Intravital multiphoton microscopy has provided powerful mechanistic insights into health and disease and has become a common instrument in the modern biological laboratory. The requisite high numerical aperture and exogenous contrast agents that enable multiphoton microscopy, however, limit the ability to investigate substantial tissue volumes or to probe dynamic changes repeatedly over prolonged periods. Here we introduce optical frequency domain imaging (OFDI) as an intravital microscopy that circumvents the technical limitations of multiphoton microscopy and, as a result, provides unprecedented access to previously unexplored, crucial aspects of tissue biology. Using unique OFDI-based approaches and entirely intrinsic mechanisms of contrast, we present rapid and repeated measurements of tumor angiogenesis, lymphangiogenesis, tissue viability and both vascular and cellular responses to therapy, thereby demonstrating the potential of OFDI to facilitate the exploration of physiological and pathological processes and the evaluation of treatment strategies.

We study the impact of correlated neuronal firing rate variability on the accuracy with which an encoded quantity can be extracted from a population of neurons. Contrary to widespread belief, correlations in the variabilities of neuronal firing rates do not, in general, limit the increase in coding accuracy provided by using large populations of encoding neurons. Furthermore, in some cases, but not all, correlations improve the accuracy of a population code.

DNA barcoding and DNA taxonomy have recently been proposed as solutions to the crisis of taxonomy and received significant attention from scientific journals, grant agencies, natural history museums, and mainstream media. Here, we test two key claims of molecular taxonomy using 1333 mitochondrial COI sequences for 449 species of Diptera. We investigate whether sequences can be used for species identification ("DNA barcoding") and find a relatively low success rate (< 70%) based on tree-based and newly proposed species identification criteria. Misidentifications are due to wide overlap between intra- and interspecific genetic variability, which causes 6.5% of all query sequences to have allospecific or a mixture of allo- and conspecific (3.6%) best-matching barcodes. Even when two COI sequences are identical, there is a 6% chance that they belong to different species. We also find that 21% of all species lack unique barcodes when consensus sequences of all conspecific sequences are used. Lastly, we test whether DNA sequences yield an unambiguous species-level taxonomy when sequence profiles are assembled based on pairwise distance thresholds. We find many sequence triplets for which two of the three pairwise distances remain below the threshold, whereas the third exceeds it; i.e., it is impossible to consistently delimit species based on pairwise distances. Furthermore, for species profiles based on a 3% threshold, only 47% of all profiles are consistent with currently accepted species limits, 20% contain more than one species, and 33% only some sequences from one species; i.e., adopting such a DNA taxonomy would require the redescription of a large proportion of the known species, thus worsening the taxonomic impediment. We conclude with an outlook on the prospects of obtaining complete barcode databases and the future use of DNA sequences in a modern integrative taxonomy.

OBJECTIVE

The purpose of this study was to assess the reliability, construct validity, and sensitivity to change of the Lower Extremity Functional Scale (LEFS).

METHODS

The LEFS was administered to 107 patients with lower-extremity musculoskeletal dysfunction referred to 12 outpatient physical therapy clinics.

METHODS

The LEFS was administered during the initial assessment, 24 to 48 hours following the initial assessment, and then at weekly intervals for 4 weeks. The SF-36 (acute version) was administered during the initial assessment and at weekly intervals. A type 2,1 intraclass correlation coefficient was used to estimate test-retest reliability. Pearson correlations and one-way analyses of variance were used to examine construct validity. Spearman rank-order correlation coefficients were used to examine the relationship between an independent prognostic rating of change for each patient and change in the LEFS and SF-36 scores.

RESULTS

Test-retest reliability of the LEFS scores was excellent (R = .94 [95% lower limit confidence interval (CI) = .89]). Correlations between the LEFS and the SF-36 physical function subscale and physical component score were r=.80 (95% lower limit CI = .73) and r = .64 (95% lower limit CI = .54), respectively. There was a higher correlation between the prognostic rating of change and the LEFS than between the prognostic rating of change and the SF-36 physical function score. The potential error associated with a score on the LEFS at a given point in time is +/-5.3 scale points (90% CI), the minimal detectable change is 9 scale points (90% CI), and the minimal clinically important difference is 9 scale points (90% CI).

