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The present work represents a Fluorescence Resonance Energy Transfer (FRET) based sensing method for detecting Gunshot Residue (GSR) components. Two laser dyes Acf and RhB have been used as donor and acceptor respectively in the FRET pair. The real sample was collected after test firing in a forensic science laboratory. On the other hand, a standard GSR solution has been prepared in the laboratory. For the preparation of standard GSR solutions, we used the water solutions of the salts BaCl2, SbCl3, and Pb(NO3)2. The FRET efficiency was measured between Acf and RhB to sense the presence of GSR components (Pb+2, Ba+2, and Sb+3) in both real sample and standard solution by mixing the salts in aqueous solution. It has been observed that the FRET efficiency systematically decreases in the presence of GSR components. To amplify the FRET efficiency of the dye pair, inorganic clay dispersion (laponite) was used. The enhancement in FRET efficiency represents a better sensitivity of the proposed sensor. The current sensor is useful for the quantification of concentrations of the GSR components in a real sample.
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Pluripotent stem cells can be differentiated into all three germ-layers including ecto-, endo-, and mesoderm in vitro. However, the early identification and rapid characterization of each germ-layer in response to chemical and physical induction of differentiation is limited. This is a long-standing issue for rapid and high-throughput screening to determine lineage specification efficiency. Here, we present deep learning (DL) methodologies for predicting and classifying early mesoderm cells differentiated from embryoid bodies (EBs) based on cellular and nuclear morphologies. Using a transgenic murine embryonic stem cell (mESC) line, namely OGTR1, we validated the upregulation of mesodermal genes (Brachyury (T): DsRed) in cells derived from EBs for the deep learning model training. Cells were classified into mesodermal and non-mesodermal (representing endo- and ectoderm) classes using a convolutional neural network (CNN) model called InceptionV3 which achieved a very high classification accuracy of 97% for phase images and 90% for nuclei images. In addition, we also performed image segmentation using an Attention U-Net CNN and obtained a mean intersection over union of 61% and 69% for phase-contrast and nuclear images, respectively. This work highlights the potential of integrating cell culture, imaging technologies, and deep learning methodologies in identifying lineage specification, thus contributing to the advancements in regenerative medicine. Collectively, our trained deep learning models can predict the mesoderm cells with high accuracy based on cellular and nuclear morphologies.
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Aprendizaje Profundo , Células Madre Pluripotentes , Animales , Ratones , Diferenciación Celular/fisiología , Estratos Germinativos/metabolismo , Mesodermo/metabolismoRESUMEN
Human coronaviruses like MERS CoV are known to utilize dipeptidyl peptidase 4 (DPP4), apart from angiotensin-converting enzyme 2(ACE2) as a potential co-receptor for viral cell entry. DPP4, the ubiquitous membrane-bound aminopeptidase, is closely associated with elevation of disease severity in comorbidities. In SARS-CoV-2, there is inadequate evidence for combination of spike protein variants with DPP4, and underlying adversity in COVID-19. To elucidate this mechanistic basis, we have investigated interaction of spike protein variants with DPP4 through molecular docking and simulation studies. The possible binding interactions between the receptor binding domain (RBD) of different spike variants of SARS-CoV-2 and DPP4 have been compared with interactions observed in the experimentally determined structure of the complex of MERS-CoV with DPP4. Comparative binding affinity confers that Delta-CoV-2: DPP4 shows close proximity with MERS-CoV:DPP4, as depicted from accessible surface area, radius of gyration and number of hydrogen bonding in the interface. Mutations in the delta variant, L452R and T478K directly participate in DPP4 interaction, enhancing DPP4 binding. E484K in alpha and gamma variants of spike protein is also found to interact with DPP4. Hence, DPP4 interaction with spike protein becomes more suitable due to mutation, especially due to L452R, T478K and E484K. Furthermore, perturbation in the nearby residues Y495, Q474 and Y489 is evident due to L452R, T478K and E484K, respectively. Virulent strains of spike protein are more susceptible to DPP4 interaction and are prone to be victimized in patients due to comorbidities. Our results will aid the rational optimization of DPP4 as a potential therapeutic target to manage COVID-19 disease severity.
