Attenuated total reflectance fourier transform infrared spectroscopy: an analytical technique to understand therapeutic responses at the molecular level

Rapid monitoring of the response to treatment in cancer patients is essential to predict the outcome of the therapeutic regimen early in the course of the treatment. The conventional methods are laborious, time-consuming, subjective and lack the ability to study different biomolecules and their interactions, simultaneously. Since; mechanisms of cancer and its response to therapy is dependent on molecular interactions and not on single biomolecules, an assay capable of studying molecular interactions as a whole, is preferred. Fourier Transform Infrared (FTIR) spectroscopy has become a popular technique in the field of cancer therapy with an ability to elucidate molecular interactions. The aim of this study, was to explore the utility of the FTIR technique along with multivariate analysis to understand whether the method has the resolution to identify the differences in the mechanism of therapeutic response. Towards achieving the aim, we utilized the mouse xenograft model of retinoblastoma and nanoparticle mediated targeted therapy. The results indicate that the mechanism underlying the response differed between the treated and untreated group which can be elucidated by unique spectral signatures generated by each group. The study establishes the efficiency of non-invasive, label-free and rapid FTIR method in assessing the interactions of nanoparticles with cellular macromolecules towards monitoring the response to cancer therapeutics

Direct differentiation of adult ocular progenitors into striatal dopaminergic neurons.

Parkinson\u27s disease, characterized by motor dysfunction due to the loss of nigrostriatal dopaminergic neurons, is one of the most prevalent age-related neurodegenerative disorders. Given there is no current cure, the stem cell approach has emerged as a viable therapeutic option to replace the dopaminergic neurons that are progressively lost to the disease. The success of the approach is likely to depend upon accessible, renewable, immune compatible, and non-tumorigenic sources of neural progenitors from which stable dopaminergic neurons can be generated efficaciously. Here, we demonstrate that neural progenitors derived from limbus, a regenerative and accessible ocular tissue, represent a safe source of dopaminergic neurons. When the limbus-derived neural progenitors were subjected to a well-established protocol of directed differentiation under the influence of Shh and FGF8, they acquired the biochemical and functional phenotype of dopaminergic neurons that included the ability to synthesize dopamine. Their intrastriatal transplantation in the rat model of hemi-Parkinsonism was associated with a reduction in the amphetamine-induced rotation. No tumor formation was observed 6 weeks post-transplantation. Together, these observations posit limbus-derived neural progenitors as an accessible and safe source of dopaminergic neurons for a potential autologous ex-vivo stem cell approach to Parkinson\u27s disease

Nucleic acid and non-nucleic acid-based reprogramming of adult limbal progenitors to pluripotency

Reprogramming somatic cells to a pluripotent state by nucleic acid based (NAB) approaches, involving the ectopic expression of transcription factors, has emerged as a standard method. We recently demonstrated that limbal progenitors that regenerate cornea are reprogrammable to pluripotency by a non-NAB approach through simple manipulation of microenvironment thus extending the possible therapeutic use of these readily accessible cells beyond the proven treatment of corneal diseases and injury. Therefore, to determine the validity and robustness of non-cell autonomous reprogramming of limbal progenitors for a wider clinical use, here, we have compared their reprogramming by non-NAB and NAB approaches. We observed that both approaches led to (1) the emergence of colonies displaying pluripotency markers, accompanied by a temporal reciprocal changes in limbal-specific and pluripotency gene expression, and (2) epigenetic alterations of Oct4 and Nanog, associated with the de-novo activation of their expression. While the efficiency of reprogramming and passaging of re-programmed cells were significantly better with the NAB approach, the non-NAB approach, in contrast, led to a regulated reprogramming of gene expression, and a significant decrease in the expression of Hormad1, a gene associated with immunogenic responses. The reprogramming efficiency by non-NAB approach was influenced by exosomes present in conditioned medium. Cells reprogrammed by both approaches were capable of differentiating along the three germ lineages and generating chimeras. The analysis suggests that both approaches are effective in reprogramming limbal progenitors but the non-NAB approach may be more suitable for potential clinical applications by averting the risk of insertional mutagenesis and immune responses associated with the NAB approach