CONCLUSIONS

The LEFS is reliable, and construct validity was supported by comparison with the SF-36. The sensitivity to change of the LEFS was superior to that of the SF-36 in this population. The LEFS is efficient to administer and score and is applicable for research purposes and clinical decision making for individual patients.

Because the mucosal epithelia are in constant contact with large numbers of microorganisms, these surfaces must be armed with efficient microbial control systems. Here, we show that the Drosophila nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme, dual oxidase (dDuox), is indispensable for gut antimicrobial activities. Adult flies in which dDuox expression is silenced showed a marked increase in mortality rate even after a minor infection through ingestion of microbe-contaminated food. This could be restored by the specific reintroduction of dDuox, demonstrating that this oxidase generates a unique epithelial oxidative burst that limits microbial proliferation in the gut. Thus, oxidant-mediated antimicrobial responses are not restricted to the phagocytes, but rather are used more broadly, including in mucosal barrier epithelia.

Today's concept of vulnerable plaque has evolved primarily from the early pioneering work uncovering the pivotal role of plaque rupture and coronary thrombosis as the major cause of acute myocardial infarction and sudden cardiac death. Since the first historical description of plaque rupture in 1844, several key studies by leading researchers and clinicians have lead to the current accepted views on lesion instability. Important to the complex paradigm of plaque destabilization and thrombosis are many discoveries beginning with the earliest descriptions of advanced plaques, reminiscent of abscesses encapsulated by fibrous tissue capable of rupture. It was not until the late 1980s that studies of remodeling provided keen insight into the growth of advanced plaques, beyond the simple accumulation of lipid. The emphasis in the next decade, however, was on a focused shift toward the mechanisms of lesion vulnerability based on the contribution of tissue proteolysis by matrix metalloproteinases as an essential factor responsible for thinning and rupture of the fibrous cap. In an attempt to unify the understanding of what constitutes a vulnerable plaque, morphological studies, mostly from autopsy, suggest the importance of necrotic core size, inflammation, and fibrous cap thickness. Definitive proof of the vulnerable plaque, however, remains elusive because animal or human data supporting a cause-and-effect relationship are lacking. Although emerging imagining technologies involving optical coherence tomography, high-resolution MRI, molecular biomarkers, and other techniques have far surpassed the limits of the early days of angiography, advancing the field will require establishing relevant translational animal models that produce vulnerable plaques at risk for rupture and further testing of these modalities in large prospective clinical trials.

Biomarker discovery produces lists of candidate markers whose presence and level must be subsequently verified in serum or plasma. Verification represents a paradigm shift from unbiased discovery approaches to targeted, hypothesis-driven methods and relies upon specific, quantitative assays optimized for the selective detection of target proteins. Many protein biomarkers of clinical currency are present at or below the nanogram/milliliter range in plasma and have been inaccessible to date by MS-based methods. Using multiple reaction monitoring coupled with stable isotope dilution mass spectrometry, we describe here the development of quantitative, multiplexed assays for six proteins in plasma that achieve limits of quantitation in the 1-10 ng/ml range with percent coefficients of variation from 3 to 15% without immunoaffinity enrichment of either proteins or peptides. Sample processing methods with sufficient throughput, recovery, and reproducibility to enable robust detection and quantitation of candidate biomarker proteins were developed and optimized by addition of exogenous proteins to immunoaffinity depleted plasma from a healthy donor. Quantitative multiple reaction monitoring assays were designed and optimized for signature peptides derived from the test proteins. Based upon calibration curves using known concentrations of spiked protein in plasma, we determined that each target protein had at least one signature peptide with a limit of quantitation in the 1-10 ng/ml range and linearity typically over 2 orders of magnitude in the measurement range of interest. Limits of detection were frequently in the high picogram/milliliter range. These levels of assay performance represent up to a 1000-fold improvement compared with direct analysis of proteins in plasma by MS and were achieved by simple, robust sample processing involving abundant protein depletion and minimal fractionation by strong cation exchange chromatography at the peptide level prior to LC-multiple reaction monitoring/MS. The methods presented here provide a solid basis for developing quantitative MS-based assays of low level proteins in blood.

