Binding of human growth hormone (hGH) to its receptor is required for regulation of normal human growth and development. Examination of the 2.8 angstrom crystal structure of the complex between the hormone and the extracellular domain of its receptor (hGHbp) showed that the complex consists of one molecule of growth hormone per two molecules of receptor. The hormone is a four-helix bundle with an unusual topology. The binding protein contains two distinct domains, similar in some respects to immunoglobulin domains. The relative orientation of these domains differs from that found between constant and variable domains in immunoglobulin Fab fragments. Both hGHbp domains contribute residues that participate in hGH binding. In the complex both receptors donate essentially the same residues to interact with the hormone, even though the two binding sites on hGH have no structural similarity. Generally, the hormone-receptor interfaces match those identified by previous mutational analyses. In addition to the hormone-receptor interfaces, there is also a substantial contact surface between the carboxyl-terminal domains of the receptors. The relative extents of the contact areas support a sequential mechanism for dimerization that may be crucial for signal transduction.

Alendronate, an inhibitor of bone resorption, is widely used in osteoporosis treatment. However, concerns have been raised about potential oversuppression of bone turnover during long-term use. We report on nine patients who sustained spontaneous nonspinal fractures while on alendronate therapy, six of whom displayed either delayed or absent fracture healing for 3 months to 2 yr during therapy. Histomorphometric analysis of the cancellous bone showed markedly suppressed bone formation, with reduced or absent osteoblastic surface in most patients. Osteoclastic surface was low or low-normal in eight patients, and eroded surface was decreased in four. Matrix synthesis was markedly diminished, with absence of double-tetracycline label and absent or reduced single-tetracycline label in all patients. The same trend was seen in the intracortical and endocortical surfaces. Our findings raise the possibility that severe suppression of bone turnover may develop during long-term alendronate therapy, resulting in increased susceptibility to, and delayed healing of, nonspinal fractures. Although coadministration of estrogen or glucocorticoids appears to be a predisposing factor, this apparent complication can also occur with monotherapy. Our observations emphasize the need for increased awareness and monitoring for the potential development of excessive suppression of bone turnover during long-term alendronate therapy.

1. We stimulated the motor cortex of normal subjects (transcranial magnetic stimulation) while they 1) observed an experimenter grasping 3D-objects, 2) looked at the same 3D-objects, 3) observed an experimenter tracing geometrical figures in the air with his arm, and 4) detected the dimming of a light. Motor evoked potentials (MEPs) were recorded from hand muscles. 2. We found that MEPs significantly increased during the conditions in which subjects observed movements. The MEP pattern reflected the pattern of muscle activity recorded when the subjects executed the observed actions. 3. We conclude that in humans there is a system matching action observation and execution. This system resembles the one recently described in the monkey.

In 1903, Leishman and Donovan separately described the protozoan now called Leishmania donovani in splenic tissue from patients in India with the life-threatening disease now called visceral leishmaniasis. Almost a century later, many features of leishmaniasis and its major syndromes (ie, visceral, cutaneous, and mucosal) have remained the same; but also much has changed. As before, epidemics of this sandfly-borne disease occur periodically in India and elsewhere; but leishmaniasis has also emerged in new regions and settings, for example, as an AIDS-associated opportunistic infection. Diagnosis still typically relies on classic microbiological methods, but molecular-based approaches are being tested. Pentavalent antimony compounds have been the mainstay of antileishmanial therapy for half a century, but lipid formulations of amphotericin B (though expensive and administered parenterally) represent a major advance for treating visceral leishmaniasis. A pressing need is for the technological advances in the understanding of the immune response to leishmania and the pathogenesis of leishmaniasis to be translated into field-applicable and affordable methods for diagnosis, treatment, and prevention of this disease.

