Web- and app-based tools for remote hearing assessment: a scoping review.

OBJECTIVE: Remote hearing screening and assessment may improve access to, and uptake of, hearing care. This review, the most comprehensive to date, aimed to (i) identify and assess functionality of remote hearing assessment tools on smartphones and online platforms, (ii) determine if assessed tools were also evaluated in peer-reviewed publications and (iii) report accuracy of existing validation data. DESIGN: Protocol was registered in INPLASY and reported according to PRISMA-Extension for Scoping Reviews. STUDY SAMPLE: In total, 187 remote hearing assessment tools (using tones, speech, self-report or a combination) and 101 validation studies met the inclusion criteria. Quality, functionality, bias and applicability of each app were assessed by at least two authors. RESULTS: Assessed tools showed considerable variability in functionality. Twenty-two (12%) tools were peer-reviewed and 14 had acceptable functionality. The validation results and their quality varied greatly, largely depending on the category of the tool. CONCLUSION: The accuracy and reliability of most tools are unknown. Tone-producing tools provide approximate hearing thresholds but have calibration and background noise issues. Speech and self-report tools are less affected by these issues but mostly do not provide an estimated pure tone audiogram. Predicting audiograms using filtered language-independent materials could be a universal solution.

Assessing the pool activity level (PAL) checklist for use with people with hearing and vision loss.

BACKGROUND: The PAL is a career-completed assessment that indexes cognitive functional ability to inform individualised support. As hearing and vision loss are prevalent, we assessed the PAL for potential bias with hearing or vision impairment. METHODS: We collected PAL responses for 333 adults aged over 60 years in the UK, France, Canada, Greece and Cyprus. All participants had normal cognition based on self-reported status and normal range scores on a cognitive screening test. Using a Kruskal-Wallis test, we compared PAL item response distributions for people with assessed hearing or vision loss compared to those with normal sensory function. RESULTS: There were no differences in response distributions between hearing or vision impaired groups versus those with normal sensory function on any PAL item. CONCLUSION: The PAL reliably indexes cognitive functional ability and may be used to inform support tailored to individual cognitive level amongst older adults with prevalent hearing and vision impairments.

Cardiac Output Measurement in Neonates and Children Using Noninvasive Electrical Bioimpedance Compared With Standard Methods: A Systematic Review and Meta-Analysis.

OBJECTIVE: To systematically review and meta-analyze the validity of electrical bioimpedance-based noninvasive cardiac output monitoring in pediatrics compared with standard methods such as thermodilution and echocardiography. DATA SOURCES: Systematic searches were conducted in MEDLINE and EMBASE (2000-2019). STUDY SELECTION: Method-comparison studies of transthoracic electrical velocimetry or whole body electrical bioimpedance versus standard cardiac output monitoring methods in children (0-18 yr old) were included. DATA EXTRACTION: Two reviewers independently performed study selection, data extraction, and risk of bias assessment. Mean differences of cardiac output, stroke volume, or cardiac index measurements were pooled using a random-effects model (R Core Team, R Foundation for Statistical Computing, Vienna, Austria, 2019). Bland-Altman statistics assessing agreement between devices and author conclusions about inferiority/noninferiority were extracted. DATA SYNTHESIS: Twenty-nine of 649 identified studies were included in the qualitative analysis, and 25 studies in the meta-analyses. No significant difference was found between means of cardiac output, stroke volume, and cardiac index measurements, except in exclusively neonatal/infant studies reporting stroke volume (mean difference, 1.00 mL; 95% CI, 0.23-1.77). Median percentage error in child/adolescent studies approached acceptability (percentage error less than or equal to 30%) for cardiac output in L/min (31%; range, 13-158%) and stroke volume in mL (26%; range, 14-27%), but not in neonatal/infant studies (45%; range, 29-53% and 45%; range, 28-70%, respectively). Twenty of 29 studies concluded that transthoracic electrical velocimetry/whole body electrical bioimpedance was noninferior. Transthoracic electrical velocimetry was considered inferior in six of nine studies with heterogeneous congenital heart disease populations. CONCLUSIONS: The meta-analyses demonstrated no significant difference between means of compared devices (except in neonatal stroke volume studies). The wide range of percentage error reported may be due to heterogeneity of study designs, devices, and populations included. Transthoracic electrical velocimetry/whole body electrical bioimpedance may be acceptable for use in child/adolescent populations, but validity in neonates and congenital heart disease patients remains uncertain. Larger studies in specific clinical contexts with standardized methodologies are required.

