CRISPR-Cas9 a boon or bane: the bumpy road ahead to cancer therapeutics.

Genome editing allows for the precise manipulation of DNA sequences in a cell making this technology essential for understanding gene function. CRISPR/Cas9 is a targeted genome-editing platform derived from bacterial adaptive immune system and has been repurposed into a genome-editing tool. The RNA-guided DNA endonuclease, Cas9 can be easily programmed to target new sites by altering its guide RNA sequence, making this technology easier, more efficient, scalable and an indispensable tool in biological research. This technology has helped genetically engineer animal models to understand disease mechanisms and elucidate molecular details that can be exploited for improved therapeutic outcomes. In this review, we describe the CRISPR-Cas9 gene-editing mechanism, CRISPR-screening methods, therapeutic targeting of CRISPR in animal models and in cancer immunotherapy. We also discuss the ongoing clinical trials using this tool, limitations of this tool that might impede the clinical applicability of CRISPR-Cas9 and future directions for developing effective CRISPR-Cas9 delivery systems that may improve cancer therapeutics.

Networks of coupled oscillators: From phase to amplitude chimeras.

We show that amplitude-mediated phase chimeras and amplitude chimeras can occur in the same network of nonlocally coupled identical oscillators. These are two different partial synchronization patterns, where spatially coherent domains coexist with incoherent domains and coherence/incoherence referring to both amplitude and phase or only the amplitude of the oscillators, respectively. By changing the coupling strength, the two types of chimera patterns can be induced. We find numerically that the amplitude chimeras are not short-living transients but can have a long lifetime. Also, we observe variants of the amplitude chimeras with quasiperiodic temporal oscillations. We provide a qualitative explanation of the observed phenomena in the light of symmetry breaking bifurcation scenarios. We believe that this study will shed light on the connection between two disparate chimera states having different symmetry-breaking properties.

Assessing the cellulase enzyme heterogeneity of bacterial strains and their feedback to cattle manure degradation in a greenhouse model of in vivo pond ecosystem.

The responses of cellulase enzymes of three bacterial isolates and their impacts on cattle manure decomposition were assessed in a greenhouse model in vivo pond ecosystem. Fifty grams of fresh cattle manure was placed in a fastened nylon bag (mesh size ~ 50 µm dia.) and placed in triplicate in a plastic bucket with 10 l of pond water which was hung inside the enclosed polyhouse, semi-closed and open systems for 4 weeks. Samples of manure residue directly from nylon bag and water from manure leached bucket water, water, and soil from the enclosed polyhouse were collected for enzymatic assays, enumeration of aerobic cellulose decomposing and heterotrophic bacteria, and determination of water and soil quality parameters. Responses of cellulases to different temperatures in situ were also elucidated. The values of test bacteria, endoglucanase, exoglucanase and ß-glucosidase, and organic carbon were significantly (P Ë‚ 0.05) higher in the closed system compared to semi-closed or open system. Priming of all the enzymes coupled with the peak of aerobic cellulose decomposing bacteria and heterotrophic bacterial populations occurred on the day 14 or 21 in vivo. Since the peaks of three cellulases of bacterial isolates (KUPH1, KUPH6, and KUPH8) were demonstrated between 35 and 40 °C, and that temperature coincided with temperature of the greenhouse model, this temperature range appeared to favor the growth of cellulose decomposing bacterial populations and involved cellulase enzymes.

Experimental evidence for quantum cutting co-operative energy transfer process in Pr3+/Yb3+ ions co-doped fluorotellurite glass: dispute over energy transfer mechanism.

Pr3+/Yb3+ doped materials have been widely reported as quantum-cutting materials in recent times. However, the question of the energy transfer mechanism in the Pr3+/Yb3+ pair in light of the quantum-cutting phenomenon still remains unanswered. In view of that, we explored a series of Pr3+/Yb3+ co-doped low phonon fluorotellurite glass systems to estimate the probability of different energy transfer mechanisms. Indeed, a novel and simple way to predict the probability of the proper energy transfer mechanism in the Pr3+/Yb3+ pair is possible by considering the donor Pr3+ ion emission intensities and the relative ratio dependence in the presence of acceptor Yb3+ ions. Moreover, the observed results are very much in accordance with other estimated results that support the quantum-cutting phenomena in Pr3+/Yb3+ pairs, such as sub-linear power dependence of Yb3+ NIR emission upon visible ∼450 nm laser excitation, integrated area of the donor Pr3+ ion's visible excitation spectrum recorded by monitoring the acceptor Yb3+ ion's NIR emission, and the experimentally obtained absolute quantum yield values using an integrating sphere setup. Our results give a simple way of estimating the probability of an energy transfer mechanism and the factors to be considered, particularly for the Pr3+/Yb3+ pair.

