Validation of the Equidyn protocol for evaluation of dynamic balance in older adults through a smartphone application.

BACKGROUND: Different tasks and proxy measurements have been employed to evaluate dynamic balance in older individuals. However, due to inherent limitations, results from most evaluations could hardly be taken as valid measurements of dynamic balance. RESEARCH QUESTION: Is the Equidyn smartphone application-based protocol valid and sensitive for assessment of dynamic balance in older adults? METHODS: Dynamic balance was evaluated in 52 physically active individuals, age range 60-80 years (M = 69.36). The dynamic tasks were one-leg sway either in the mediolateral (ML) or anteroposterior (AP) direction while supported on the contralateral leg, and cyclic sit-to-stand with a narrow support base. These tasks were performed under standardized movement amplitude and rhythm. Outcomes were correlated with unipedal quiet standing. A smartphone was attached to the trunk backside, and a custom-made application (Equidyn) was employed to provide guidance throughout evaluation, timed beeps to pace the movements, and three-dimensional trunk acceleration measurement for balance evaluation. RESULTS: Our data showed (a) that both ML and AP leg sway tasks were sensitive to aging and to direction of leg sway movements; (b) referenced to quiet unipedal stance, moderate/strong correlations for the ML/AP leg sway tasks and moderate correlations for the sit-to-stand task; and (c) moderate/strong correlations between the ML and AP leg sway tasks, and moderate correlations between the sit-to-stand and the two unipedal dynamic tasks in the ML acceleration direction. SIGNIFICANCE: The current results support the conclusion that the Equidyn protocol is a sensitive and valid tool to evaluate dynamic balance in healthy older individuals. The protocol tasks standardized in amplitude and rhythm favor their reproducibility and trunk acceleration data interpretation. As the whole assessment is made through a smartphone application, this dynamic balance evaluation could be made in a low-cost simple way both in the laboratory and clinical settings.

Relationship Between Aerobic Capacity, Mobility, and Spatial Navigation in Healthy Individuals and Older Adults With Mild Cognitive Impairment: A Cross-Sectional Study.

This study aimed to investigate the relationship between physical ability and spatial navigation in older adults with mild cognitive impairment and healthy controls, using the floor maze test. Study participants (n = 58) were subjected to the following tests: floor maze test, sit-to-stand, 8-foot up-and-go, and aerobic steps. Factorial analyses showed that performance of the physical tests combined explained approximately 87% of the sample variability. Mobility (R2 = .22, p ≤ .001) and aerobic capacity (R2 = .27, p ≤ .001) were both associated with delayed maze time in the floor maze test. Low levels of aerobic capacity were also associated with an increased odds to perform poorly in the delayed maze time after controlling for age, sex, and mild cognitive impairment diagnosis (odds ratio = 3.1; 95% confidence interval [1.0, 9.5]; p = .04). Aerobic capacity and mobility are associated with spatial navigation in patients with mild cognitive impairment and healthy older adults.

Beyond the Mini-Mental State Examination: The Use of Physical and Spatial Navigation Tests to Help to Screen for Mild Cognitive Impairment and Alzheimer's Disease.

BACKGROUND: Spatial navigation and dual-task (DT) performance may represent a low-cost approach to the identification of the cognitive decline in older adults and may support the clinical diagnosis of mild cognitive impairment (MCI) and Alzheimer's disease (AD). OBJECTIVE: To assess the accuracy of different types of motor tasks in differentiating older persons with MCI and AD from healthy peers. METHODS: Older adults aged 60 years or over (n = 105; healthy = 39; MCI = 23; AD = 43) were evaluated by the floor maze test (FMT), the senior fitness test, and DT performance. Receiver operating characteristic curve (ROC) analysis was used to evaluate the accuracy of the tests. We also performed principal component analysis (PCA) and logistic regression analysis to explore the variance and possible associations of the variables within the sample. RESULTS: FMT (AUC = 0.84, sensitivity = 75.7%, specificity = 76.1%, p < 0.001) and DT (AUC = 0.87, sensitivity = 80.4%, specificity = 86.9%, p < 0.001) showed the highest performance for distinguishing MCI from AD individuals. Moreover, FMT presented better sensitivity in distinguishing AD patients from their healthy peers (AUC = 0.93, sensitivity = 94%, specificity = 85.6%, p < 0.001) when compared to the Mini-Mental State Examination. PCA revealed that the motor test performance explains a total of 73.9% of the variance of the sample. Additionally, the results of the motor tests were not influenced by age and education. CONCLUSION: Spatial navigation tests showed better accuracy than usual cognitive screening tests in distinguishing patients with neurocognitive disorders.

Hidden by the name: A new fluorescent pumpkin toadlet from the Brachycephalus ephippium group (Anura: Brachycephalidae).