Change in cell stiffness is a new characteristic of cancer cells that affects the way they spread. Despite several studies on architectural changes in cultured cell lines, no ex vivo mechanical analyses of cancer cells obtained from patients have been reported. Using atomic force microscopy, we report the stiffness of live metastatic cancer cells taken from the body (pleural) fluids of patients with suspected lung, breast and pancreas cancer. Within the same sample, we find that the cell stiffness of metastatic cancer cells is more than 70% softer, with a standard deviation over five times narrower, than the benign cells that line the body cavity. Different cancer types were found to display a common stiffness. Our work shows that mechanical analysis can distinguish cancerous cells from normal ones even when they show similar shapes. These results show that nanomechanical analysis correlates well with immunohistochemical testing currently used for detecting cancer.

Innate immunity is an evolutionarily ancient system that provides organisms with immediately available defense mechanisms through recognition of pathogen-associated molecular patterns. We show that in the CNS, specific activation of innate immunity through a Toll-like receptor 4 (TLR4)-dependent pathway leads to neurodegeneration. We identify microglia as the major lipopolysaccharide (LPS)-responsive cell in the CNS. TLR4 activation leads to extensive neuronal death in vitro that depends on the presence of microglia. LPS leads to dramatic neuronal loss in cultures prepared from wild-type mice but does not induce neuronal injury in CNS cultures derived from tlr4 mutant mice. In an in vivo model of neurodegeneration, stimulating the innate immune response with LPS converts a subthreshold hypoxic-ischemic insult from no discernable neuronal injury to severe axonal and neuronal loss. In contrast, animals bearing a loss-of-function mutation in the tlr4 gene are resistant to neuronal injury in the same model. The present study demonstrates a mechanistic link among innate immunity, TLRs, and neurodegeneration.

Mutations in CUC1 and CUC2 (for CUP-SHAPED COTYLEDON), which are newly identified genes of Arabidopsis, caused defects in the separation of cotyledons (embryonic organs), sepals, and stamens (floral organs) as well as in the formation of shoot apical meristems. These defects were most apparent in the double mutant. Phenotypes of the mutants suggest a common mechanism for separating adjacent organs within the same whorl in both embryos and flowers. We cloned the CUC2 gene and found that the encoded protein was homologous to the petunia NO APICAL MERISTEM (NAM) protein, which is thought to act in the development of embryos and flowers.

Prediabetes (intermediate hyperglycaemia) is a high-risk state for diabetes that is defined by glycaemic variables that are higher than normal, but lower than diabetes thresholds. 5-10% of people per year with prediabetes will progress to diabetes, with the same proportion converting back to normoglycaemia. Prevalence of prediabetes is increasing worldwide and experts have projected that more than 470 million people will have prediabetes by 2030. Prediabetes is associated with the simultaneous presence of insulin resistance and β-cell dysfunction-abnormalities that start before glucose changes are detectable. Observational evidence shows associations between prediabetes and early forms of nephropathy, chronic kidney disease, small fibre neuropathy, diabetic retinopathy, and increased risk of macrovascular disease. Multifactorial risk scores using non-invasive measures and blood-based metabolic traits, in addition to glycaemic values, could optimise estimation of diabetes risk. For prediabetic individuals, lifestyle modification is the cornerstone of diabetes prevention, with evidence of a 40-70% relative-risk reduction. Accumulating data also show potential benefits from pharmacotherapy.

A roentgenographic knee evaluation system endorsed by The Knee Society is included in this year's proceedings to encourage uniform reporting of the results of total knee arthroplasty. No rating system is ideal, but if many surgeons and centers use the same reporting system, then relative comparisons will at least become possible. Agreement on a new system by many experienced surgeons and institutions with a large clinical volume represents a sacrifice because old rating system data will have to be discarded.

Stress is a condition of human existence and a factor in the expression of disease. A broader view of stress is that it is not just the dramatic stressful events that exact their toll but rather the many events of daily life that elevate activities of physiological systems to cause some measure of wear and tear. We call this wear and tear "allostatic load," and it reflects not only the impact of life experiences but also of genetic load; individual habits reflecting items such as diet, exercise, and substance abuse; and developmental experiences that set life-long patterns of behavior and physiological reactivity (see McEwen). Hormones associated with stress and allostatic load protect the body in the short run and promote adaptation, but in the long run allostatic load causes changes in the body that lead to disease. This will be illustrated for the immune system and brain. Among the most potent of stressors are those arising from competitive interactions between animals of the same species, leading to the formation of dominance hierarchies. Psychosocial stress of this type not only impairs cognitive function of lower ranking animals, but it can also promote disease (e.g. atherosclerosis) among those vying for the dominant position. Social ordering in human society is also associated with gradients of disease, with an increasing frequency of mortality and morbidity as one descends the scale of socioeconomic status that reflects both income and education. Although the causes of these gradients of health are very complex, they are likely to reflect, with increasing frequency at the lower end of the scale, the cumulative burden of coping with limited resources and negative life events and the allostatic load that this burden places on the physiological systems involved in coping and adaptation.