Cellular signaling in glioblastoma: A molecular and clinical perspective.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor with an average life expectancy of less than 15 months. Such high patient mortality in GBM is pertaining to the presence of clinical and molecular heterogeneity attributed to various genetic and epigenetic alterations. Such alterations in critically important signaling pathways are attributed to aberrant gene signaling. Different subclasses of GBM show predominance of different genetic alterations and therefore, understanding the complex signaling pathways and their key molecular components in different subclasses of GBM is extremely important with respect to clinical management. In this book chapter, we summarize the common and important signaling pathways that play a significant role in different subclasses and discuss their therapeutic targeting approaches in terms of preclinical studies and clinical trials.

Improved immunostaining of nanostructures and cells in human brain specimens through expansion-mediated protein decrowding.

Proteins are densely packed in cells and tissues, where they form complex nanostructures. Expansion microscopy (ExM) variants have been used to separate proteins from each other in preserved biospecimens, improving antibody access to epitopes. Here, we present an ExM variant, decrowding expansion pathology (dExPath), that can expand proteins away from each other in human brain pathology specimens, including formalin-fixed paraffin-embedded (FFPE) clinical specimens. Immunostaining of dExPath-expanded specimens reveals, with nanoscale precision, previously unobserved cellular structures, as well as more continuous patterns of staining. This enhanced molecular staining results in observation of previously invisible disease marker-positive cell populations in human glioma specimens, with potential implications for tumor aggressiveness. dExPath results in improved fluorescence signals even as it eliminates lipofuscin-associated autofluorescence. Thus, this form of expansion-mediated protein decrowding may, through improved epitope access for antibodies, render immunohistochemistry more powerful in clinical science and, perhaps, diagnosis.

Paper analytical devices for fast field screening of beta lactam antibiotics and antituberculosis pharmaceuticals.

Reports of low-quality pharmaceuticals have been on the rise in the past decade, with the greatest prevalence of substandard medicines in developing countries, where lapses in manufacturing quality control or breaches in the supply chain allow substandard medicines to reach the marketplace. Here, we describe inexpensive test cards for fast field screening of pharmaceutical dosage forms containing beta lactam antibiotics or combinations of the four first-line antituberculosis (TB) drugs. The devices detect the active pharmaceutical ingredients (APIs) ampicillin, amoxicillin, rifampicin, isoniazid, ethambutol, and pyrazinamide and also screen for substitute pharmaceuticals, such as acetaminophen and chloroquine that may be found in counterfeit pharmaceuticals. The tests can detect binders and fillers such as chalk, talc, and starch not revealed by traditional chromatographic methods. These paper devices contain 12 lanes, separated by hydrophobic barriers, with different reagents deposited in the lanes. The user rubs some of the solid pharmaceutical across the lanes and dips the edge of the paper into water. As water climbs up the lanes by capillary action, it triggers a library of different chemical tests and a timer to indicate when the tests are completed. The reactions in each lane generate colors to form a "color bar code" which can be analyzed visually by comparison with standard outcomes. Although quantification of the APIs is poor compared with conventional analytical methods, the sensitivity and selectivity for the analytes is high enough to pick out suspicious formulations containing no API or a substitute API as well as formulations containing APIs that have been "cut" with inactive ingredients.

Electron Transfer Dynamics from CsPbBr3 Nanocrystals to Au144 Clusters.

Lead halide perovskite nanocrystals have received significant attention as an absorber material for designing efficient optoelectronic devices. The fundamental understanding of the hot carrier (HC) dynamics as well as its extraction in hybrid systems is essential to further boost the performance of solar cells. Herein, we have explored the electron transfer dynamics in the CsPbBr3-Au144 cluster hybrid using ultrafast transient absorption spectroscopy. Our analysis reveals faster HC cooling time (from 515 to 334 fs) and a significant drop in HC temperature from 1055 to 860 K in hybrid, suggesting the hot electron transfer from CsPbBr3 nanocrystals to the Au nanoclusters (NCs). Eventually, we observe a much faster hot electron transfer compared to the band-edge electron transfer, and 45% hot-electron transfer efficiency was achieved at 0.64 eV, above band-edge photoexcitation. Furthermore, the significant enhancement of the photocurrent to the dark current ratio in this hybrid system confirms the charge separation via the electron transfer from CsPbBr3 nanocrystals to Au144 NCs. These findings on HC dynamics could be beneficial for optoelectronic devices.