Species of Brachycephalus has been having taxonomical issues due its morphological similarity and genetic conservatism. Herein, we describe a new species of Brachycephalus from the south Mantiqueira mountain range and semidecidual forests in the municipalities of Mogi das Cruzes, Campinas and Jundiaí, state of São Paulo, Brazil, based on an integrative approach. It can be distinguished from all species of the B. ephippium species group based on morphological characters (especially osteology and head shape), advertisement call and divergence in partial mitochondrial DNA gene sequences (16S). The new species is genetically similar to B. margaritatus and morphologically similar to B. ephippium. It can be differentiated from B. ephippium by the presence of dark faded spots on skull and post-cranial plates, presence of black connective tissue connective tissue scattered over dorsal musculature, parotic plate morphology, smaller snout-vent length (adult SVL: males 13.46-15.92 mm; females 16.04-17.69 mm) and 3% genetic distance. We also present natural history data and discuss the robustness of the integrative approach, geographic distribution, genetic data, behaviour, fluorescence in ontogeny, and conservation status.

Centesimal composition and meat yield of Hoplias malabaricus: association with intestinal parasites.

Hoplias malabaricus is a non-migratory fish commonly found in the Mogi Guaçu River basin, mainly feeding on fish, small crustaceans and insects. It forms part of the diet for humans, birds and some mammals. This fish has great nutritional value, with both good quality and good quantities of essential vitamins and amino acids. Regarding parasitic fauna, this fish can host different species of helminths in its gastrointestinal tract. The aim of the present study was to investigate the possible interference of parasitism in the meat yield from H. malabaricus and the centesimal composition. For this purpose, fish specimens were collected from marginal lagoons of the Mogi Guaçu River (Pirassununga, state of São Paulo, Brazil) using hooks and fishing nets. We found that all specimens of H. malabaricus were parasitized by at least one species, including larvae of Contracaecum sp. (Nematoda: Anisakidae). Parasitism did not have any significant influence on centesimal composition, but meat yield was negatively correlated with the abundance of larvae.

An IMiD-inducible degron provides reversible regulation for chimeric antigen receptor expression and activity.

The recent development of successful CAR (chimeric antigen receptor) T cell therapies has been accompanied by a need to better control potentially fatal toxicities that can arise from adverse immune reactions. Here we present a ligand-controlled CAR system, based on the IKZF3 ZF2 ß-hairpin IMiD-inducible degron, which allows for the reversible control of expression levels of type I membrane proteins, including CARs. Testing this system in an established mouse xenotransplantation model for acute lymphoblastic leukemia, we validate the ability of the CAR19-degron to target and kill CD19-positive cells displaying complete control/clearance of the tumor. We also demonstrate that the activity of CAR19-degron can be regulated in vivo when dosing a US Food and Drug Administration-approved drug, lenalidomide.

Gait analysis with videogrammetry can differentiate healthy elderly, mild cognitive impairment, and Alzheimer's disease: A cross-sectional study.

Gait parameters have been investigated as an additional tool for differential diagnosis in neurocognitive disorders, especially among healthy elderly (HE), those with mild cognitive impairment (MCI), and Alzheimer's disease (AD) patients. A videogrammetry system could be used as a low-cost and clinically practical equipment to capture and analyze gait in older adults. The aim of this study was to select the better gait parameter to differentiate these groups among different motor test conditions with videogrammetry analyses. Different motor conditions were used in three specific assessments: 10-meter walk test (10mWT), timed up and go test (TUGT), and treadmill walk test (TWT). These tasks were compared among HE (n=17), MCI (n=23), and AD (n=23) groups. One-way ANOVA, Kruskal-Wallis, and Bonferroni post-hoc tests were used to compare variables among groups. Then, an effect size (ES) and a linear regression analysis were calculated. The gait parameters showed significant differences among groups in all conditions, but not in TWT. Controlled by confounding variables, the gait velocity in 10mWT at usual speed, and TUGT in dual-task condition, predicts 39% and 53% of the difference among diagnoses, respectively. Finally, these results suggest that a low-cost and practical video analysis could be able to differentiate HE, those with MCI, and AD patients in clinical assessments.

Stages of mild cognitive impairment and Alzheimer's disease can be differentiated by declines in timed up and go test: A systematic review and meta-analysis.