FAK displacement from focal adhesions: a promising strategy to target processes implicated in cancer progression and metastasis.

BACKGROUND: Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is overexpressed or activated in several advanced-stage solid cancers. It is known to play both kinase-dependent and -independent roles in promoting tumor progression and metastasis. Numerous inhibitors, targeting either the enzymatic or scaffolding activities of FAK have been generated, with varying degree of success. Here, we describe a novel approach to site-specifically target both kinase-dependent and -independent FAK functions at focal adhesions (FAs), the primary sites at which the kinase exerts its activity. METHODS: We took advantage of the well-characterized interactions between the paxillin LD motifs and the FAK FAT domain and generated a polypeptide (LD2-LD3-LD4) expected to compete with interactions with paxillin. Co-immunoprecipitation experiments were performed to examine the interaction between the LD2-LD3-LD4 polypeptide and FAK. The effects of LD2-LD3-LD4 in the localization and functions of FAK, as well as FA composition, were evaluated using quantitative immunofluorescence, cell fractionation, FA isolation and Western Blot analysis. Live cell imaging, as well as 2-D migration and cell invasion assays were used to examine the effects on FA turnover and tumor cell migration and invasion. RESULTS: Expression of the LD2-LD3-LD4 polypeptide prevents FAK localization at FAs, in a controlled and dose-dependent manner, by competing with endogenous paxillin for FAK binding. Importantly, the LD2-LD3-LD4 peptide did not otherwise affect FA composition or integrin activation. LD2-LD3-LD4 inhibited FAK-dependent downstream integrin signaling and, unlike existing inhibitors, also blocked FAK's scaffolding functions. We further show that LD2-LD3-LD4 expression markedly reduces FA turnover and inhibits tumor cell migration and invasion. Finally, we show that dimers of a single motif, linked through a flexible linker of the proper size, are sufficient for the displacement of FAK from FAs and for inhibition of tumor cell migration. This work raises the possibility of using a synthetic peptide as an antimetastatic agent, given that effective displacement of FAK from FAs only requires dimers of a single LD motif linked by a short flexible linker. CONCLUSION: In conclusion, these results suggest that FAK displacement from FAs is a promising new strategy to target critical processes implicated in cancer progression and metastasis. Video abstract.

Impact of an intervention to support hearing and vision in dementia: The SENSE-Cog Field Trial.

OBJECTIVES: Hearing, vision, and cognitive impairment commonly co-occur in older adults. Improving sensory function may positively impact outcomes in people with dementia (PwD). We developed a "sensory intervention" (SI) to support hearing and vision in PwD. Here, we report the findings of an international open-label field trial, and nested case series, to explore the impact of the SI on dementia-related outcomes. METHODS: This was a home-based trial conducted in France, England, and Cyprus. Participants were people with mild-to-moderate dementia and hearing and/or vision impairment (n = 19) and their study partners (unpaid carers; n = 19). The "basic" SI included a hearing and vision assessment and provision of glasses and/or hearing aids. A subsample received the "extended" SI with additional weekly visits from a sensory support therapist (SST). Exploratory analyses of dementia-related, health utility and resource utilisation outcomes were performed. RESULTS: Quality of life (QoL) and sensory functional ability improved. Change in QoL exceeded the threshold for a minimum clinically important difference. There was a modest improvement (in absolute terms) post intervention in behavioural disturbance, self-efficacy, and relationship satisfaction. Study partner time assisting instrumental activities of daily living (iADL) and supervision decreased by about 22 and 38 hours per month, respectively, although time for personal ADL support increased. Qualitative data supported effectiveness of the intervention: PwD were more socially engaged, less isolated, less dependent on study partners, and had improved functional ability and communication. CONCLUSIONS: These findings support the need for a definitive randomised controlled trial (RCT) to evaluate the effectiveness of the intervention.

Tools for App- and Web-Based Self-Testing of Cognitive Impairment: Systematic Search and Evaluation.

