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.

Loss of telomere function can induce cell cycle arrest and apoptosis. To investigate the processes that trigger cellular responses to telomere dysfunction, we crossed mTR-/- G6 mice that have short telomeres with mice heterozygous for telomerase (mTR+/-) that have long telomeres. The phenotype of the telomerase null offspring was similar to that of the late generation parent, although only half of the chromosomes were short. Strikingly, spectral karyotyping (SKY) analysis revealed that loss of telomere function occurred preferentially on chromosomes with critically short telomeres. Our data indicate that, while average telomere length is measured in most studies, it is not the average but rather the shortest telomeres that constitute telomere dysfunction and limit cellular survival in the absence of telomerase.

BACKGROUND

The last nationally representative assessment of kidney stone prevalence in the United States occurred in 1994. After a 13-yr hiatus, the National Health and Nutrition Examination Survey (NHANES) reinitiated data collection regarding kidney stone history.

OBJECTIVE

Describe the current prevalence of stone disease in the United States, and identify factors associated with a history of kidney stones.

METHODS

A cross-sectional analysis of responses to the 2007-2010 NHANES (n=12 110).

METHODS

Self-reported history of kidney stones. Percent prevalence was calculated and multivariable models were used to identify factors associated with a history of kidney stones.

CONCLUSIONS

The prevalence of kidney stones was 8.8% (95% confidence interval [CI], 8.1-9.5). Among men, the prevalence of stones was 10.6% (95% CI, 9.4-11.9), compared with 7.1% (95% CI, 6.4-7.8) among women. Kidney stones were more common among obese than normal-weight individuals (11.2% [95% CI, 10.0-12.3] compared with 6.1% [95% CI, 4.8-7.4], respectively; p<0.001). Black, non-Hispanic and Hispanic individuals were less likely to report a history of stone disease than were white, non-Hispanic individuals (black, non-Hispanic: odds ratio [OR]: 0.37 [95% CI, 0.28-0.49], p<0.001; Hispanic: OR: 0.60 [95% CI, 0.49-0.73], p<0.001). Obesity and diabetes were strongly associated with a history of kidney stones in multivariable models. The cross-sectional survey design limits causal inference regarding potential risk factors for kidney stones.

CONCLUSIONS

Kidney stones affect approximately 1 in 11 people in the United States. These data represent a marked increase in stone disease compared with the NHANES III cohort, particularly in black, non-Hispanic and Hispanic individuals. Diet and lifestyle factors likely play an important role in the changing epidemiology of kidney stones.

Although density functional theory is widely used in the computational chemistry community, the most popular density functional, B3LYP, has some serious shortcomings: (i) it is better for main-group chemistry than for transition metals; (ii) it systematically underestimates reaction barrier heights; (iii) it is inaccurate for interactions dominated by medium-range correlation energy, such as van der Waals attraction, aromatic-aromatic stacking, and alkane isomerization energies. We have developed a variety of databases for testing and designing new density functionals. We used these data to design new density functionals, called M06-class (and, earlier, M05-class) functionals, for which we enforced some fundamental exact constraints such as the uniform-electron-gas limit and the absence of self-correlation energy. Our M06-class functionals depend on spin-up and spin-down electron densities (i.e., spin densities), spin density gradients, spin kinetic energy densities, and, for nonlocal (also called hybrid) functionals, Hartree-Fock exchange. We have developed four new functionals that overcome the above-mentioned difficulties: (a) M06, a hybrid meta functional, is a functional with good accuracy "across-the-board" for transition metals, main group thermochemistry, medium-range correlation energy, and barrier heights; (b) M06-2X, another hybrid meta functional, is not good for transition metals but has excellent performance for main group chemistry, predicts accurate valence and Rydberg electronic excitation energies, and is an excellent functional for aromatic-aromatic stacking interactions; (c) M06-L is not as accurate as M06 for barrier heights but is the most accurate functional for transition metals and is the only local functional (no Hartree-Fock exchange) with better across-the-board average performance than B3LYP; this is very important because only local functionals are affordable for many demanding applications on very large systems; (d) M06-HF has good performance for valence, Rydberg, and charge transfer excited states with minimal sacrifice of ground-state accuracy. In this Account, we compared the performance of the M06-class functionals and one M05-class functional (M05-2X) to that of some popular functionals for diverse databases and their performance on several difficult cases. The tests include barrier heights, conformational energy, and the trend in bond dissociation energies of Grubbs' ruthenium catalysts for olefin metathesis. Based on these tests, we recommend (1) the M06-2X, BMK, and M05-2X functionals for main-group thermochemistry and kinetics, (2) M06-2X and M06 for systems where main-group thermochemistry, kinetics, and noncovalent interactions are all important, (3) M06-L and M06 for transition metal thermochemistry, (4) M06 for problems involving multireference rearrangements or reactions where both organic and transition-metal bonds are formed or broken, (5) M06-2X, M05-2X, M06-HF, M06, and M06-L for the study of noncovalent interactions, (6) M06-HF when the use of full Hartree-Fock exchange is important, for example, to avoid the error of self-interaction at long-range, (7) M06-L when a local functional is required, because a local functional has much lower cost for large systems.

Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated. We describe a multilaboratory study to assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRM-based assays, conducted by NCI-CPTAC. Using common materials and standardized protocols, we demonstrate that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low mug/ml protein concentrations in unfractionated plasma. We provide data and benchmarks against which individual laboratories can compare their performance and evaluate new technologies for biomarker verification in plasma.

Assessment of epithelial dysplasia in ulcerative colitis has been hindered by inconsistencies in and disagreements about nomenclature and interpretation. To resolve these issues, pathologists from ten institutions participated in three exchanges of multiple slides and, following each exchange, in discussions of the results. A classification system for the epithelial changes that occur in ulcerative colitis was developed, which should be applicable to other forms of inflammatory bowel disease as well. The classification makes use of standardized terminology, addresses specific problem areas, and offers practical solutions. The reproducibility of the system was studied by means of examinations of both inter- and intra-observer variations. The clinical implications of the findings were incorporated into suggestions for patient management. The basis of the classification is that the term "dysplasia" is reserved for epithelial changes that are unequivocally neoplastic and may therefore give rise directly to invasive carcinoma. Specimens are categorized as negative, indefinite, or positive for dysplasia. The negative category includes all inflammatory and regenerative lesions and indicates that only continued regular surveillance is required. The indefinite category is applied to epithelial changes that appear to exceed the limits of ordinary regeneration but are insufficient for an unequivocal diagnosis of dysplasia or are associated with other features that prevent such unequivocal diagnosis. Clinically, it indicates that early repeat biopsy is often required to assess the changes more accurately. The positive category is divided into two subcategories: 1) high-grade dysplasia, for which colectomy should be strongly considered after confirmation of the diagnosis, and 2) low-grade dysplasia, which also requires confirmation and early repeat biopsy or colectomy, depending on other findings.

Cisplatin is one of the most widely used and most potent chemotherapy drugs. However, side effects in normal tissues and organs, notably nephrotoxicity in the kidneys, limit the use of cisplatin and related platinum-based therapeutics. Recent research has shed significant new lights on the mechanism of cisplatin nephrotoxicity, especially on the signaling pathways leading to tubular cell death and inflammation. Renoprotective approaches are being discovered, but the protective effects are mostly partial, suggesting the need for combinatorial strategies. Importantly, it is unclear whether these approaches would limit the anticancer effects of cisplatin in tumors. Examination of tumor-bearing animals and identification of novel renoprotective strategies that do not diminish the anticancer efficacy of cisplatin are essential to the development of clinically applicable interventions.