Motor dysfunction increases in the moderate and severe stages of dementia. However, there is still no consensus on changes in mobility during its early stages. This meta-analysis aimed to measure the level of single-task functional mobility in older subjects with mild cognitive impairment (MCI) and/or Alzheimer's disease (AD). In a search of the PubMed, ISI Web of Knowledge, and Scopus databases, 2728 articles were identified. At the end of the selection, a total of 18 studies were included in the meta-analysis. Functional mobility was investigated using the timed up and go (TUG) test in all studies. When compared to healthy elderly (HE) adults, the following mean differences (MD) in seconds were found for the investigated subgroups: no amnestic MCI (MD = 0.26; CI95% = -0.77, 1.29), amnestic MCI (MD = 0.86; CI95% = -0.02, 1.73), very mild AD (MD = 1.32; CI95% = 0.63, 2.02), mild AD (MD = 2.43; CI95% = 1.84, 3.01), mild-moderate AD (MD = 3.01; CI95% = 2.47, 3.55), and mild-severe AD (MD = 4.51; CI95% = 1.14, 7.88); for the groups, the following MD were found: MCI (MD = 0.97; CI95% = 0.51, 1.44) and AD (MD = 2.66; CI95% = 2.16, 3.15). These results suggest a transition period in motor capacity between healthy aging and dementia, wherein functional mobility analysis in a single-task (TUG) can contribute to the diagnosis and staging of predementia states and AD.

The dark side of pumpkin toadlet: a new species of Brachycephalus (Anura: Brachycephalidae) from Serra do Brigadeiro, southeastern Brazil.

Brachycephalus is a frog genus endemic to the Brazilian Atlantic Forest and characterized by the bright yellow-orange aposematic colors and the high degree of miniaturization. Herein, we describe a new species of Brachycephalus from Serra do Brigadeiro, Municipality of Ervália, Minas Gerais State, southeastern Brazil. Specimens were collected at high altitudes (i.e., 1266-1498 m above sea level) amidst the leaf litter. The new species is characterized by the presence of black connective tissue covering all dorsal muscles, body completely yellow-orange in life, presence of skull and post-cranial plates, large size (SVL of adults: 14.8-18.5 mm), bufoniform body, absence of metacarpal and metatarsal tubercles, and presence of harmonics in its advertisement call.

Site-specific protein modification using immobilized sortase in batch and continuous-flow systems.

Transpeptidation catalyzed by sortase A allows the preparation of proteins that are site-specifically and homogeneously modified with a wide variety of functional groups, such as fluorophores, PEG moieties, lipids, glycans, bio-orthogonal reactive groups and affinity handles. This protocol describes immobilization of sortase A on a solid support (Sepharose beads). Immobilization of sortase A simplifies downstream purification of a protein of interest after labeling of its N or C terminus. Smaller batch and larger-scale continuous-flow reactions require only a limited amount of enzyme. The immobilized enzyme can be reused for multiple cycles of protein modification reactions. The described protocol also works with a Ca(2+)-independent variant of sortase A with increased catalytic activity. This heptamutant variant of sortase A (7M) was generated by combining previously published mutations, and this immobilized enzyme can be used for the modification of calcium-senstive substrates or in instances in which low temperatures are needed. Preparation of immobilized sortase A takes 1-2 d. Batch reactions take 3-12 h and flow reactions proceed at 0.5 ml h(-1), depending on the geometry of the reactor used.

Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation.

While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%-99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a ∼1,000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retrotranslocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways.

Site-specific chemoenzymatic labeling of aerolysin enables the identification of new aerolysin receptors.

Aerolysin is a secreted bacterial toxin that perforates the plasma membrane of a target cell with lethal consequences. Previously explored native and epitope-tagged forms of the toxin do not allow site-specific modification of the mature toxin with a probe of choice. We explore sortase-mediated transpeptidation reactions (sortagging) to install fluorophores and biotin at three distinct sites in aerolysin, without impairing binding of the toxin to the cell membrane and with minimal impact on toxicity. Using a version of aerolysin labeled with different fluorophores at two distinct sites we followed the fate of the C-terminal peptide independently from the N-terminal part of the toxin, and show its loss in the course of intoxication. Making use of the biotinylated version of aerolysin, we identify mesothelin, urokinase plasminogen activator surface receptor (uPAR, CD87), glypican-1, and CD59 glycoprotein as aerolysin receptors, all predicted or known to be modified with a glycosylphosphatidylinositol anchor. The sortase-mediated reactions reported here can be readily extended to other pore forming proteins.

GPR107, a G-protein-coupled receptor essential for intoxication by Pseudomonas aeruginosa exotoxin A, localizes to the Golgi and is cleaved by furin.