Recent RNA interference screens have identified several proteins that are essential for store-operated Ca2+ influx and Ca2+ release-activated Ca2+ (CRAC) channel activity in Drosophila and in mammals, including the transmembrane proteins Stim (stromal interaction molecule) and Orai. Stim probably functions as a sensor of luminal Ca2+ content and triggers activation of CRAC channels in the surface membrane after Ca2+ store depletion. Among three human homologues of Orai (also known as olf186-F), ORAI1 on chromosome 12 was found to be mutated in patients with severe combined immunodeficiency disease, and expression of wild-type Orai1 restored Ca2+ influx and CRAC channel activity in patient T cells. The overexpression of Stim and Orai together markedly increases CRAC current. However, it is not yet clear whether Stim or Orai actually forms the CRAC channel, or whether their expression simply limits CRAC channel activity mediated by a different channel-forming subunit. Here we show that interaction between wild-type Stim and Orai, assessed by co-immunoprecipitation, is greatly enhanced after treatment with thapsigargin to induce Ca2+ store depletion. By site-directed mutagenesis, we show that a point mutation from glutamate to aspartate at position 180 in the conserved S1-S2 loop of Orai transforms the ion selectivity properties of CRAC current from being Ca2+-selective with inward rectification to being selective for monovalent cations and outwardly rectifying. A charge-neutralizing mutation at the same position (glutamate to alanine) acts as a dominant-negative non-conducting subunit. Other charge-neutralizing mutants in the same loop express large inwardly rectifying CRAC current, and two of these exhibit reduced sensitivity to the channel blocker Gd3+. These results indicate that Orai itself forms the Ca2+-selectivity filter of the CRAC channel.

OBJECTIVE

To assess whether the association of serum homocysteine concentration with ischaemic heart disease, deep vein thrombosis and pulmonary embolism, and stroke is causal and, if so, to quantify the effect of homocysteine reduction in preventing them.

METHODS

Meta-analyses of the above three diseases using (a) 72 studies in which the prevalence of a mutation in the MTHFR gene (which increases homocysteine) was determined in cases (n=16 849) and controls, and (b) 20 prospective studies (3820 participants) of serum homocysteine and disease risk.

METHODS

Odds ratios of the three diseases for a 5 micromol/l increase in serum homocysteine concentration.

RESULTS

There were significant associations between homocysteine and the three diseases. The odds ratios for a 5 micromol/l increase in serum homocysteine were, for ischaemic heart disease, 1.42 (95% confidence interval 1.11 to 1.84) in the genetic studies and 1.32 (1.19 to 1.45) in the prospective studies; for deep vein thrombosis with or without pulmonary embolism, 1.60 (1.15 to 2.22) in the genetic studies (there were no prospective studies); and, for stroke, 1.65 (0.66 to 4.13) in the genetic studies and 1.59 (1.29 to 1.96) in the prospective studies.

CONCLUSIONS

The genetic studies and the prospective studies do not share the same potential sources of error, but both yield similar highly significant results-strong evidence that the association between homocysteine and cardiovascular disease is causal. On this basis, lowering homocysteine concentrations by 3 micromol/l from current levels (achievable by increasing folic acid intake) would reduce the risk of ischaemic heart disease by 16% (11% to 20%), deep vein thrombosis by 25% (8% to 38%), and stroke by 24% (15% to 33%).