Discerning non-disjunction in Down syndrome patients by means of GluK1-(AGAT)(n) and D21S2055-(GATA)(n) microsatellites on chromosome 21.

INTRODUCTION: Down syndrome (DS), the leading genetic cause of mental retardation, stems from non-disjunction of chromosome 21. AIM: Our aim was to discern non-disjunction in DS patients by genotyping GluK1-(AGAT)(n) and D21S2055-(GATA)(n) microsatellites on chromosome 21 using a family-based study design. MATERIALS AND METHODS: We have used a PCR and automated DNA sequencing followed by appropriate statistical analysis of genotype data for the present study RESULTS AND DISCUSSION: We show that a high power of discrimination and a low probability of matching indicate that both markers may be used to distinguish between two unrelated individuals. That the D21S2055-(GATA)(n) allele distribution is evenly balanced, is indicated by a high power of exclusion [PE=0.280]. The estimated values of observed heterozygosity and polymorphism information content reveal that relative to GluK1-(AGAT)(n)[H(obs)=0.286], the D21S2055- (GATA)(n)[H(obs)=0.791] marker, is more informative. Though allele frequencies for both polymorphisms do not conform to Hardy-Weinberg equilibrium proportions, we were able to discern the parental origin of non-disjunction and also garnered evidence for triallelic (1:1:1) inheritance. The estimated proportion of meiosis-I to meiosis-II errors is 2:1 in maternal and 4:1 in paternal cases for GluK1-(AGAT)(n), whereas for D21S2055-(GATA)(n), the ratio is 2:1 in both maternal and paternal cases. Results underscore a need to systematically evaluate additional chromosome 21-specific markers in the context of non-disjunction DS.

No Evidence for Mutations that Deregulate GARS-AIRS-GART Protein Levels in Children with Down Syndrome.

UNLABELLED: GARS-AIRS-GART is crucial in studies of Down syndrome (DS)-related mental retardation due to its chromosomal location (21q22.1), involvement in de novo purine biosynthesis and over-expression in fetal DS brain postmortem samples. GARS-AIRS-GART regions important for structure-function were screened for mutations that might alter protein levels in DS patients. Mutation screening relied on multiplex/singleplex PCR-based amplification of genomic targets followed by amplicon size determination/fingerprinting. Serum protein samples were resolved by SDS-PAGE and immunoblotted with a GARS-AIRS-GART monoclonal antibody. No variation in amplicon size/fingerprints was observed in regions encoding the ATP-binding, active site residues of GARS, the structurally important glycine-rich loops of AIRS, substrate-binding, flexible and folate-binding loops of GART or the poly-adenylation signal sequences. The de novo occurrence or inheritance of large insertion/deletion/rearrangement-type mutations is therefore excluded. Immunoblots show presence of GARS-AIRS-GART protein in all patient samples, with no change in expression levels with respect to either sex or developmental age. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12291-011-0183-6) contains supplementary material, which is available to authorized users.

Drug repurposing for stroke intervention.

Despite the availability of advanced interventions, stroke remains one of the most significant causes of mortality and morbidity worldwide. US Food and Drug Administration (FDA)-approved treatment options for stroke include tissue plasminogen activators (tPAs) and mechanical thrombectomy (MT). However, these are limited by a narrow therapeutic time window. Additionally, poststroke rehabilitation therapies can provide functional recovery but take a long time to show benefits. Drug repurposing could be a novel approach to broaden treatment options in this scenario. In this review, we summarize marketed drugs that could be repurposed based on their safety and efficacy data. We also briefly discuss their mechanisms of action and provide a list of repurposed drugs under trials for ischemic stroke therapy.

Combination therapy to checkmate Glioblastoma: clinical challenges and advances.

Combination therapy is increasingly becoming the cornerstone of current day antitumor therapy. Glioblastoma multiforme is an aggressive brain tumor with a dismal median survival post diagnosis and a high rate of disease recurrence. The poor prognosis can be attributed to unique treatment limitations, which include the infiltrative nature of tumor cells, failure of anti-glioma drugs to cross the blood-brain barrier, tumor heterogeneity and the highly metastatic and angiogenic nature of the tumor making cells resistant to chemotherapy. Combination therapy approach is being developed against glioblastoma with new innovative combination drug regimens being tested in preclinical and clinical trials. In this review, we discuss the pathophysiology of glioblastoma, diagnostic markers, therapeutic targeting strategies, current treatment limitations, novel combination therapies in the context of current treatment options and the ongoing clinical trials for glioblastoma therapy.