This review article summarizes how frailty can be considered in relation to deficit accumulation. Recalling that frailty is an age-associated, nonspecific vulnerability, we consider symptoms, signs, diseases, and disabilities as deficits, which are combined in a frailty index. An individual's frailty index score reflects the proportion of potential deficits present in that person, and indicates the likelihood that frailty is present. Although based on a simple count, the frailty index shows several interesting properties, including a characteristic rate of accumulation, a submaximal limit, and characteristic changes with age in its distribution. The frailty index, as a state variable, is able to quantitatively summarize vulnerability. Future studies include the application of network analyses and stochastic analytical techniques to the evaluation of the frailty index and the description of other state variables in relation to frailty.

Limits on the storage capacity of working memory significantly affect cognitive abilities in a wide range of domains, but the nature of these capacity limits has been elusive. Some researchers have proposed that working memory stores a limited set of discrete, fixed-resolution representations, whereas others have proposed that working memory consists of a pool of resources that can be allocated flexibly to provide either a small number of high-resolution representations or a large number of low-resolution representations. Here we resolve this controversy by providing independent measures of capacity and resolution. We show that, when presented with more than a few simple objects, human observers store a high-resolution representation of a subset of the objects and retain no information about the others. Memory resolution varied over a narrow range that cannot be explained in terms of a general resource pool but can be well explained by a small set of discrete, fixed-resolution representations.

Endothelial cells of human capillary blood vessels at the blood-brain and other blood-tissue barrier sites express P-glycoprotein as detected by mouse monoclonal antibodies against the human multidrug-resistance gene product. This pattern of endothelial cell expression may indicate a physiological role for P-glycoprotein in regulating the entry of certain molecules into the central nervous system and other anatomic compartments, such as the testes. These tissues, which limit the access of systemic drugs, are known pharmacologic sanctuaries for metastatic cancer. P-glycoprotein expression in capillary endothelium of brain and testes and not other tissues (i.e., kidney and placenta) may in part explain this phenomenon and could have important implications in cancer chemotherapy.

BACKGROUND

Fish (finfish or shellfish) may have health benefits and also contain contaminants, resulting in confusion over the role of fish consumption in a healthy diet.

METHODS

We searched MEDLINE, governmental reports, and meta-analyses, supplemented by hand reviews of references and direct investigator contacts, to identify reports published through April 2006 evaluating (1) intake of fish or fish oil and cardiovascular risk, (2) effects of methylmercury and fish oil on early neurodevelopment, (3) risks of methylmercury for cardiovascular and neurologic outcomes in adults, and (4) health risks of dioxins and polychlorinated biphenyls in fish. We concentrated on studies evaluating risk in humans, focusing on evidence, when available, from randomized trials and large prospective studies. When possible, meta-analyses were performed to characterize benefits and risks most precisely.

RESULTS

Modest consumption of fish (eg, 1-2 servings/wk), especially species higher in the n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), reduces risk of coronary death by 36% (95% confidence interval, 20%-50%; P<.001) and total mortality by 17% (95% confidence interval, 0%-32%; P = .046) and may favorably affect other clinical outcomes. Intake of 250 mg/d of EPA and DHA appears sufficient for primary prevention. DHA appears beneficial for, and low-level methylmercury may adversely affect, early neurodevelopment. Women of childbearing age and nursing mothers should consume 2 seafood servings/wk, limiting intake of selected species. Health effects of low-level methylmercury in adults are not clearly established; methylmercury may modestly decrease the cardiovascular benefits of fish intake. A variety of seafood should be consumed; individuals with very high consumption >> or =5 servings/wk) should limit intake of species highest in mercury levels. Levels of dioxins and polychlorinated biphenyls in fish are low, and potential carcinogenic and other effects are outweighed by potential benefits of fish intake and should have little impact on choices or consumption of seafood (women of childbearing age should consult regional advisories for locally caught freshwater fish).

CONCLUSIONS

For major health outcomes among adults, based on both the strength of the evidence and the potential magnitudes of effect, the benefits of fish intake exceed the potential risks. For women of childbearing age, benefits of modest fish intake, excepting a few selected species, also outweigh risks.

