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.

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.

Sortase-mediated modification of αDEC205 affords optimization of antigen presentation and immunization against a set of viral epitopes.

A monoclonal antibody against the C-type lectin DEC205 (αDEC205) is an effective vehicle for delivery of antigens to dendritic cells through creation of covalent αDEC205-antigen adducts. These adducts can induce antigen-specific T-cell immune responses or tolerance. We exploit the transpeptidase activity of sortase to install modified peptides and protein-sized antigens onto the heavy chain of αDEC205, including linkers that contain nonnatural amino acids. We demonstrate stoichiometric site-specific labeling on a scale not easily achievable by genetic fusions (49 distinct fusions in this report). We conjugated a biotinylated version of a class I MHC-restricted epitope to unlabeled αDEC205 and monitored epitope generation upon binding of the adduct to dendritic cells. Our results show transfer of αDEC205 heavy chain to the cytoplasm, followed by proteasomal degradation. Introduction of a labile dipeptide linker at the N terminus of a T-cell epitope improves proteasome-dependent class I MHC-restricted peptide cross-presentation when delivered by αDEC205 in vitro and in vivo. We also conjugated αDEC205 with a linker-optimized peptide library of known CD8 T-cell epitopes from the mouse γ-herpes virus 68. Animals immunized with such conjugates displayed a 10-fold reduction in viral load.

Intact sphingomyelin biosynthetic pathway is essential for intracellular transport of influenza virus glycoproteins.

Cells genetically deficient in sphingomyelin synthase-1 (SGMS1) or blocked in their synthesis pharmacologically through exposure to a serine palmitoyltransferase inhibitor (myriocin) show strongly reduced surface display of influenza virus glycoproteins hemagglutinin (HA) and neuraminidase (NA). The transport of HA to the cell surface was assessed by accessibility of HA on intact cells to exogenously added trypsin and to HA-specific antibodies. Rates of de novo synthesis of viral proteins in wild-type and SGMS1-deficient cells were equivalent, and HA negotiated the intracellular trafficking pathway through the Golgi normally. We engineered a strain of influenza virus to allow site-specific labeling of HA and NA using sortase. Accessibility of both HA and NA to sortase was blocked in SGMS1-deficient cells and in cells exposed to myriocin, with a corresponding inhibition of the release of virus particles from infected cells. Generation of influenza virus particles thus critically relies on a functional sphingomyelin biosynthetic pathway, required to drive influenza viral glycoproteins into lipid domains of a composition compatible with virus budding and release.

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.

Cholera toxin activates nonconventional adjuvant pathways that induce protective CD8 T-cell responses after epicutaneous vaccination.

The ability to induce humoral and cellular immunity via antigen delivery through the unbroken skin (epicutaneous immunization, EPI) has immediate relevance for vaccine development. However, it is unclear which adjuvants induce protective memory CD8 T-cell responses by this route, and the molecular and cellular requirements for priming through intact skin are not defined. We report that cholera toxin (CT) is superior to other adjuvants in its ability to prime memory CD8 T cells that control bacterial and viral challenges. Epicutaneous immunization with CT does not require engagement of classic toll-like receptor (TLR) and inflammasome pathways and, surprisingly, is independent of skin langerin-expressing cells (including Langerhans cells). However, CT adjuvanticity required type-I IFN sensitivity, participation of a Batf3-dependent dendritic cell (DC) population and engagement of CT with suitable gangliosides. Chemoenzymatic generation of CT-antigen fusion proteins led to efficient priming of the CD8 T-cell responses, paving the way for development of this immunization strategy as a therapeutic option.

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.

M13 bacteriophage display framework that allows sortase-mediated modification of surface-accessible phage proteins.

We exploit bacterial sortases to attach a variety of moieties to the capsid proteins of M13 bacteriophage. We show that pIII, pIX, and pVIII can be functionalized with entities ranging from small molecules (e.g., fluorophores, biotin) to correctly folded proteins (e.g., GFP, antibodies, streptavidin) in a site-specific manner, and with yields that surpass those of any reported using phage display technology. A case in point is modification of pVIII. While a phage vector limits the size of the insert into pVIII to a few amino acids, a phagemid system limits the number of copies actually displayed at the surface of M13. Using sortase-based reactions, a 100-fold increase in the efficiency of display of GFP onto pVIII is achieved. Taking advantage of orthogonal sortases, we can simultaneously target two distinct capsid proteins in the same phage particle and maintain excellent specificity of labeling. As demonstrated in this work, this is a simple and effective method for creating a variety of structures, thus expanding the use of M13 for materials science applications and as a biological tool.