Staphylococcus aureus is an important nosocomial and community-acquired pathogen. Its genetic plasticity has facilitated the evolution of many virulent and drug-resistant strains, presenting a major and constantly changing clinical challenge. We sequenced the approximately 2.8-Mbp genomes of two disease-causing S. aureus strains isolated from distinct clinical settings: a recent hospital-acquired representative of the epidemic methicillin-resistant S. aureus EMRSA-16 clone (MRSA252), a clinically important and globally prevalent lineage; and a representative of an invasive community-acquired methicillin-susceptible S. aureus clone (MSSA476). A comparative-genomics approach was used to explore the mechanisms of evolution of clinically important S. aureus genomes and to identify regions affecting virulence and drug resistance. The genome sequences of MRSA252 and MSSA476 have a well conserved core region but differ markedly in their accessory genetic elements. MRSA252 is the most genetically diverse S. aureus strain sequenced to date: approximately 6% of the genome is novel compared with other published genomes, and it contains several unique genetic elements. MSSA476 is methicillin-susceptible, but it contains a novel Staphylococcal chromosomal cassette (SCC) mec-like element (designated SCC(476)), which is integrated at the same site on the chromosome as SCCmec elements in MRSA strains but encodes a putative fusidic acid resistance protein. The crucial role that accessory elements play in the rapid evolution of S. aureus is clearly illustrated by comparing the MSSA476 genome with that of an extremely closely related MRSA community-acquired strain; the differential distribution of large mobile elements carrying virulence and drug-resistance determinants may be responsible for the clinically important phenotypic differences in these strains.

The bacterium Listeria monocytogenes is ubiquitous in the environment and can lead to severe food-borne infections. It has recently emerged as a multifaceted model in pathogenesis. However, how this bacterium switches from a saprophyte to a pathogen is largely unknown. Here, using tiling arrays and RNAs from wild-type and mutant bacteria grown in vitro, ex vivo and in vivo, we have analysed the transcription of its entire genome. We provide the complete Listeria operon map and have uncovered far more diverse types of RNAs than expected: in addition to 50 small RNAs (<500 nucleotides), at least two of which are involved in virulence in mice, we have identified antisense RNAs covering several open-reading frames and long overlapping 5' and 3' untranslated regions. We discovered that riboswitches can act as terminators for upstream genes. When Listeria reaches the host intestinal lumen, an extensive transcriptional reshaping occurs with a SigB-mediated activation of virulence genes. In contrast, in the blood, PrfA controls transcription of virulence genes. Remarkably, several non-coding RNAs absent in the non-pathogenic species Listeria innocua exhibit the same expression patterns as the virulence genes. Together, our data unravel successive and coordinated global transcriptional changes during infection and point to previously unknown regulatory mechanisms in bacteria.

LAP, a transcriptional activator, and LIP, a transcriptional repressor, are translated from a single mRNA species by using two AUGs within the same reading frame. These two proteins share the 145 C-terminal amino acids that contain the basic DNA-binding domain and the leucine zipper dimerization helix. Probably owing to its higher affinity for its DNA cognate sequences, LIP can attenuate the transcriptional stimulation by LAP in substoichiometric amounts. As revealed by transient transfection experiments, a moderate increase in the LAP/LIP ratio results in a significantly higher transcriptional activation of an appropriate target gene. The LAP/LIP ratio increases about 5-fold during terminal rat liver differentiation and is thus likely to modulate the activity of LAP in the intact animal.

Diffusion tensor imaging (DTI) tractography allows perform virtual dissections of white matter pathways in the living human brain. In 2002, Catani et al. published a method to reconstruct white matter pathways using a region of interest (ROI) approach. The method produced virtual representations of white matter tracts faithful to classical post-mortem descriptions but it required detailed a priori anatomical knowledge. Here, using the same approach, we provide a template to guide the delineation of ROIs for the reconstruction of the association, projection and commissural pathways of the living human brain. The template can be used for single case studies and case-control comparisons. An atlas of the 3D reconstructions of the single tracts is also provided as anatomical reference in the Montreal Neurological Institute (MNI) space.

BACKGROUND

The SF-6D and EQ-5D are both preference-based measures of health. Empirical work is required to determine what the smallest change is in utility scores that can be regarded as important and whether this change in utility value is constant across measures and conditions.