BACKGROUND: Tools for app- and Web-based self-testing for identification of cognitive impairment are widely available but are of uncertain quality. OBJECTIVE: The objective of this study was to undertake a scoping review of app- and Web-based self-tests for cognitive impairment and determine the validity of these tests. METHODS: We conducted systematic searches in electronic databases, including Google search, Google Play Store, and iPhone Operating System App Store, using the search terms "Online OR Internet-based AND Memory OR Brain OR Dementia OR mild cognitive impairment OR MCI AND Test OR Screen OR Check." RESULTS: We identified 3057 tools, of which 25 were included in the review. Most tools meeting the inclusion criteria assessed multiple cognitive domains. The most frequently assessed domains were memory, attention, and executive function. We then conducted an electronic survey with the developers of the tools to identify data relating to development and validation of each tool. If no response to the survey was received, Google (to identify gray literature), Google Scholar, and Medical Literature Analysis and Retrieval System Online were searched using key terms "(name of developer, if available)" AND "(the name of the tool)" to identify any additional data. Only 7 tools had any information concerning psychometric quality, and only 1 tool reported data on performance norms, reliability, validity, sensitivity, and specificity for the detection of cognitive impairment. CONCLUSIONS: The number of cognitive self-assessment electronic health tools for cognitive impairment is increasing, but most are of uncertain quality. There is a need for well-validated tools and guidance for users concerning which tools provide reliable information about possible cognitive impairment that could warrant further investigation.

Assessing and managing concurrent hearing, vision and cognitive impairments in older people: an international perspective from healthcare professionals.

BACKGROUND: there is a significant gap in the understanding, assessment and management of people with dementia and concurrent hearing and vision impairments. OBJECTIVE: from the perspective of professionals in dementia, hearing and vision care, we aimed to: (1) explore the perceptions of gaps in assessment and service provision in ageing-related hearing, vision and cognitive impairment; (2) consider potential solutions regarding this overlap and (3) ascertain the attitudes, awareness and practice, with a view to implementing change. METHODS: our two-part investigation with hearing, vision, and dementia care professionals involved: (1) an in-depth, interdisciplinary, international Expert Reference Group (ERG; n = 17) and (2) a wide-scale knowledge, attitudes and practice survey (n = 653). The ERG involved consensus discussions around prototypic clinical vignettes drawn from a memory centre, an audiology clinic, and an optometry clinic, analysed using an applied content approach. RESULTS: the ERG revealed several gaps in assessment and service provision, including a lack of validated assessment tools for concurrent impairments, poor interdisciplinary communication and care pathways, and a lack of evidence-based interventions. Consensus centred on the need for flexible, individualised, patient-centred solutions, using an interdisciplinary approach. The survey data validated these findings, highlighting the need for clear guidelines for assessing and managing concurrent impairments. CONCLUSIONS: this is the first international study exploring professionals' views of the assessment and care of individuals with age-related hearing, vision and hearing impairment. The findings will inform the adaptation of assessments, the development of supportive interventions, and the new provision of services.

The need for improved cognitive, hearing and vision assessments for older people with cognitive impairment: a qualitative study.

BACKGROUND: Hearing and vision (sensory) impairments are highly prevalent in people with dementia (PwD) and exacerbate the impact of living with dementia. Assessment of sensory or cognitive function may be difficult if people have concurrent dual or triple impairments. Most standard cognitive assessment tests are heavily dependent on having intact hearing and vision, and impairments in these domains may render the assessments unreliable or even invalid. Likewise, dementia may impede on the accurate reporting of symptoms that is required for most hearing and vision assessments. Thus, there is an urgent need for hearing, vision and cognitive assessment strategies to be adapted to ensure that appropriate management and support can be provided. OBJECTIVE: To explore the perspectives of PwD and the care partners regarding the need for accurate hearing, vision and cognitive assessments. METHODS: We conducted focus groups and semi-structured interviews regarding the clinical assessment for cognitive, hearing and visual impairment. Participants (n = 18) were older adults with mild to moderate dementia and a sensory impairment as well as their care partners (e.g. a family member) (n = 15) at three European sites. The qualitative material was analysed according to Mayring's summative content analysis approach. RESULTS: Participants reported that hearing, vision and cognitive assessments were not appropriate to the complex needs of PwD and sensory comorbidity and that challenges in communication with professionals and conveying unmet needs and concerns by PwD were common in all three types of clinical assessments. They felt that information about and guidance regarding support for the condition was not adequate in the assessments and that information sharing among the professionals regarding the concurrent problems was limited. Professionals were reported as being concerned only with problems related to their own discipline and had limited regard for problems in other domains which might impact on their own assessments. CONCLUSIONS: The optimal assessment and support for PwD with multiple impairments, more comprehensive, yet easy to understand, information regarding these linked to conditions and corrective device use is needed. Communication among health care professionals relevant to hearing, vision and cognition needs to be improved.