Transition from homogeneous to inhomogeneous limit cycles: Effect of local filtering in coupled oscillators.

We report an interesting symmetry-breaking transition in coupled identical oscillators, namely, the continuous transition from homogeneous to inhomogeneous limit cycle oscillations. The observed transition is the oscillatory analog of the Turing-type symmetry-breaking transition from amplitude death (i.e., stable homogeneous steady state) to oscillation death (i.e., stable inhomogeneous steady state). This novel transition occurs in the parametric zone of occurrence of rhythmogenesis and oscillation death as a consequence of the presence of local filtering in the coupling path. We consider paradigmatic oscillators, such as Stuart-Landau and van der Pol oscillators, under mean-field coupling with low-pass or all-pass filtered self-feedback and through a rigorous bifurcation analysis we explore the genesis of this transition. Further, we experimentally demonstrate the observed transition, which establishes its robustness in the presence of parameter fluctuations and noise.

Role of Yb(3+) ions on enhanced ~2.9 µm emission from Ho(3+) ions in low phonon oxide glass system.

The foremost limitation of an oxide based crystal or glass host to demonstrate mid- infrared emissions is its high phonon energy. It is very difficult to obtain radiative mid-infrared emissions from these hosts which normally relax non-radiatively between closely spaced energy levels of dopant rare earth ions. In this study, an intense mid-infrared emission around 2.9 µm has been perceived from Ho(3+) ions in Yb(3+)/Ho(3+) co-doped oxide based tellurite glass system. This emission intensity has increased many folds upon Yb(3+): 985 nm excitation compared to direct Ho(3+) excitations due to efficient excited state resonant energy transfer through Yb(3+): (2)F5/2 → Ho(3+): (5)I5 levels. The effective bandwidth (FWHM) and cross-section (σem) of measured emission at 2.9 µm are assessed to be 180 nm and 9.1 × 10(-21) cm(2) respectively which are comparable to other crystal/glass hosts and even better than ZBLAN fluoride glass host. Hence, this Ho(3+)/Yb(3+) co-doped oxide glass system has immense potential for the development of solid state mid-infrared laser sources operating at 2.9 µm region.

Mixed-mode oscillation suppression states in coupled oscillators.

We report a collective dynamical state, namely the mixed-mode oscillation suppression state where the steady states of the state variables of a system of coupled oscillators show heterogeneous behaviors. We identify two variants of it: The first one is a mixed-mode death (MMD) state, which is an interesting oscillation death state, where a set of variables show dissimilar values, while the rest arrive at a common value. In the second mixed death state, bistable and monostable nontrivial homogeneous steady states appear simultaneously to a different set of variables (we refer to it as the MNAD state). We find these states in the paradigmatic chaotic Lorenz system and Lorenz-like system under generic coupling schemes. We identify that while the reflection symmetry breaking is responsible for the MNAD state, the breaking of both the reflection and translational symmetries result in the MMD state. Using a rigorous bifurcation analysis we establish the occurrence of the MMD and MNAD states, and map their transition routes in parameter space. Moreover, we report experimental observation of the MMD and MNAD states that supports our theoretical results. We believe that this study will broaden our understanding of oscillation suppression states; subsequently, it may have applications in many real physical systems, such as laser and geomagnetic systems, whose mathematical models mimic the Lorenz system.

Revival of oscillation from mean-field-induced death: Theory and experiment.

The revival of oscillation and maintaining rhythmicity in a network of coupled oscillators offer an open challenge to researchers as the cessation of oscillation often leads to a fatal system degradation and an irrecoverable malfunctioning in many physical, biological, and physiological systems. Recently a general technique of restoration of rhythmicity in diffusively coupled networks of nonlinear oscillators has been proposed in Zou et al. [Nat. Commun. 6, 7709 (2015)], where it is shown that a proper feedback parameter that controls the rate of diffusion can effectively revive oscillation from an oscillation suppressed state. In this paper we show that the mean-field diffusive coupling, which can suppress oscillation even in a network of identical oscillators, can be modified in order to revoke the cessation of oscillation induced by it. Using a rigorous bifurcation analysis we show that, unlike other diffusive coupling schemes, here one has two control parameters, namely the density of the mean-field and the feedback parameter that can be controlled to revive oscillation from a death state. We demonstrate that an appropriate choice of density of the mean field is capable of inducing rhythmicity even in the presence of complete diffusion, which is a unique feature of this mean-field coupling that is not available in other coupling schemes. Finally, we report the experimental observation of revival of oscillation from the mean-field-induced oscillation suppression state that supports our theoretical results.