OBJECTIVE

To use distribution and anchor-based methods to determine and compare the minimally important difference (MID) for the SF-6D and EQ-5D for various datasets.

METHODS

The SF-6D is scored on a 0.29-1.00 scale and the EQ-5D on a -0.59-1.00 scale, with a score of 1.00 on both, indicating 'full health'. Patients were followed for a period of time, then asked, using question 2 of the SF-36 as our anchor, if their general health is much better (5), somewhat better (4), stayed the same (3), somewhat worse (2) or much worse (1) compared to the last time they were assessed. We considered patients whose global rating score was 4 or 2 as having experienced some change equivalent to the MID. This paper describes and compares the MID and standardised response mean (SRM) for the SF-6D and EQ-5D from eight longitudinal studies in 11 patient groups that used both instruments.

RESULTS

From the 11 reviewed studies, the MID for the SF-6D ranged from 0.011 to 0.097, mean 0.041. The corresponding SRMs ranged from 0.12 to 0.87, mean 0.39 and were mainly in the 'small to moderate' range using Cohen's criteria, supporting the MID results. The mean MID for the EQ-5D was 0.074 (range -0.011-0.140) and the SRMs ranged from -0.05 to 0.43, mean 0.24. The mean MID for the EQ-SD was almost double that of the mean MID for the SF-6D.

CONCLUSIONS

There is evidence that the MID for these two utility measures are not equal and differ in absolute values. The EQ-5D scale has approximately twice the range of the SF-6D scale. Therefore, the estimates of the MID for each scale appear to be proportionally equivalent in the context of the range of utility scores for each scale. Further empirical work is required to see whether or not this holds true for other utility measures, patient groups and populations.

OBJECTIVE

To assess the additional resource utilization associated with an adverse drug event (ADE).

METHODS

Nested case-control study within a prospective cohort study.

METHODS

The cohort included 4108 admissions to a stratified random sample of 11 medical and surgical units in 2 tertiary-care hospitals over a 6-month period. Cases were patients with an ADE, and the control for each case was the patient on the same unit as the case with the most similar pre-event length of stay.

METHODS

Postevent length of stay and total costs.

METHODS

Incidents were detected by self-report stimulated by nurses and pharmacists and by daily chart review, and were classified as to whether they represented ADEs. Information on length of stay and charges was obtained from billing data, and costs were estimated by multiplying components of charges times hospital-specific ratios of costs to charges.

RESULTS

During the study period, there were 247 ADEs among 207 admissions. After outliers and multiple episodes were excluded, there were 190 ADEs, of which 60 were preventable. In paired regression analyses adjusting for multiple factors, including severity, comorbidity, and case mix, the additional length of stay associated with an ADE was 2.2 days (P=.04), and the increase in cost associated with an ADE was $3244 (P=.04). For preventable ADEs, the increases were 4.6 days in length of stay (P=.03) and $5857 in total cost (P=.07). After adjusting for our sampling strategy, the estimated postevent costs attributable to an ADE were $2595 for all ADEs and $4685 for preventable ADEs. Based on these costs and data about the incidence of ADEs, we estimate that the annual costs attributable to all ADEs and preventable ADEs for a 700-bed teaching hospital are $5.6 million and $2.8 million, respectively.

CONCLUSIONS

The substantial costs of ADEs to hospitals justify investment in efforts to prevent these events. Moreover, these estimates are conservative because they do not include the costs of injuries to patients or malpractice costs.

Familial advanced sleep phase syndrome (FASPS) is an autosomal dominant circadian rhythm variant; affected individuals are "morning larks" with a 4-hour advance of the sleep, temperature, and melatonin rhythms. Here we report localization of the FASPS gene near the telomere of chromosome 2q. A strong candidate gene (hPer2), a human homolog of the period gene in Drosophila, maps to the same locus. Affected individuals have a serine to glycine mutation within the casein kinase Iepsilon (CKIepsilon) binding region of hPER2, which causes hypophosphorylation by CKIepsilon in vitro. Thus, a variant in human sleep behavior can be attributed to a missense mutation in a clock component, hPER2, which alters the circadian period.