Screening tools for the identification of dementia for adults with age-related acquired hearing or vision impairment: a scoping review.

BACKGROUND: Cognitive screening tests frequently rely on items being correctly heard or seen. We aimed to identify, describe, and evaluate the adaptation, validity, and availability of cognitive screening and assessment tools for dementia which have been developed or adapted for adults with acquired hearing and/or vision impairment. METHOD: Electronic databases were searched using subject terms "hearing disorders" OR "vision disorders" AND "cognitive assessment," supplemented by exploring reference lists of included papers and via consultation with health professionals to identify additional literature. RESULTS: 1,551 papers were identified, of which 13 met inclusion criteria. Four papers related to tests adapted for hearing impairment; 11 papers related to tests adapted for vision impairment. Frequently adapted tests were the Mini-Mental State Examination (MMSE) and the Montreal Cognitive Assessment (MOCA). Adaptations for hearing impairment involved deleting or creating written versions for hearing-dependent items. Adaptations for vision impairment involved deleting vision-dependent items or spoken/tactile versions of visual tasks. No study reported validity of the test in relation to detection of dementia in people with hearing/vision impairment. Item deletion had a negative impact on the psychometric properties of the test. CONCLUSIONS: While attempts have been made to adapt cognitive tests for people with acquired hearing and/or vision impairment, the primary limitation of these adaptations is that their validity in accurately detecting dementia among those with acquired hearing or vision impairment is yet to be established. It is likely that the sensitivity and specificity of the adapted versions are poorer than the original, especially if the adaptation involved item deletion. One solution would involve item substitution in an alternative sensory modality followed by re-validation of the adapted test.

A cross-cultural study of the Montreal Cognitive Assessment for people with hearing impairment.

BACKGROUND: Cognitive screening tools enable the detection of cognitive impairment, facilitate timely intervention, inform clinical care, and allow long-term planning. The Montreal Cognitive Assessment for people with hearing impairment (MoCA-H) was developed as a reliable cognitive screening tool for people with hearing loss. Using the same methodology across four languages, this study examined whether cultural or linguistic factors affect the performance of the MoCA-H. METHODS: The current study investigated the performance of the MoCA-H across English, German, French, and Greek language groups (n = 385) controlling for demographic factors known to affect the performance of the MoCA-H. RESULTS: In a multiple regression model accounting for age, sex, and education, cultural-linguistic group accounted for 6.89% of variance in the total MoCA-H score. Differences between languages in mean score of up to 2.6 points were observed. CONCLUSIONS: Cultural or linguistic factors have a clinically significant impact on the performance of the MoCA-H such that optimal performance cut points for identification of cognitive impairment derived in English-speaking populations are likely inappropriate for use in non-English speaking populations. To ensure reliable identification of cognitive impairment, it is essential that locally appropriate performance cut points are established for each translation of the MoCA-H.

Dietary Stimuli, Intestinal Bacteria and Peptide Hormones Regulate Female Drosophila Defecation Rate.

Peptide hormones control Drosophila gut motility, but the intestinal stimuli and the gene networks coordinating this trait remain poorly defined. Here, we customized an assay to quantify female Drosophila defecation rate as a proxy of intestinal motility. We found that bacterial infection with the human opportunistic bacterial pathogen Pseudomonas aeruginosa (strain PA14) increases defecation rate in wild-type female flies, and we identified specific bacteria of the fly microbiota able to increase defecation rate. In contrast, dietary stress, imposed by either water-only feeding or high ethanol consumption, decreased defecation rate and the expression of enteroendocrine-produced hormones in the fly midgut, such as Diuretic hormone 31 (Dh31). The decrease in defecation due to dietary stress was proportional to the impact of each stressor on fly survival. Furthermore, we exploited the Drosophila Genetic Reference Panel wild type strain collection and identified strains displaying high and low defecation rates. We calculated the narrow-sense heritability of defecation rate to be 91%, indicating that the genetic variance observed using our assay is mostly additive and polygenic in nature. Accordingly, we performed a genome-wide association (GWA) analysis revealing 17 candidate genes linked to defecation rate. Downregulation of four of them (Pmp70, CG11307, meso18E and mub) in either the midgut enteroendocrine cells or in neurons reduced defecation rate and altered the midgut expression of Dh31, that in turn regulates defecation rate via signaling to the visceral muscle. Hence, microbial and dietary stimuli, and Dh31-controlling genes, regulate defecation rate involving signaling within and among neuronal, enteroendocrine, and visceral muscle cells.

