most common known causes were myocarditis (46%) and neuromuscular disease (26%). The 1- and 5-year rates of death or transplantation were 31% and 46%, respectively. Independent risk factors at DCM diagnosis for subsequent death or transplantation were older age, congestive heart failure, lower left ventricular fractional shortening Z score, and cause of DCM (P<.001 for all).

CONCLUSIONS

In children, DCM is a diverse disorder with outcomes that depend largely on cause, age, and heart failure status at presentation. Race, sex, and age affect the incidence of disease. Most children do not have a known cause of DCM, which limits the potential for disease-specific therapies.

Some recent articles have discussed biased methods for estimating risk ratios from adjusted odds ratios when the outcome is common, and the problem of setting confidence limits for risk ratios. These articles have overlooked the extensive literature on valid estimation of risks, risk ratios, and risk differences from logistic and other models, including methods that remain valid when the outcome is common, and methods for risk and rate estimation from case-control studies. The present article describes how most of these methods can be subsumed under a general formulation that also encompasses traditional standardization methods and methods for projecting the impact of partially successful interventions. Approximate variance formulas for the resulting estimates allow interval estimation; these intervals can be closely approximated by rapid simulation procedures that require only standard software functions.

We optimally localized isolated fluorescent beads and molecules imaged as diffraction-limited spots, determined the orientation of molecules and present reliable formulas for the precision of various localization methods. Both theory and experimental data showed that unweighted least-squares fitting of a Gaussian squanders one-third of the available information, a popular formula for its precision exaggerates beyond Fisher's information limit, and weighted least-squares may do worse, whereas maximum-likelihood fitting is practically optimal.