A number of toxins, including exotoxin A (PE) of Pseudomonas aeruginosa, kill cells by inhibiting protein synthesis. PE kills by ADP-ribosylation of the translation elongation factor 2, but many of the host factors required for entry, membrane translocation, and intracellular transport remain to be elucidated. A genome-wide genetic screen in human KBM7 cells was performed to uncover host factors used by PE, several of which were confirmed by CRISPR/Cas9-gene editing in a different cell type. Several proteins not previously implicated in the PE intoxication pathway were identified, including GPR107, an orphan G-protein-coupled receptor. GPR107 localizes to the trans-Golgi network and is essential for retrograde transport. It is cleaved by the endoprotease furin, and a disulfide bond connects the two cleaved fragments. Compromising this association affects the function of GPR107. The N-terminal region of GPR107 is critical for its biological function. GPR107 might be one of the long-sought receptors that associates with G-proteins to regulate intracellular vesicular transport.

A natural bacterial-derived product, the metalloprotease arazyme, inhibits metastatic murine melanoma by inducing MMP-8 cross-reactive antibodies.

The increased incidence, high rates of mortality and few effective means of treatment of malignant melanoma, stimulate the search for new anti-tumor agents and therapeutic targets to control this deadly metastatic disease. In the present work the antitumor effect of arazyme, a natural bacterial-derived metalloprotease secreted by Serratia proteomaculans, was investigated. Arazyme significantly reduced the number of pulmonary metastatic nodules after intravenous inoculation of B16F10 melanoma cells in syngeneic mice. In vitro, the enzyme showed a dose-dependent cytostatic effect in human and murine tumor cells, and this effect was associated to the proteolytic activity of arazyme, reducing the CD44 expression at the cell surface, and also reducing in vitro adhesion and in vitro/in vivo invasion of these cells. Arazyme treatment or immunization induced the production of protease-specific IgG that cross-reacted with melanoma MMP-8. In vitro, this antibody was cytotoxic to tumor cells, an effect increased by complement. In vivo, arazyme-specific IgG inhibited melanoma lung metastasis. We suggest that the antitumor activity of arazyme in a preclinical model may be due to a direct cytostatic activity of the protease in combination with the elicited anti-protease antibody, which cross-reacts with MMP-8 produced by tumor cells. Our results show that the bacterial metalloprotease arazyme is a promising novel antitumor chemotherapeutic agent.

A meta-analysis of the effects of fragmentation on herbivorous insects.

We reviewed the evidence for the effects of fragmentation on insects and plants by conducting a meta-analysis for the effects of artificial forest edge formation on insect herbivore abundance, herbivore richness, and plant herbivory, with data pooled from 31 studies and 159 independent comparisons. Hedge's d was used as the metric to combine all studies. Edge formation exhibited strong effects on plant herbivory rates, as edge plants exhibited 70% more damage than interior plants. Edges also increased herbivore abundance by 14% and herbivore richness by almost 65%, and effects of edge formation were stronger for Lepidoptera (mainly caterpillars) and Orthoptera. Edge effects were also stronger for forested ecosystems compared with open habitats and for temperate regions. Because the studies here evaluated did not simultaneously evaluate bottom-up and top-down factors, the mechanisms responsible for the patterns found cannot be properly addressed, although variation in host plant chemistry, relaxation of pressure exerted by natural enemies, or both, can be suggested as potential factors explaining variation in herbivory between edge and interior habitats. Higher herbivory rates on edge habitats, as shown by our meta-analytical review, have the potential to alter community composition and should be studied in detail to unravel their effects on ecosystem functioning.

Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions.

Methods for site-specific modification of proteins should be quantitative and versatile with respect to the nature and size of the biological or chemical targets involved. They should require minimal modification of the target, and the underlying reactions should be completed in a reasonable amount of time under physiological conditions. Sortase-mediated transpeptidation reactions meet these criteria and are compatible with other labeling methods. Here we describe the expression and purification conditions for two sortase A enzymes that have different recognition sequences. We also provide a protocol that allows the functionalization of any given protein at its C terminus, or, for select proteins, at an internal site. The target protein is engineered with a sortase-recognition motif (LPXTG) at the place where modification is desired. Upon recognition, sortase cleaves the protein between the threonine and glycine residues, facilitating the attachment of an exogenously added oligoglycine peptide modified with the functional group of choice (e.g., fluorophore, biotin, protein or lipid). Expression and purification of sortase takes ∼3 d, and sortase-mediated reactions take only a few minutes, but reaction times can be extended to increase yields.

Site-specific N-terminal labeling of proteins using sortase-mediated reactions.

This protocol describes the use of sortase-mediated reactions to label the N terminus of any given protein of interest. The sortase recognition sequence, LPXTG (for Streptococcus aureus sortase A) or LPXTA (for Staphylococcus pyogenes sortase A), can be appended to a variety of probes such as fluorophores, biotin or even to other proteins. The protein to be labeled acts as a nucleophile by attacking the intermediate formed between the probe containing the LPXTG/A motif and the sortase enzyme. If sortase, the protein of interest and a suitably functionalized label are available, the reactions usually require less than 3 h.