Sortase A as a tool for high-yield histatin cyclization.

Cyclic peptides are highly valued tools in biomedical research. In many cases, they show higher receptor affinity, enhanced biological activity, and improved serum stability. Technical difficulties in producing cyclic peptides, especially larger ones, in appreciable yields have precluded a prolific use in biomedical research. Here, we describe a novel and efficient cyclization method that uses the peptidyl-transferase activity of the Staphylococcus aureus enzyme sortase A to cyclize linear synthetic precursor peptides. As a model, we used histatin 1, a 38-mer salivary peptide with motogenic activity. Chemical cyclization of histatin 1 resulted in ≤ 3% yields, whereas sortase-mediated cyclization provided a yield of >90%. The sortase-cyclized peptide displayed a maximum wound closure activity at 10 nM, whereas the linear peptide displayed maximal activity at 10 µM. Circular dichroism and NMR spectroscopic analysis of the linear and cyclic peptide in solution showed no evidence for conformational changes, suggesting that structural differences due to cyclization only became manifest when these peptides were located in the binding domain of the receptor. The sortase-based cyclization technology provides a general method for easy and efficient manufacturing of large cyclic peptides.

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.

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.

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.

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.

Dose-rate distribution of 32P-glass microspheres for intra-arterial brachytherapy.

PURPOSE: The intra-arterial administration of radioactive glass microspheres is an alternative therapy option for treating primary hepatocellular carcinoma, the main cause of liver cancer death, and metastatic liver cancer, another important kind of cancer induced in the liver. The technique involves the administration of radioactive microspheres in the hepatic artery, which are trapped preferentially in the tumor. METHODS: In this work the GEANT4 toolkit was used to calculate the radial dose-rate distributions in water from 32P-loaded glass microspheres and also from 90Y-loaded glass microspheres. To validate the toolkit for this application, the authors compared the dose-rate distribution of 32P and 90Y point sources in water with data from the International Commission on Radiation Units and Measurements report 72. RESULTS: Tables of radial dose-rate distributions are provided for practical use in brachytherapy planning with these microspheres. CONCLUSIONS: The simulations with the microspheres show that the shape of the beta ray energy spectra with respect to the 32P and 90Y sources is significantly modified by the glass matrix.

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.

Site-specific N- and C-terminal labeling of a single polypeptide using sortases of different specificity.

The unique reactivity of two sortase enzymes, SrtA(staph) from Staphylococcus aureus and SrtA(strep) from Streptococcus pyogenes, is exploited for site-specific labeling of a single polypeptide with different labels at its N and C termini. SrtA(strep) is used to label the protein's C terminus at an LPXTG site with a fluorescently labeled dialanine nucleophile. Selective N-terminal labeling of proteins containing N-terminal glycine residues is achieved using SrtA(staph) and LPXT derivatives. The generality of N-terminal labeling with SrtA(staph) is demonstrated by near-quantitative labeling of multiple protein substrates with excellent site specificity.

Physical models, cross sections, and numerical approximations used in MCNP and GEANT4 Monte Carlo codes for photon and electron absorbed fraction calculation.

PURPOSE: Radiopharmaceutical applications in nuclear medicine require a detailed dosimetry estimate of the radiation energy delivered to the human tissues. Over the past years, several publications addressed the problem of internal dose estimate in volumes of several sizes considering photon and electron sources. Most of them used Monte Carlo radiation transport codes. Despite the widespread use of these codes due to the variety of resources and potentials they offered to carry out dose calculations, several aspects like physical models, cross sections, and numerical approximations used in the simulations still remain an object of study. Accurate dose estimate depends on the correct selection of a set of simulation options that should be carefully chosen. This article presents an analysis of several simulation options provided by two of the most used codes worldwide: MCNP and GEANT4. METHODS: For this purpose, comparisons of absorbed fraction estimates obtained with different physical models, cross sections, and numerical approximations are presented for spheres of several sizes and composed as five different biological tissues. RESULTS: Considerable discrepancies have been found in some cases not only between the different codes but also between different cross sections and algorithms in the same code. Maximum differences found between the two codes are 5.0% and 10%, respectively, for photons and electrons. CONCLUSION: Even for simple problems as spheres and uniform radiation sources, the set of parameters chosen by any Monte Carlo code significantly affects the final results of a simulation, demonstrating the importance of the correct choice of parameters in the simulation.

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.