Transitions among the diverse oscillation quenching states induced by the interplay of direct and indirect coupling.

We report the transitions among different oscillation quenching states induced by the interplay of diffusive (direct) coupling and environmental (indirect) coupling in coupled identical oscillators. This coupling scheme was introduced by Resmi et al. [Phys. Rev. E 84, 046212 (2011)] as a general scheme to induce amplitude death (AD) in nonlinear oscillators. Using a detailed bifurcation analysis we show that, in addition to AD, which actually occurs only in a small region of parameter space, this coupling scheme can induce other oscillation quenching states, namely oscillation death (OD) and a novel nontrvial AD (NAD) state, which is a nonzero bistable homogeneous steady state; more importantly, this coupling scheme mediates a transition from the AD state to the OD state and a new transition from the AD state to the NAD state. We identify diverse routes to the NAD state and map all the transition scenarios in the parameter space for periodic oscillators. Finally, we present the first experimental evidence of oscillation quenching states and their transitions induced by the interplay of direct and indirect coupling.

Transition from amplitude to oscillation death under mean-field diffusive coupling.

We study the transition from the amplitude death (AD) to the oscillation death (OD) state in limit-cycle oscillators coupled through mean-field diffusion. We show that this coupling scheme can induce an important transition from AD to OD even in identical limit cycle oscillators. We identify a parameter region where OD and a nontrivial AD (NTAD) state coexist. This NTAD state is unique in comparison with AD owing to the fact that it is created by a subcritical pitchfork bifurcation and parameter mismatch does not support this state, but destroys it. We extend our study to a network of mean-field coupled oscillators to show that the transition scenario is preserved and the oscillators form a two-cluster state.

Experimental observation of a transition from amplitude to oscillation death in coupled oscillators.

We report the experimental evidence of an important transition scenario, namely the transition from amplitude death (AD) to oscillation death (OD) state in coupled limit cycle oscillators. We consider two Van der Pol oscillators coupled through mean-field diffusion and show that this system exhibits a transition from AD to OD, which was earlier shown for Stuart-Landau oscillators under the same coupling scheme [T. Banerjee and D. Ghosh, Phys. Rev. E 89, 052912 (2014)]. We show that the AD-OD transition is governed by the density of mean-field and beyond a critical value this transition is destroyed; further, we show the existence of a nontrivial AD state that coexists with OD. Next, we implement the system in an electronic circuit and experimentally confirm the transition from AD to OD state. We further characterize the experimental parameter zone where this transition occurs. The present study may stimulate the search for the practical systems where this important transition scenario can be observed experimentally.

SNaPshot Assay in Quantitative Detection of Allelic Nondisjunction in Down Syndrome.

AIM: We wished to identify markers associated with allelic nondisjunction in nuclear families with Down syndrome (DS) offspring. Since the GRIK1 and GARS-AIRS-GART genes, mapping to chromosome 21q22.1, may be informative in this regard, we genotyped four single-nucleotide polymorphisms [30952599(A/G) rs363484; 30924733(A/G) rs363506; 34901423(A/G) rs2834235; 34877070(A/G) rs7283354] present in these genes using the SNaPshot(™) assay protocol. RESULTS: We have reported 30952599(A/G)-rs363484 to be monomorphic in our sample population. Genotyping revealed 35/65 families to be informative for 34877070(A/G)-rs7283354 (GARS-AIRS-GART), whereas only 25/65 and 11/65 are informative for 34901423(A/G)-rs2834235 (GARS-AIRS-GART) and 30924733(A/G)-rs363506 (GRIK1) polymorphisms, respectively. The parent- and stage-of-origin of nondisjunction could be traced in 48/65 families using at least one polymorphic marker. A single trio provided internal validation for assignment of the parent- and stage-of-origin of nondisjunction whereby the nondisjoining alleles were independently identified as G-rs363506, G-rs2834235, and G-rs7283354, respectively. An enhanced ratio of meiosis-I to meiosis-II errors during maternal or paternal meioses accounts for allelic nondisjunction. CONCLUSIONS: The SNaPshot assay is quantitative and permits multiplexing for detection of allelic nondisjunction. Inclusion of additional informative chromosome 21-specific markers may aid rapid aneuploidy detection, screening, and prenatal counseling of parents at risk of having babies with DS.