Because ethical restrictions limit in vivo studies of the human hemato-lymphoid system, substitute human to small animal xenotransplantation models have been employed. Existing models, however, sustain only limited development and maintenance of human lymphoid cells and rarely produce immune responses. Here we show that intrahepatic injection of CD34+ human cord blood cells into conditioned newborn Rag2-/-gammac-/- mice leads to de novo development of B, T, and dendritic cells; formation of structured primary and secondary lymphoid organs; and production of functional immune responses. This provides a valuable model to study development and function of the human adaptive immune system in vivo.

After entry into lymph nodes (LNs), B cells migrate to follicles, whereas T cells remain in the paracortex, with each lymphocyte type showing apparently random migration within these distinct areas. Other than chemokines, the factors contributing to this spatial segregation and to the observed patterns of lymphocyte movement are poorly characterized. By combining confocal, electron, and intravital microscopy, we showed that the fibroblastic reticular cell network regulated naive T cell access to the paracortex and also supported and defined the limits of T cell movement within this domain, whereas a distinct follicular dendritic cell network similarly served as the substratum for movement of follicular B cells. These results highlight the central role of stromal microanatomy in orchestrating cell migration within the LN.

Chemotherapy and radiation therapy for cancer often have severe side effects that limit their efficacy. Because these effects are in part determined by p53-mediated apoptosis, temporary suppression of p53 has been suggested as a therapeutic strategy to prevent damage of normal tissues during treatment of p53-deficient tumors. To test this possibility, a small molecule was isolated for its ability to reversibly block p53-dependent transcriptional activation and apoptosis. This compound, pifithrin-alpha, protected mice from the lethal genotoxic stress associated with anticancer treatment without promoting the formation of tumors. Thus, inhibitors of p53 may be useful drugs for reducing the side effects of cancer therapy and other types of stress associated with p53 induction.

It has been previously shown that intrinsic metabolites can be imaged based on their water proton exchange rates using saturation transfer techniques. The goal of this study was to identify an appropriate chemical exchange site that could be developed for use as an exogenous chemical exchange dependent saturation transfer (CEST) contrast agent under physiological conditions. These agents would function by reducing the water proton signal through a chemical exchange site on the agent via saturation transfer. The ideal chemical exchange site would have a large chemical shift from water. This permits a high exchange rate without approaching the fast exchange limit at physiological pH (6.5-7.6) and temperature (37 degrees C), as well as minimizing problems associated with magnetic field susceptibility. Numerous candidate chemicals (amino acids, sugars, nucleotides, heterocyclic ring chemicals) were evaluated in this preliminary study. Of these, barbituric acid and 5, 6-dihydrouracil were more fully characterized with regard to pH, temperature, and concentration CEST effects. The best chemical exchange site found was the 5.33-ppm indole ring -NH site of 5-hydroxytryptophan. These data demonstrate that a CEST-based exogenous contrast agent for MRI is feasible.

The intrinsic limits of conventional cancer therapies prompted the development and application of various nanotechnologies for more effective and safer cancer treatment, herein referred to as cancer nanomedicine. Considerable technological success has been achieved in this field, but the main obstacles to nanomedicine becoming a new paradigm in cancer therapy stem from the complexities and heterogeneity of tumour biology, an incomplete understanding of nano-bio interactions and the challenges regarding chemistry, manufacturing and controls required for clinical translation and commercialization. This Review highlights the progress, challenges and opportunities in cancer nanomedicine and discusses novel engineering approaches that capitalize on our growing understanding of tumour biology and nano-bio interactions to develop more effective nanotherapeutics for cancer patients.