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COVID-19 , Coronavirus del Síndrome Respiratorio de Oriente Medio , Humanos , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Coronavirus del Síndrome Respiratorio de Oriente Medio/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Glicoproteína de la Espiga del Coronavirus/metabolismo , Unión Proteica , MutaciónRESUMEN
Ataxin-7 maintains the integrity of Spt-Ada-Gcn5-Acetyltransferase (SAGA), an evolutionarily conserved coactivator in stimulating preinitiation complex (PIC) formation for transcription initiation, and thus, its upregulation or downregulation is associated with various diseases. However, it remains unknown how ataxin-7 is regulated that could provide new insights into disease pathogenesis and therapeutic interventions. Here, we show that ataxin-7's yeast homologue, Sgf73, undergoes ubiquitylation and proteasomal degradation. Impairment of such regulation increases Sgf73's abundance, which enhances recruitment of TATA box-binding protein (TBP) (that nucleates PIC formation) to the promoter but impairs transcription elongation. Further, decreased Sgf73 level reduces PIC formation and transcription. Thus, Sgf73 is fine-tuned by ubiquitin-proteasome system (UPS) in orchestrating transcription. Likewise, ataxin-7 undergoes ubiquitylation and proteasomal degradation, alteration of which changes ataxin-7's abundance that is associated with altered transcription and cellular pathologies/diseases. Collectively, our results unveil a novel UPS regulation of Sgf73/ataxin-7 for normal cellular health and implicate alteration of such regulation in diseases.
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Proteínas de Saccharomyces cerevisiae , Ubiquitina , Ataxina-7/genética , Ataxina-7/metabolismo , Ubiquitina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Histona Acetiltransferasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Mechanoregulation of cell-extracellular matrix (ECM) interactions are crucial for dictating pluripotent stem cell differentiation. However, not all pluripotent cells respond homogeneously which results in heterogeneous cell populations. When cells, such as mouse epiblast stem cells (EpiSCs), are cultured in clusters, the heterogeneity effect during differentiation is even more pronounced. While past studies implicated variations in signaling pathways to be the root cause of heterogeneity, the biophysical aspects of differentiation have not been thoroughly considered. Here, we demonstrate that the heterogeneity of EpiSC differentiation arises from differences in the colony size and varying degrees of interactions between cells within the colonies and the ECM. Confocal imaging demonstrates that cells in the colony periphery established good contact with the surface while the cells in the colony center were separated by an average of 1-2 µm from the surface. Traction force measurements of the cells within the EpiSC colonies show that peripheral cells generate large tractions while the colony center cells do not. A finite element modeling of EpiSC colonies shows that tractions generated by the cells at the colony periphery lift off the colony center preventing the colony center from undergoing differentiation. Together, our results demonstrate a biophysical regulation of heterogeneous EpiSC colony differentiation.
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Células Madre Pluripotentes , Ratones , Animales , Diferenciación Celular , Células Madre Pluripotentes/metabolismo , Estratos Germinativos/metabolismo , Transducción de SeñalRESUMEN
The engineering of exceptional points (EPs) in open optical systems has lately attracted much attention for developing future all-optical devices. However, investigation of the fascinating features of EPs in fiber geometries is lacking. We design a fabrication feasible dual-core optical fiber segment, where non-Hermiticity in terms of a symmetric customized gain-loss profile is introduced to modulate the interaction between two corresponding coupled modes toward hosting a dynamical EP encirclement scheme in the gain-loss parameter space. An asymmetric conversion process between two supported modes is reported by exploiting the chirality of the encountered EP. The proposed scheme can lead to an advanced platform to design mode-manipulative all-optical components in communication and all-fiber photonic devices.
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A new methodology to enhance the sensitivity of a long period fiber grating sensor (LPFG) at the Turn Around Point (TAP) is here presented. The LPFG sensor has been fabricated by etching the fiber up to 20.4 µm, until the sidelobes of dispersed LP0,2 cladding mode appeared near TAP in aqueous medium. The dual peak sensitivity of the sidelobes was found to be 16,044 nm/SRIU (surrounding refractive index units) in the RI range from 1.333 to 1.3335.