In 52 patients with rectal adenocarcinoma whole-mount sections of the entire operative specimen were examined by transverse slicing. There was spread to the lateral resection margin in 14 of 52 (27%) patients and 12 of these proceeded to local pelvic recurrence. The specificity, sensitivity, and positive predictive values were 92%, 95%, and 85%, respectively. In a retrospective stage-matched and grade-matched control group there was local recurrence in the same proportion of patients, but in this series no patient had been shown by routine sampling to have lateral spread. In rectal adenocarcinoma, local recurrence is mainly due to lateral spread of the tumour and has previously been underestimated.

Are faces recognized using more holistic representations than other types of stimuli? Taking holistic representation to mean representation without an internal part structure, we interpret the available evidence on this issue and then design new empirical tests. Based on previous research, we reasoned that if a portion of an object corresponds to an explicitly represented part in a hierarchical visual representation, then when that portion is presented in isolation it will be identified relatively more easily than if it did not have the status of an explicitly represented part. The hypothesis that face recognition is holistic therefore predicts that a part of a face will be disproportionately more easily recognized in the whole face than as an isolated part, relative to recognition of the parts and wholes of other kinds of stimuli. This prediction was borne out in three experiments: subjects were more accurate at identifying the parts of faces, presented in the whole object, than they were at identifying the same part presented in isolation, even though both parts and wholes were tested in a forced-choice format and the whole faces differed only by one part. In contrast, three other types of stimuli--scrambled faces, inverted faces, and houses--did not show this advantage for part identification in whole object recognition.

We describe a suffix-tree algorithm that can align the entire genome sequences of eukaryotic and prokaryotic organisms with minimal use of computer time and memory. The new system, MUMmer 2, runs three times faster while using one-third as much memory as the original MUMmer system. It has been used successfully to align the entire human and mouse genomes to each other, and to align numerous smaller eukaryotic and prokaryotic genomes. A new module permits the alignment of multiple DNA sequence fragments, which has proven valuable in the comparison of incomplete genome sequences. We also describe a method to align more distantly related genomes by detecting protein sequence homology. This extension to MUMmer aligns two genomes after translating the sequence in all six reading frames, extracts all matching protein sequences and then clusters together matches. This method has been applied to both incomplete and complete genome sequences in order to detect regions of conserved synteny, in which multiple proteins from one organism are found in the same order and orientation in another. The system code is being made freely available by the authors.

All available antidepressant medications are based on serendipitous discoveries of the clinical efficacy of two classes of antidepressants more than 50 years ago. These tricyclic and monoamine oxidase inhibitor antidepressants were subsequently found to promote serotonin or noradrenaline function in the brain. Newer agents are more specific but have the same core mechanisms of action in promoting these monoamine neurotransmitters. This is unfortunate, because only approximately 50% of individuals with depression show full remission in response to these mechanisms. This review summarizes the obstacles that have hindered the development of non-monoamine-based antidepressants, and provides a progress report on some of the most promising current strategies.

Interleukin-17A (IL-17A) is a cytokine produced by T helper 17 (Th17) cells and plays important roles in the development of inflammatory diseases. Although IL-17F is highly homologous to IL-17A and binds the same receptor, the functional roles of this molecule remain largely unknown. Here, we demonstrated with Il17a(-/-), Il17f(-/-), and Il17a(-/-)Il17f(-/-) mice that IL-17F played only marginal roles, if at all, in the development of delayed-type and contact hypersensitivities, autoimmune encephalomyelitis, collagen-induced arthritis, and arthritis in Il1rn(-/-) mice. In contrast, both IL-17F and IL-17A were involved in host defense against mucoepithelial infection by Staphylococcus aureus and Citrobacter rodentium. IL-17A was produced mainly in T cells, whereas IL-17F was produced in T cells, innate immune cells, and epithelial cells. Although only IL-17A efficiently induced cytokines in macrophages, both cytokines activated epithelial innate immune responses. These observations indicate that IL-17A and IL-17F have overlapping yet distinct roles in host immune and defense mechanisms.