In our opinion, all of the phenomena that are inhibited by cytochalasin can be thought of as resulting from contractile activity of cellular organelles. Smooth muscle contraction, clot retraction, beat of heart cells, and shortening of the tadpole tail are all cases in which no argument of substance for alternative causes can be offered. The morphogenetic processes in epithelia, contractile ring function during cytokinesis, migration of cells on a substratum, and streaming in plant cells can be explained most simply on the basis of contractility being the causal event in each process. The many similarities between the latter cases and the former ones in which contraction is certain argue for that conclusion. For instance, platelets probably contract, possess a microfilament network, and behave like undulating membrane organelles. Migrating cells possess undulating membranes and contain a similar network. It is very likely, therefore, that their network is also contractile. In all of the cases that have been examined so far, microfilaments of some type are observed in the cells; furthermore, those filaments are at points where contractility could cause the respective phenomenon. The correlations from the cytochalasin experiments greatly strengthen the case; microfilaments are present in control and "recovered" cells and respective biological phenomena take place in such cells; microfilaments are absent or altered in treated cells and the phenomena do not occur. The evidence seems overwhelming that microfilaments are the contractile machinery of nonmuscle cells. The argument is further strengthened if we reconsider the list of processes insensitive to cytochalasin (Table 2). Microtubules and their sidearms, plasma membrane, or synthetic machinery of cells are presumed to be responsible for such processes, and colchicine, membrane-active drugs, or inhibitors of protein synthesis are effective at inhibiting the respective phenomena. These chemical agents would not necessarily be expected to affect contractile apparatuses over short periods of time, they either do not or only secondarily interfere with the processes sensitive to cytochalasin (Table 1). It is particularly noteworthy in this context that microtubules are classed as being insensitive to cytochalasin and so are not considered as members of the "contractile microfilament" family. The overall conclusion is that a broad spectrum of cellular and developmental processes are caused by contractile apparatuses that have at least the common feature of being sensitive to cytochalasin. Schroeder's important insight (3) has, then, led to the use of cytochalasin as a diagnostic tool for such contracile activity: the prediction is that sensitivity to the drug implies presence of some type of contractile microfilament system. Only further work will define the limits of confidence to be placed upon such diagnoses. The basis of contraction in microfilament systems is still hypothetical. Contraction of glycerol-extracted cells in response to adenosine triphosphate (53), extraction of actin-like or actomyosin-like proteins from cells other than muscle cells (54), and identification of activity resembling that of the actomyosin-adenosine triphosphatase system in a variety of nonmuscle tissues (40, 54) are consistent with the idea that portions of the complex, striated muscle contractile system may be present in more primitive contractile machinery. In the case of the egg cortex, calcium-activated contractions can be inhibited by cytochalasin. If, as seems likely, microfilaments are the agents activated by calcium, then it will be clear that they have the same calcium requirement as muscle. Biochemical analyses of primitive contractile systems are difficult to interpret. Ishikawa's important observation (31), that heavy meromyosin complexes with fine filaments oriented parallel to the surface of chondrocytes and perpendicular to the surface of intestinal epithelial cells, implies that both types of filaments are "actin-like" in this one respect. Yet, it is very likely that these actin-like filaments correspond respectively to the cytochalasin-insensitive sheath of glial and heart fibroblasts and the core filaments of oviduct microvilli. No evidence from our studies links contractility directly to these meromyosin-binding filaments. Apart from this problem, activity resembling that of the myosin-adenosine triphosphatase has been associated with the microtubule systems of sperm tails and cilia (55), but those organelles are insensitive to cytochalasin in structure and function. Clearly, a means must be found to distinguish between enzymatic activities associated with microfilament networks, microfilament bundles, microtubules, and the sheath filaments of migratory cells. Until such distinctions are possible, little of substance can be said about the molecular bases of primitive contractile systems. Three variables are important for the control of cellular processes