Licensed vaccines against viral diseases generate antibodies that neutralize the infecting virus and protect against infection or disease. Similarly, an effective vaccine against HIV-1 will likely need to induce antibodies that prevent initial infection of host cells or that limit early events of viral dissemination. Such antibodies must target the surface envelope glycoproteins of HIV-1, which are highly variable in sequence and structure. The first subunit vaccines to enter clinical trails were safe and immunogenic but unable to elicit antibodies that neutralized most circulating strains of HIV-1. However, potent virus neutralizing antibodies (NAbs) can develop during the course of HIV-1 infection, and this is the type of antibody response that researchers seek to generate with a vaccine. Thus, current vaccine design efforts have focused on a more detailed understanding of these broadly neutralizing antibodies and their epitopes to inform the design of improved vaccines.

Reperfusion is a pre-requisite to salvaging viable myocardium, following an acute myocardial infarction. Reperfusion of ischaemic myocardium, however, is not without risk, as the act of reperfusion itself can paradoxically result in myocyte death: a phenomenon termed lethal reperfusion-induced injury. Therapeutic strategies that target and attenuate reperfusion-induced cell death may provide novel pharmacological agents, which can be used as an adjunct to current reperfusion therapy, to limit myocardial infarction. Recent evidence has implicated apoptotic cell death during the phase of reperfusion as an important contributor to lethal reperfusion-induced injury. Targeting anti-apoptotic mechanisms of cellular protection at the time of reperfusion may therefore offer a potential approach to attenuating reperfusion-induced cell death. In this regard, ischaemia-reperfusion has been shown to activate the anti-apoptotic pro-survival kinase signalling cascades, phosphatidylinositol-3-OH kinase (PI3K)-Akt and p42/p44 extra-cellular signal-regulated kinases (Erk 1/2), both of which have been implicated in cellular survival. Activating these pro-survival kinase cascades at the time of reperfusion has been demonstrated to confer protection against reperfusion-induced injury. We and others have shown that insulin, insulin-like growth factor-1 (IGF-1), transforming growth factor-beta1 (TGF-beta1), cardiotrophin-1 (CT-1), urocortin, atorvastatin and bradykinin protect the heart, by activating the PI3K-Akt and/or Erk 1/2 kinase cascades, when given at the commencement of reperfusion, following a lethal ischaemic insult. Pharmacological manipulation and up-regulation of these pro-survival kinase cascades, which we refer to as the Reperfusion Injury Salvage Kinase (RISK) pathway, as an adjunct to reperfusion may therefore protect the myocardium from lethal reperfusion-induced cell death and provide a novel strategy to salvaging viable myocardium and limiting infarct size.

In a contemporary clinical laboratory it is very common to have to assess the agreement between two quantitative methods of measurement. The correct statistical approach to assess this degree of agreement is not obvious. Correlation and regression studies are frequently proposed. However, correlation studies the relationship between one variable and another, not the differences, and it is not recommended as a method for assessing the comparability between methods. In 1983 Altman and Bland (B&A) proposed an alternative analysis, based on the quantification of the agreement between two quantitative measurements by studying the mean difference and constructing limits of agreement. The B&A plot analysis is a simple way to evaluate a bias between the mean differences, and to estimate an agreement interval, within which 95% of the differences of the second method, compared to the first one, fall. Data can be analyzed both as unit differences plot and as percentage differences plot. The B&A plot method only defines the intervals of agreements, it does not say whether those limits are acceptable or not. Acceptable limits must be defined a priori, based on clinical necessity, biological considerations or other goals. The aim of this article is to provide guidance on the use and interpretation of Bland Altman analysis in method comparison studies.

OBJECTIVE

This paper reviews the literature on obstetric complications as a risk factor for schizophrenia. The authors trace the evolution of this literature through different methods and carry out a quantitative review of the results from prospective, population-based studies.

METHODS

Relevant papers were identified by a MEDLINE search, by examination of reference lists of published papers, and through personal contact with researchers in the field. Studies were grouped in chronological order according to common themes or methods. Meta-analytic techniques were used to summarize the findings of prospective population-based studies.