Development and validation of the Montreal cognitive assessment for people with hearing impairment (MoCA-H).

BACKGROUND: Hearing impairment is common among older adults and affects cognitive assessments for identification of dementia which rely on good hearing function. We developed and validated a version of the Montreal Cognitive Assessment (MoCA) for people with hearing impairment. METHODS: We adapted existing MoCA 8.1 items for people with hearing impairment by presenting instructions and stimuli in written rather than spoken format. One Attention domain and two Language domain items required substitution by alternative items. Three and four candidate items respectively were constructed and field-tested along with the items adapted to written form. We used a combination of individual item analysis and item substitution to select the set of alternative items to be included in the final form of the MoCA-H in place of the excluded original items. We then evaluated the performance and reliability of the final tool, including making any required adjustments for demographic factors. RESULTS: One hundred and fifty-nine hearing-impaired participants, including 76 with normal cognition and 83 with dementia, completed the adapted version of the MoCA. A further 97 participants with normal hearing completed the standard MoCA as well as the novel items developed for the MoCA-H to assess score equivalence between the existing and alternative MoCA items and for independence from hearing impairment. Twenty-eight participants were retested between 2-4 weeks after initial testing. After the selection of optimal item set, the final MoCA-H had an area under the curve of 0.973 (95% CI 0.952-0.994). At a cut-point of 24 points or less sensitivity and specificity for dementia was 92.8% and 90.8%, respectively. The intraclass correlation for test-retest reliability was 0.92 (95%CI 0.78-0.97). CONCLUSION: The MoCA-H is a sensitive and reliable means of identifying dementia among adults with acquired hearing impairment.

High-resolution whole-mount in situ hybridization using Quantum Dot nanocrystals.

The photostability and narrow emission spectra of nanometer-scale semiconductor crystallites (QDs) make them desirable candidates for whole-mount fluorescent in situ hybridization to detect mRNA transcripts in morphologically preserved intact embryos. We describe a method for direct QD labeling of modified oligonucleotide probes through streptavidin-biotin and antibody-mediated interactions (anti-FITC and anti-digoxigenin). To overcome permeability issues and allow QD conjugate penetration, embryos were treated with proteinase K. The use of QDs dramatically increased sensitivity of whole-mount in situ hybridization (WISH) in comparison with organic fluorophores and enabled fluorescent detection of specific transcripts within cells without the use of enzymatic amplification. Therefore, this method offers significant advantages both in terms of sensitivity, as well as resolution. Specifically, the use of QDs alleviates issues of photostability and limited brightness plaguing organic fluorophores and allows fluorescent imaging of cleared embryos. It also offers new imaging possibilities, including intracellular localization of mRNAs, simultaneous multiple-transcript detection, and visualization of mRNA expression patterns in 3D.

Proteobacteria and Firmicutes Secreted Factors Exert Distinct Effects on Pseudomonas aeruginosa Infection under Normoxia or Mild Hypoxia.

Microbiota may alter a pathogen's virulence potential at polymicrobial infection sites. Here, we developed a multi-modal Drosophila assay, amenable to the assessment of human bacterial interactions using fly survival or midgut regeneration as a readout, under normoxia or mild hypoxia. Deploying a matrix of 12 by 33 one-to-one Drosophila co-infections via feeding, we classified bacterial interactions as neutral, synergistic, or antagonistic, based on fly survival. Twenty six percent of these interactions were antagonistic, mainly occurring between Proteobacteria. Specifically, Pseudomonas aeruginosa infection was antagonized by various Klebsiella strains, Acinetobacter baumannii, and Escherichia coli. We validated these interactions in a second screen of 7 by 34 one-to-one Drosophila co-infections based on assessments of midgut regeneration, and in bacterial co-culture test tube assays, where antagonistic interactions depended on secreted factors produced upon high sugar availability. Moreover, Enterococci interacted synergistically with P. aeruginosa in flies and in test tubes, enhancing the virulence and pyocyanin production by P. aeruginosa. However, neither lactic acid bacteria nor their severely hypoxic culture supernatants provided a survival benefit upon P. aeruginosa infection of flies or mice, respectively. We propose that at normoxic or mildly hypoxic sites, Firmicutes may exacerbate, whereas Proteobacteria secreted factors may ameliorate, P. aeruginosa infections.