dependent upon microfilaments: (i) which cells of a population shall manufacture and assemble the filaments; (ii) where filaments shall be assembled in cells; and (iii) when contractility shall occur. With respect to distribution among cells, the networks involved in cell locomotion are presumed to be present in all cells that have the potential to move in cell culture. In this respect, the networks can be regarded as a common cellular organelle in the sense that cytoplasmic microtubules are so regarded. In some developing systems, all cells of an epithelium possess microfilament bundles (7, 13), whereas, in others, only discrete subpopulations possess the bundles (5, 6). In these cases the filaments can be regarded as being differentiation products associated only with certain cell types. These considerations may be related to the fact that microfilament networks are associated with behavior of individual cells (such as migration, wound healing, and cytokinesis), whereas the bundles are present in cells that participate in coordinated changes in shape of cell populations. With respect to placement in cells, two alternatives are apparent, namely, localized or ubiquitous association with the plasma membrane. Microfilament bundles of epithelial cells are only found extending across the luminal and basal ends of cells. In this respect they contrast with desmosomal tonofilaments and with microtubules, each of which can curve in a variety of directions through the cell. The strict localization of microfilament bundles probably rests upon their association with special junctional complex insertion regions that are only located near the ends of cells. In the case of mitotically active cells, the orientation of the spindle apparatus may determine the site at which the contractile ring of microfilaments will form (4, 56); this raises the question of what sorts of cytoplasmic factors can influence the process of association between filament systems and plasma membranes. In contrast to such cases of localized distribution, contractile networks responsible for cell locomotion are probably found beneath all of the plasma membrane, just as the network of thrombosthenin may extend to all portions of the periphery of a blood platelet. This ubiquitous distribution probably accounts for the ability of a fibroblast or glial cell to establish an undulating membrane at any point on its edge, or of an axon to form lateral microspikes along its length. The third crucial aspect of control of these contractile apparatuses involves the choice of when contraction shall occur (and as a corollary the degree or strength of contraction that will occur). In the simplest situation, contraction would follow automatically upon assembly of the microfilament bundles or networks. In cleavage furrows of marine embryos (4), for instance, microfilaments are seen beneath the central cleavage furrow and at its ends, but not beyond, under the portion of plasma membrane that will subsequently become part of the furrow. This implies that the furrow forms very soon after the contractile filaments are assembled in the egg cortex. In other cases, microfilaments are apparently assembled but not in a state of (maximal?) contraction. Thus, networks are seen along the sides of migratory cells, although such regions are not then active as undulating membrane organelles. Similarly, microfilament bundles occur in all epithelial cells of the salivary gland (13), or pancreatic anlage (7), although only the ones at discrete points are thought to generate morphogenetic tissue movements. Likewise, bundles begin to appear as early as 12 hours after estrogen administration to oviduct, although visible tubular gland formation does not start until 24 to 30 hours. Finally, streaming in plant cells can wax and wane, depending upon external factors such as auxin (57). All of these cases imply a control mechanism other than mere assembly of the microfilament systems and even raise the possibility that within one cell some filaments may be contracting while others are not. In discussing this problem, it must be emphasized that different degrees of contraction or relaxation cannot as yet be recognized with the electron microscope. In fact, every one of the cases cited above could be explained by contraction following immediately upon some subtle sort of "assembly." Inclusive in the latter term are relations between individual filaments, relations of the filaments and their insertion points on plasma membrane, and quantitative alterations in filament systems. Furthermore, the critical role of calcium and high-energy compounds in muscle contraction suggest that equivalent factors may be part of primitive, cytochalasinsensitive systems. The finding that calcium-induced contraction in the cortex of eggs is sensitive to cytochalasin strengthens that supposition and emphasizes the importance of compartmentalization of cofactors as a means of controlling microfilaments in cells.