RESULTS

The meta-analytic synthesis of the prospective population-based studies revealed that three groups of complications were significantly associated with schizophrenia: 1) complications of pregnancy (bleeding, diabetes, rhesus incompatibility, preeclampsia); 2) abnormal fetal growth and development: (low birthweight, congenital malformations, reduced head circumference), and 3) complications of delivery (uterine atony, asphyxia, emergency Cesarean section). Pooled estimates of effect sizes were generally less than 2.

CONCLUSIONS

Current methods of investigating the relationship between obstetric complications and schizophrenia are reaching the limit of their usefulness. Lack of statistical power to measure small and interactive effects and lack of detailed information about the prenatal period are major problems with current approaches. A combination of disciplines and approaches will be needed to elucidate the mechanisms underlying these small but important associations.

Crosstalk and complexity within signaling pathways and their perturbation by oncogenes limit component-by-component approaches to understanding human disease. Network analysis of how normal and oncogenic signaling can be rewired by drugs may provide opportunities to target tumors with high specificity and efficacy. Using targeted inhibition of oncogenic signaling pathways, combined with DNA-damaging chemotherapy, we report that time-staggered EGFR inhibition, but not simultaneous coadministration, dramatically sensitizes a subset of triple-negative breast cancer cells to genotoxic drugs. Systems-level analysis-using high-density time-dependent measurements of signaling networks, gene expression profiles, and cell phenotypic responses in combination with mathematical modeling-revealed an approach for altering the intrinsic state of the cell through dynamic rewiring of oncogenic signaling pathways. This process converts these cells to a less tumorigenic state that is more susceptible to DNA damage-induced cell death by reactivation of an extrinsic apoptotic pathway whose function is suppressed in the oncogene-addicted state.

Salinity tolerance comes from genes that limit the rate of salt uptake from the soil and the transport of salt throughout the plant, adjust the ionic and osmotic balance of cells in roots and shoots, and regulate leaf development and the onset of senescence. This review lists some candidate genes for salinity tolerance, and draws together hypotheses about the functions of these genes and the specific tissues in which they might operate. Little has been revealed by gene expression studies so far, perhaps because the studies are not tissue-specific, and because the treatments are often traumatic and unnatural. Suggestions are made to increase the value of molecular studies in identifying genes that are important for salinity tolerance.

We have discovered that three longevity mutants of the nematode Caenorhabditis elegans also exhibit increased intrinsic thermotolerance (Itt) as young adults. Mutation of the age-1 gene causes not only 65% longer life expectancy but also Itt. The Itt phenotype cosegregates with age-1. Long-lived spe-26 and daf-2 mutants also exhibit Itt. We investigated the relationship between increased thermotolerance and increased life-span by developing conditions for environmental induction of thermotolerance. Such pretreatments at sublethal temperatures induce significant increases in thermotolerance and small but statistically highly significant increases in life expectancy, consistent with a causal connection between these two traits. Thus, when an animal's resistance to stress is increased, by either genetic or environmental manipulation, we also observe an increase in life expectancy. These results suggest that ability to respond to stress limits the life expectancy of C. elegans and might do so in other metazoa as well.

In recent years, a tremendous effort has been devoted to the detailed characterization of the phenotype and function of distinct T cell subpopulations in humans, as well as to their pathway(s) of differentiation and role in immune responses. But these studies seem to have generated more questions than definitive answers. To clarify issues related to the function and differentiation of T cell subsets, one session of the MASIR 2008 conference was dedicated to this topic. Several points of consensus and discord were highlighted in the work presented during this session. We provide here an account of these points, including the relative heterogeneity of T cell subpopulations during infections with distinct pathogens, the relationship between phenotypic and functional T cell attributes, and the pathway(s) of T cell differentiation. Finally, we discuss the problems which still limit general agreement.