Multicenter Evaluation of Diagnostic Circulating Biomarkers to Detect Sight-Threatening Diabetic Retinopathy

Importance: It is a global challenge to provide regular retinal screening for all people with diabetes to detect sight-threatening diabetic retinopathy (STDR). Objective: To determine if circulating biomarkers could be used to prioritize people with type 2 diabetes for retinal screening to detect STDR. Design, Setting, and Participants: This cross-sectional study collected data from October 22, 2018, to December 31, 2021. All laboratory staff were masked to the clinical diagnosis, assigned a study cohort, and provided with the database containing the clinical data. This was a multicenter study conducted in parallel in 3 outpatient ophthalmology clinics in the UK and 2 centers in India. Adults 40 years and older were categorized into 4 groups: (1) no history of diabetes, (2) type 2 diabetes of at least 5 years' duration with no evidence of DR, (3) nonproliferative DR with diabetic macular edema (DME), or (4) proliferative DR. STDR comprised groups 3 and 4. Exposures: Thirteen previously verified biomarkers were measured using enzyme-linked immunosorbent assay. Main Outcomes and Measures: Severity of DR and presence of DME were diagnosed using fundus photographs and optical coherence tomography. Weighted logistic regression and receiver operating characteristic curve analysis (ROC) were performed to identify biomarkers that discriminate STDR from no DR beyond the standard clinical parameters of age, disease duration, ethnicity (in the UK) and hemoglobin A1c. Results: A total of 538 participants (mean [SD] age, 60.8 [9.8] years; 319 men [59.3%]) were recruited into the study. A total of 264 participants (49.1%) were from India (group 1, 54 [20.5%]; group 2, 53 [20.1%]; group 3, 52 [19.7%]; group 4, 105 [39.8%]), and 274 participants (50.9%) were from the UK (group 1, 50 [18.2%]; group 2, 70 [25.5%]; group 3, 55 [20.1%]; group 4, 99 [36.1%]). ROC analysis (no DR vs STDR) showed that in addition to age, disease duration, ethnicity (in the UK) and hemoglobin A1c, inclusion of cystatin C had near-acceptable discrimination power in both countries (area under the receiver operating characteristic curve [AUC], 0.779; 95% CI, 0.700-0.857 in 215 patients in the UK with complete data; AUC, 0.696; 95% CI, 0.602-0.791 in 208 patients in India with complete data). Conclusions and Relevance: Results of this cross-sectional study suggest that serum cystatin C had good discrimination power in the UK and India. Circulating cystatin-C levels may be considered as a test to identify those who require prioritization for retinal screening for STDR

Notch and Wnt Signaling Mediated Rod Photoreceptor Regeneration by Müller Cells in Adult Mammalian Retina

Background: Evidence emerging from a variety of approaches used in different species suggests that Müller cell function may extend beyond its role of maintaining retinal homeostasis to that of progenitors in the adult retina. Enriched Müller cells in vitro or those that re-enter cell cycle in response to neurotoxin-damage to retina in vivo display multipotential and self-renewing capacities, the cardinal features of stem cells. Methodology/Principal Findings: We demonstrate that Notch and Wnt signaling activate Müller cells through their canonical pathways and that a rare subset of activated Müller cells differentiates along rod photoreceptor lineage in the outer nuclear layer. The differentiation of activated Müller cells along photoreceptor lineage is confirmed by multiple approaches that included Hoechst dye efflux analysis, genetic analysis using retina from Nrl-GFP mice, and lineage tracing using GS-GFP lentivirus in wild type and rd mice in vitro and S334ter rats in vivo. Examination of S334ter rats for head-neck tracking of visual stimuli, a behavioral measure of light perception, demonstrates a significant improvement in light perception in animals treated to activate Müller cells. The number of activated Müller cells with rod photoreceptor phenotype in treated animals correlates with the improvement in their light perception. Conclusion/Significance: In summary, our results provide a proof of principle for non-neurotoxin-mediated activation o