Support care needs of people with hearing and vision impairment in dementia: a European cross-national perspective.

PURPOSE: Aging-related sensory impairments are among the most common and disabling comorbidities in people with dementia (PwD). This study explored the unmet support care needs (SCNs) from the perspectives of people with hearing and/or vision impairment in dementia (PwD), and their care partners in Europe. METHODS: This was a two-phase mixed methods study. We administered standardized questionnaires of SCNs and quality of life (QoL) to PwD with hearing and/or vision impairment (n = 97), and their care partners (n = 97) in the UK, France, and Cyprus. Next, a purposive sub-sample of 34 participants (PwD and care partners) participated as focus groups (FGs) or semi-structured interviews to explore their SCNs in depth. RESULTS: Over 94% of the participants reported unmet SCNs (median, 13 (range 5-23)). Nearly three-quarters reported SCNs in the moderate to high range, with the most prevalent unmet SCNs for PwD being in the psychological (>60%) and physical domains (>56%), followed by the need for health information (>46%). Emergent qualitative themes were: (1) the need for tailored support care interventions; (2) care burden, social isolation, and loneliness arising from the combined problems; (3) the need for adequate support from professionals from the different fields, including education around the use of sensory aids. Both study phases revealed that SCNs were highly individualized. CONCLUSIONS: This cross-national study revealed that PwD with sensory impairment and their care partners experience a wide range of unmet SCNs, the interactions between sensory impairments, SCNs and QoL are also complex. A tailored intervention could address these unmet SCNs, including additional support with sensory aids, psychological support, more information about concurrent impairments, and joined up health systems providing care.Implications for rehabilitationA majority of participants with combined age-related hearing, vision, and cognitive impairment had unmet SCNs.The needs of care partners including the risk of loneliness and social isolation, need to be considered.Individually tailored, specific interventions for hearing, vision, and cognitive impairment should incorporate physical and psychological support, as well as education.

Innate NKT lymphocytes confer superior adaptive immunity via tumor-capturing dendritic cells.

If irradiated tumor cells could be rendered immunogenic, they would provide a safe, broad, and patient-specific array of antigens for immunotherapies. Prior approaches have emphasized genetic transduction of live tumor cells to express cytokines, costimulators, and surrogate foreign antigens. We asked if immunity could be achieved by delivering irradiated, major histocompatibility complex-negative plasmacytoma cells to maturing mouse dendritic cells (DCs) within lymphoid organs. Tumor cells injected intravenously (i.v.) were captured by splenic DCs, whereas subcutaneous (s.c.) injection led only to weak uptake in lymph node or spleen. The natural killer T (NKT) cells mobilizing glycolipid alpha-galactosyl ceramide, used to mature splenic DCs, served as an effective adjuvant to induce protective immunity. This adjuvant function was mimicked by a combination of poly IC and agonistic alphaCD40 antibody. The adjuvant glycolipid had to be coadministered with tumor cells i.v. rather than s.c. Specific resistance was generated both to a plasmacytoma and lymphoma. The resistance afforded by a single vaccination lasted >2 mo and required both CD4+ and CD8+ T cells. Mature tumor capturing DCs stimulated the differentiation of P1A tumor antigen-specific, CD8+ T cells and uniquely transferred tumor resistance to naive mice. Therefore, the access of dying tumor cells to DCs that are maturing to activated NKT cells efficiently induces long-lived adaptive resistance.

Split-inteins for simultaneous, site-specific conjugation of quantum dots to multiple protein targets in vivo.