Persistent androgen receptor (AR) transcriptional activity underlies resistance to AR-targeted therapy and progression to lethal castration-resistant prostate cancer (CRPC). Recent success in retargeting persistent AR activity with next generation androgen/AR axis inhibitors such as enzalutamide (MDV3100) has validated AR as a master regulator during all stages of disease progression. However, resistance to next generation AR inhibitors limits therapeutic efficacy for many patients. One emerging mechanism of CRPC progression is AR gene rearrangement, promoting synthesis of constitutively active truncated AR splice variants (AR-V) that lack the AR ligand-binding domain. In this study, we show that cells with AR gene rearrangements expressing both full-length and AR-Vs are androgen independent and enzalutamide resistant. However, selective knock-down of AR-V expression inhibited androgen-independent growth and restored responsiveness to androgens and antiandrogens. In heterogeneous cell populations, AR gene rearrangements marked individual AR-V-dependent cells that were resistant to enzalutamide. Gene expression profiling following knock-down of full-length AR or AR-Vs showed that AR-Vs drive resistance to AR-targeted therapy by functioning as constitutive and independent effectors of the androgen/AR transcriptional program. Further, mitotic genes deemed previously to be unique AR-V targets were found to be biphasic targets associated with a proliferative level of signaling output from either AR-Vs or androgen-stimulated AR. Overall, these studies highlight AR-Vs as key mediators of persistent AR signaling and resistance to the current arsenal of conventional and next generation AR-directed therapies, advancing the concept of AR-Vs as therapeutic targets in advanced disease.

Currently available HIV-1 protease inhibitors are potent agents in the therapy of HIV-1 infection. However, limited oral absorption and variable tissue distribution, both of which are largely unexplained, complicate their use. We tested the hypothesis that P-glycoprotein is an important transporter for these agents. We studied the vectorial transport characteristics of indinavir, nelfinavir, and saquinavir in vitro using the model P-glycoprotein expressing cell lines L-MDR1 and Caco-2 cells, and in vivo after intravenous and oral administration of these agents to mice with a disrupted mdr1a gene. All three compounds were found to be transported by P-glycoprotein in vitro. After oral administration, plasma concentrations were elevated 2-5-fold in mdr1a (-/-) mice and with intravenous administration, brain concentrations were elevated 7-36-fold. These data demonstrate that P-glycoprotein limits the oral bioavailability and penetration of these agents into the brain. This raises the possibility that higher HIV-1 protease inhibitor concentrations may be obtained by targeted pharmacologic inhibition of P-glycoprotein transport activity.

Activation of the p53 protein protects the organism against the propagation of cells that carry damaged DNA with potentially oncogenic mutations. MDM2, a p53-specific E3 ubiquitin ligase, is the principal cellular antagonist of p53, acting to limit the p53 growth-suppressive function in unstressed cells. In unstressed cells, MDM2 constantly monoubiquitinates p53 and thus is the critical step in mediating its degradation by nuclear and cytoplasmic proteasomes. The interaction between p53 and MDM2 is conformation-based and is tightly regulated on multiple levels. Disruption of the p53-MDM2 complex by multiple routes is the pivotal event for p53 activation, leading to p53 induction and its biological response. Because the p53-MDM2 interaction is structurally and biologically well understood, the design of small lipophilic molecules that disrupt or prevent it has become an important target for cancer therapy.

Recombinant adeno-associated viruses (AAVs) have unique gene-transfer properties that speak to their potential as carriers for gene therapy or vaccine applications. However, the presence of neutralizing antibodies to AAV as a result of previous exposure can significantly limit effective gene transfer. In this study, we obtained 888 human serum samples from healthy volunteers in 10 countries around the world. Samples were assayed for neutralizing antibodies to AAV1, AAV2, AAV7, and AAV8, as well as to a novel, structurally distinct AAV vector, rh32.33, in an in vitro transduction inhibition assay. Our data revealed that neutralizing antibodies to AAV2 were the most prevalent antibodies in all regions, followed by antibodies to AAV1. The seroprevalences of antibodies to AAV7 and to AAV8 were lower than that for antibodies to AAV1, and neutralization of AAVrh32.33 was only rarely detected. Our data also indicate a strong linkage of seroreactivity between apparently distinct serotypes that has not been predicted previously in animal models.