The polarization of the fluorescence and the real-time fluorescence intensity decay of the two tryptophan residues of aspartate transcarbamylase from Escherichia coli were studied as a function of temperature. The protein was dissolved in an 80% glycerol/buffer mixture, and temperatures were varied between -40 and 20 degrees C in order to limit the depolarization to local rotations of the tryptophans. Two fluorescent species contribute to over 95% of the emission. They differ in their fluorescence lifetimes by approximately 4 ns depending upon the temperature observed and their fractional contributions to the total intensity. The Y-plot analysis of the polarization and lifetime data allows for the distinction of two rotational species by their critical amplitude of rotation, the first being component 1 and the second being component 2. We suggest that these two species correspond to the two tryptophan residues of the protein. The polarization and lifetime experiments were carried out for ATCase in presence of the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate (PALA) and in presence of the nucleotide effector molecules ATP and CTP. The binding of PALA results in an increase in the thermal coefficient of frictional resistance to rotation of tryptophan 1 and a decrease in that of tryptophan 2. ATP binding does not affect the degree to which the protein hinders tryptophan rotation but does result in a change in the critical amplitude of rotation of tryptophan 2. The results obtained in the presence of CTP are similar to those obtained with PALA.

BACKGROUND

The availability of new antiretroviral drugs and formulations, including drugs in new classes, and recent data on treatment choices for antiretroviral-naive and -experienced patients warrant an update of the International AIDS Society-USA guidelines for the use of antiretroviral therapy in adult human immunodeficiency virus (HIV) infection.

OBJECTIVE

To summarize new data in the field and to provide current recommendations for the antiretroviral management and laboratory monitoring of HIV infection. This report provides guidelines in key areas of antiretroviral management: when to initiate therapy, choice of initial regimens, patient monitoring, when to change therapy, and how best to approach treatment options, including optimal use of recently approved drugs (maraviroc, raltegravir, and etravirine) in treatment-experienced patients.

METHODS

A 14-member panel with expertise in HIV research and clinical care was appointed. Data published or presented at selected scientific conferences since the last panel report (August 2006) through June 2008 were identified.

METHODS

Data that changed the previous guidelines were reviewed by the panel (according to section). Guidelines were drafted by section writing committees and were then reviewed and edited by the entire panel. Recommendations were made by panel consensus.

CONCLUSIONS

New data and considerations support initiating therapy before CD4 cell count declines to less than 350/microL. In patients with 350 CD4 cells/microL or more, the decision to begin therapy should be individualized based on the presence of comorbidities, risk factors for progression to AIDS and non-AIDS diseases, and patient readiness for treatment. In addition to the prior recommendation that a high plasma viral load (eg, >100,000 copies/mL) and rapidly declining CD4 cell count (>100/microL per year) should prompt treatment initiation, active hepatitis B or C virus coinfection, cardiovascular disease risk, and HIV-associated nephropathy increasingly prompt earlier therapy. The initial regimen must be individualized, particularly in the presence of comorbid conditions, but usually will include efavirenz or a ritonavir-boosted protease inhibitor plus 2 nucleoside reverse transcriptase inhibitors (tenofovir/emtricitabine or abacavir/lamivudine). Treatment failure should be identified and managed promptly, with the goal of therapy, even in heavily pretreated patients, being an HIV-1 RNA level below assay detection limits.

The purpose of this review is to put into perspective the many health benefits of vitamin D and the role of vitamin D deficiency in increasing the risk of many common and serious diseases, including some common cancers, type 1 diabetes, cardiovascular disease, and osteoporosis. Numerous epidemiologic studies suggest that exposure to sunlight, which enhances the production of vitamin D(3) in the skin, is important in preventing many chronic diseases. Because very few foods naturally contain vitamin D, sunlight supplies most of our vitamin D requirement. 25-Hydroxyvitamin D [25(OH)D] is the metabolite that should be measured in the blood to determine vitamin D status. Vitamin D deficiency is prevalent in infants who are solely breastfed and who do not receive vitamin D supplementation and in adults of all ages who have increased skin pigmentation or who always wear sun protection or limit their outdoor activities. Vitamin D deficiency is often misdiagnosed as fibromyalgia. A new dietary source of vitamin D is orange juice fortified with vitamin D. Studies in both human and animal models add strength to the hypothesis that the unrecognized epidemic of vitamin D deficiency worldwide is a contributing factor of many chronic debilitating diseases. Greater awareness of the insidious consequences of vitamin D deficiency is needed. Annual measurement of serum 25(OH)D is a reasonable approach to monitoring for vitamin D deficiency. The recommended adequate intakes for vitamin D are inadequate, and, in the absence of exposure to sunlight, a minimum of 1000 IU vitamin D/d is required to maintain a healthy concentration of 25(OH)D in the blood.