Provably correct on−chip communication: A formal approach to automatic protocol converter synthesis

Hardware module reuse is a standard solution to the problems of increasing complexity of chip architectures and pressure to reduce time to market. In the absence of a single module interface standard, predesigned modules for ?plug-and-play? usually require a converter between incompatible interface protocols. Current approaches to automatic synthesis of protocol converters mostly lack formal foundations and either employ abstractions far removed from the HDL implementation level or grossly simplify the structure of the protocols considered. This work presents a state-machine-based formalism for modeling bus-based communication protocols and a notion of protocol compatibility and of correct conversion between incompatible protocols. This formalism is used to derive algorithms for checking protocol compatibility and for provably correct, automatic converter synthesis. Experiments with automatic converter synthesis between different configurations of widely used commercial bus protocols, such as AMBA AHB, ASB APB, and the Open Core Protocol (OCP) are discussed. The work here is unique in its combination of a completely formal approach and the use of a low abstraction level that enables precise modeling of protocol characteristics that is also close to HDL

A Formal Approach To The Protocol Converter Problem

In the absence of a single module interface standard, integration of pre-designed modules in System-on-Chip design often requires the use of protocol converters. Existing approaches to automatic synthesis of protocol converters mostly lack formal foundations and either employ abstractions that ignore crucial low level behaviors, or grossly simplify the structure of the protocols considered. We present a state-machine based formal model for bus based communication protocols, and precisely define protocol compatibility, and correct protocol conversion. Our model is expressive enough to capture features of commercial protocols such as bursts, pipelined transfers, wait state insertion, and data persistence, in cycle accurate detail. We show that the most general, correct converter for a pair of protocols, can be described as the greatest fixed point of a function for updating buffer states. This characterization yields a natural algorithm for automatic synthesis of a provably correct converter by iterative computation of the fixed point. We report our experience with automatic converter synthesis between widely used commercial bus protocols, such as AMBA AHB, ASB, APB, and OCP, considering features which are beyond the scope of current techniques

A Formal Approach To The Protocol Converter Problem

In the absence of a single module interface standard, integration of pre-designed modules in System-on-Chip design often requires the use of protocol converters. Existing approaches to automatic synthesis of protocol converters mostly lack formal foundations and either employ abstractions that ignore crucial low level behaviors, or grossly simplify the structure of the protocols considered. We present a state-machine based formal model for bus based communication protocols, and precisely define protocol compatibility, and correct protocol conversion. Our model is expressive enough to capture features of commercial protocols such as bursts, pipelined transfers, wait state insertion, and data persistence, in cycle accurate detail. We show that the most general, correct converter for a pair of protocols, can be described as the greatest fixed point of a function for updating buffer states. This characterization yields a natural algorithm for automatic synthesis of a provably correct converter by iterative computation of the fixed point. We report our experience with automatic converter synthesis between widely used commercial bus protocols, such as AMBA AHB, ASB, APB, and OCP, considering features which are beyond the scope of current techniques

Generation of a human induced pluripotent stem cell line (VRFi001-A) from orbital adipose tissue of a bilateral retinoblastoma patient with heterozygous RB1 gene deletion

Retinoblastoma (RB) is a pediatric intraocular tumor caused by mutations in retinoblastoma (RB1) gene. We have generated induced pluripotent stem cell line VRFi001-A from a bilateral retinoblastoma patient with heterozygous RB1 gene deletion. The iPSC line VRFi001-A retained the mutation and expressed pluripotency markers, had a normal karyotype and was capable of trilineage differentiation