As with other organs, the eye's growth is regulated by homeostatic control mechanisms. Unlike other organs, the eye relies on vision as a principal input to guide growth. In this review, we consider several implications of this visual guidance. First, we compare the regulation of eye growth to that of other organs. Second, we ask how the visual system derives signals that distinguish the blur of an eye too large from one too small. Third, we ask what cascade of chemical signals constitutes this growth control system. Finally, if the match between the length and optics of the eye is under homeostatic control, why do children so commonly develop myopia, and why does the myopia not limit itself? Long-neglected studies may provide an answer to this last question.

Herpes simplex virus (HSV)-specific proteins fall into at least three kinetic classes whose synthesis is sequentially and coordinaely regulated. Temperature-sensitive (ts) mutants of one complementation group (1-2) are defective in the transition from immediate early to early and late protein synthesis. To elucidate the function of the 1-2 gene product in the HSV type 1 replicative cycle, nine ts mutants in this group were mapped by fine-structure analysis and characterized members of the group lie within the terminally repeated sequences of the S region of the genome. Fine-structure genetic and physical mapping permitted the mutations to be ordered within these sequences. Because it has been shown that the message for VP175 and the DNA template specifying this protein extend beyond the limits of the physical map of the mutations, it follows that the mutations must lie within the structural gene for VP175. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that most members of the group overproduced the immediate early proteins VP175, -136, -110, and -63 and markedly underproduced early and late proteins at the nonpermissive temperature. In temperature shiftup experiments, it was fund that the synthesis of early and late proteins ceased, whereas the synthesis of immediate early proteins began again. Thus, it is postulated that VP175 is (i) involved in the transition from immediate early to early protein synthesis, (ii) requird continuously to maintain early protein synthesis, (iii) autoregulated, acting to inhibit immediate early protein synthesis.

This brief review examines some of the methods used to infer central control strategies from surface electromyogram (EMG) recordings. Among the many uses of the surface EMG in studying the neural control of movement, the review critically evaluates only some of the applications. The focus is on the relations between global features of the surface EMG and the underlying physiological processes. Because direct measurements of motor unit activation are not available and many factors can influence the signal, these relations are frequently misinterpreted. These errors are compounded by the counterintuitive effects that some system parameters can have on the EMG signal. The phenomenon of crosstalk is used as an example of these problems. The review describes the limitations of techniques used to infer the level of muscle activation, the type of motor unit recruited, the upper limit of motor unit recruitment, the average discharge rate, and the degree of synchronization between motor units. Although the global surface EMG is a useful measure of muscle activation and assessment, there are limits to the information that can be extracted from this signal.

Type 2 diabetes (T2D) is a major comorbidity of COVID-19. However, the impact of blood glucose (BG) control on the degree of required medical interventions and on mortality in patients with COVID-19 and T2D remains uncertain. Thus, we performed a retrospective, multi-centered study of 7,337 cases of COVID-19 in Hubei Province, China, among which 952 had pre-existing T2D. We found that subjects with T2D required more medical interventions and had a significantly higher mortality (7.8% versus 2.7%; adjusted hazard ratio [HR], 1.49) and multiple organ injury than the non-diabetic individuals. Further, we found that well-controlled BG (glycemic variability within 3.9 to 10.0 mmol/L) was associated with markedly lower mortality compared to individuals with poorly controlled BG (upper limit of glycemic variability exceeding 10.0 mmol/L) (adjusted HR, 0.14) during hospitalization. These findings provide clinical evidence correlating improved glycemic control with better outcomes in patients with COVID-19 and pre-existing T2D.

CD8+ T lymphocytes recognize antigens as short peptides bound to MHC class I molecules. Available methods cannot determine the number and distribution of these ligands on individual cells or detect antigen-presenting cells in tissues. Here we describe a method for eliciting and identifying monoclonal antibodies specific for a particular peptide-MHC class I combination. One such antibody can identify antigen complexes with a limit of detection approaching that of T cells. We used this antibody to determine the number of peptide-class I complexes generated upon viral infection, to identify antigen-presenting cells in cell mixtures, to determine the site of peptide-MHC class I interaction inside cells, and to visualize cells bearing specific peptide-MHC class I complexes after in vivo infection. Similar antibodies may prove useful for diagnostic or therapeutic purposes in cancer, infectious diseases, and autoimmune disorders.