Microbial cells operate under governing constraints that limit their range of possible functions. With the availability of annotated genome sequences, it has become possible to reconstruct genome-scale biochemical reaction networks for microorganisms. The imposition of governing constraints on a reconstructed biochemical network leads to the definition of achievable cellular functions. In recent years, a substantial and growing toolbox of computational analysis methods has been developed to study the characteristics and capabilities of microorganisms using a constraint-based reconstruction and analysis (COBRA) approach. This approach provides a biochemically and genetically consistent framework for the generation of hypotheses and the testing of functions of microbial cells.

Autophagy is a catabolic process involving self-digestion of cellular organelles during starvation as a means of cell survival; however, if it proceeds to completion, autophagy can lead to cell death. Autophagy is also a haploinsufficient tumor suppressor mechanism for mammary tumorigenesis, as the essential autophagy regulator beclin1 is monoallelically deleted in breast carcinomas. However, the mechanism by which autophagy suppresses breast cancer remains elusive. Here we show that allelic loss of beclin1 and defective autophagy sensitized mammary epithelial cells to metabolic stress and accelerated lumen formation in mammary acini. Autophagy defects also activated the DNA damage response in vitro and in mammary tumors in vivo, promoted gene amplification, and synergized with defective apoptosis to promote mammary tumorigenesis. Therefore, we propose that autophagy limits metabolic stress to protect the genome, and that defective autophagy increases DNA damage and genomic instability that ultimately facilitate breast cancer progression.

Clinically diagnosed Alzheimer's disease and other dementing illnesses were assessed in a geographically defined US community. Of 3623 persons (80.8% of all community residents over 65 years of age) who had brief memory testing in their homes, a stratified sample of 467 persons underwent neurological, neuropsychological, and laboratory examination. Prevalence rates of Alzheimer's disease were calculated for the community population from the sample undergoing clinical evaluation. Of those over the age of 65 years, an estimated 10.3% (95% confidence limits, 8.1% and 12.5%) had probable Alzheimer's disease. This prevalence rate was strongly associated with age. Of those 65 to 74 years old, 3.0% (95% confidence limits, 0.8 and 5.2) had probable Alzheimer's disease, compared with 18.7% (95% confidence limits, 13.2 and 24.2) of those 75 to 84 years old and 47.2% (95% confidence limits, 37.0 and 63.2) of those over 85 years. Other dementing conditions were uncommon. Of community residents with moderate or severe cognitive impairment, 84.1% had clinically diagnosed Alzheimer's disease as the only probable diagnosis. These data suggest that clinically diagnosed Alzheimer's disease is a common condition and that its public health impact will continue to increase with increasing longevity of the population.

Inflammasomes are multiprotein complexes that activate caspase-1, which leads to maturation of the proinflammatory cytokines interleukin 1β (IL-1β) and IL-18 and the induction of pyroptosis. Members of the Nod-like receptor (NLR) family, including NLRP1, NLRP3 and NLRC4, and the cytosolic receptor AIM2 are critical components of inflammasomes and link microbial and endogenous danger signals to the activation of caspase-1. In response to microbial infection, activation of the inflammasomes contributes to host protection by inducing immune responses that limit microbial invasion, but deregulated activation of inflammasomes is associated with autoinflammatory syndromes and other pathologies. Thus, understanding inflammasome pathways may provide insight into the mechanisms of host defense against microbes and the development of inflammatory disorders.