BACKGROUND: Proteins labelled with Quantum Dots (QDs) can be imaged over long periods of time with ultrahigh spatial and temporal resolution, yielding important information on the spatiotemporal dynamics of proteins within live cells or in vivo. However one of the major problems regarding the use of QDs for biological imaging is the difficulty of targeting QDs onto proteins. We have recently developed a DnaE split intein-based method to conjugate Quantum Dots (QDs) to the C-terminus of target proteins in vivo. In this study, we expand this approach to achieve site-specific conjugation of QDs to two or more proteins simultaneously with spectrally distinguishable QDs for multiparameter imaging of cellular functions. RESULTS: Using the DnaE split intein we target QDs to the C-terminus of paxillin and show that paxillin-QD conjugates become localized at focal adhesions allowing imaging of the formation and dissolution of these complexes. We go on to utilize a different split intein, namely Ssp DnaB mini-intein, to demonstrate N-terminal protein tagging with QDs. Combination of these two intein systems allowed us to simultaneously target two distinct proteins with spectrally distinguishable QDs, in vivo, without any cross talk between the two intein systems. CONCLUSIONS: Multiple target labeling is a unique feature of the intein based methodology which sets it apart from existing tagging methodologies in that, given the large number of characterized split inteins, the number of individual targets that can be simultaneously tagged is only limited by the number of QDs that can be spectrally distinguished within the cell. Therefore, the intein-mediated approach for simultaneous, in vivo, site-specific (N- and C-terminus) conjugation of Quantum Dots to multiple protein targets opens up new possibilities for bioimaging applications and offers an effective system to target QDs and other nanostructures to intracellular compartments as well as specific molecular complexes.

In vivo targeting of antigens to maturing dendritic cells via the DEC-205 receptor improves T cell vaccination.

The prevention and treatment of prevalent infectious diseases and tumors should benefit from improvements in the induction of antigen-specific T cell immunity. To assess the potential of antigen targeting to dendritic cells to improve immunity, we incorporated ovalbumin protein into a monoclonal antibody to the DEC-205 receptor, an endocytic receptor that is abundant on these cells in lymphoid tissues. Simultaneously, we injected agonistic alpha-CD40 antibody to mature the dendritic cells. We found that a single low dose of antibody-conjugated ovalbumin initiated immunity from the naive CD4+ and CD8+ T cell repertoire. Unexpectedly, the alphaDEC-205 antigen conjugates, given s.c., targeted to dendritic cells systemically and for long periods, and ovalbumin peptide was presented on MHC class I for 2 weeks. This was associated with stronger CD8+ T cell-mediated immunity relative to other forms of antigen delivery, even when the latter was given at a thousand times higher doses. In parallel, the mice showed enhanced resistance to an established rapidly growing tumor and to viral infection at a mucosal site. By better harnessing the immunizing functions of maturing dendritic cells, antibody-mediated antigen targeting via the DEC-205 receptor increases the efficiency of vaccination for T cell immunity, including systemic and mucosal resistance in disease models.

Intein-mediated site-specific conjugation of Quantum Dots to proteins in vivo.

We describe an intein based method to site-specifically conjugate Quantum Dots (QDs) to target proteins in vivo. This approach allows the covalent conjugation of any nanostructure and/or nanodevice to any protein and thus the targeting of such material to any intracellular compartment or signalling complex within the cells of the developing embryo. We genetically fused a pleckstrin-homology (PH) domain with the N-terminus half of a split intein (IN). The C-terminus half (IC) of the intein was conjugated to QDs in vitro. IC-QD's and RNA encoding PH-IN were microinjected into Xenopus embryos. In vivo intein-splicing resulted in fully functional QD-PH conjugates that could be monitored in real time within live embryos. Use of Near Infra Red (NIR)-emitting QDs allowed monitoring of QD-conjugates within the embryo at depths where EGFP is undetectable demonstrating the advantages of QD's for this type of experiment. In conclusion, we have developed a novel in vivo methodology for the site-specific conjugation of QD's and other artificial structures to target proteins in different intracellular compartments and signaling complexes.

Determining Temporal and Spatial Expression of Calpains in Amphibians.

Calpains are a family of calcium-dependent intracellular cysteine proteases that regulate important physiological processes by substrate cleavage. Despite the fact that Calpains have been identified in the Xenopus genome, their expression patterns and role have not been characterized. Therefore, herein, we describe two methods to determine temporal and spatial expression of Calpain 2 during Xenopus development, namely, RT-PCR and whole-mount in situ hybridization (WISH). In addition, indirect immunofluorescence (IF) is described to determine translocation to the plasma membrane, which correlates with activity levels of Calpain 2.