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A single center open label phase 2 randomised control trial (Clinical Trial Registry of India No. CTRI/2020/05/025209) was done to assess clinical and immunological benefits of passive immunization using convalescent plasma therapy. At the Infectious Diseases and Beleghata General Hospital in Kolkata, India, 80 patients hospitalized with severe COVID-19 disease and fulfilling the inclusion criteria (aged more than 18 years, with either mild ARDS having PaO2/FiO2 200-300 or moderate ARDS having PaO2/FiO2 100-200, not on mechanical ventilation) were recruited and randomized into either standard of care (SOC) arm (N = 40) or the convalescent plasma therapy (CPT) arm (N = 40). Primary outcomes were all-cause mortality by day 30 of enrolment and immunological correlates of response to therapy if any, for which plasma abundance of a large panel of cytokines was quantitated before and after intervention to assess the effect of CPT on the systemic hyper-inflammation encountered in these patients. The secondary outcomes were recovery from ARDS and time taken to negative viral RNA PCR as well as to report any adverse reaction to plasma therapy. Transfused convalescent plasma was characterized in terms of its neutralizing antibody content as well as proteome. The trial was completed and it was found that primary outcome of all-cause mortality was not significantly different among severe COVID-19 patients with ARDS randomized to two treatment arms (Mantel-Haenszel Hazard Ratio 0.6731, 95% confidence interval 0.3010-1.505, with a P value of 0.3424 on Mantel-Cox Log-rank test). No adverse effect was reported with CPT. In severe COVID-19 patients with mild or moderate ARDS no significant clinical benefit was registered in this clinical trial with convalescent plasma therapy in terms of prespecified outcomes.
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COVID-19/terapia , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/uso terapéutico , Donantes de Sangre , COVID-19/inmunología , COVID-19/virología , Citocinas/sangre , Femenino , Hospitales Generales , Humanos , Inmunidad Humoral , Inmunización Pasiva , India , Inflamación , Masculino , Filogenia , Síndrome de Dificultad Respiratoria/inmunología , Síndrome de Dificultad Respiratoria/terapia , Síndrome de Dificultad Respiratoria/virología , SARS-CoV-2/clasificación , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Análisis de Supervivencia , Resultado del Tratamiento , Carga Viral , Sueroterapia para COVID-19RESUMEN
Cap-binding complex (CBC) associates cotranscriptionally with the cap structure at the 5' end of nascent mRNA to protect it from exonucleolytic degradation. Here, we show that CBC promotes the targeting of an mRNA export adaptor, Yra1 (forming transcription export [TREX] complex with THO and Sub2), to the active genes and enhances mRNA export in Saccharomyces cerevisiae Likewise, recruitment of Npl3 (an hnRNP involved in mRNA export via formation of export-competent ribonuclear protein complex [RNP]) to the active genes is facilitated by CBC. Thus, CBC enhances targeting of the export factors and promotes mRNA export. Such function of CBC is not mediated via THO and Sub2 of TREX, cleavage and polyadenylation factors, or Sus1 (that regulates mRNA export via transcription export 2 [TREX-2]). However, CBC promotes splicing of SUS1 mRNA and, consequently, Sus1 protein level and mRNA export via TREX-2. Collectively, our results support the hypothesis that CBC promotes recruitment of Yra1 and Npl3 to the active genes, independently of THO, Sub2, or cleavage and polyadenylation factors, and enhances mRNA export via TREX and RNP, respectively, in addition to its role in facilitating SUS1 mRNA splicing to increase mRNA export through TREX-2, revealing distinct stimulatory functions of CBC in mRNA export.
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Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Proteínas de Unión a Caperuzas de ARN/metabolismo , ARN Mensajero/metabolismo , Transporte Activo de Núcleo Celular/genética , Transporte Activo de Núcleo Celular/fisiología , Complejo Proteico Nuclear de Unión a la Caperuza/genética , Proteínas Nucleares/metabolismo , Empalme del ARN , Transporte de ARN/fisiología , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
Morphology of erythrocyte membrane has been recognized as an alternative biomarker of several patho-physiological states. Numerous attempts have been made to upgrade the existing method of primitive manual counting, particularly exploring the light scattering properties of erythrocyte. All the techniques are at best semi-empirical and heavily rely on the effectiveness of the statistical correlations. Precisely, this is due to the lack of a non-empirical scale of the so-called "morphological scores". In this article, fractal dimension of erythrocyte membrane has been used to formulate a suitable scoring scale. Subsequently, the rapid experimental output of flow-cytometry has been functionally related to the mean morphological quantifier of the whole cell population via an optimum neural network model (R2 = 0.98). Moreover, the fractal dimension has been further demonstrated to be an important parameter in early detection of an abnormal patho-physiological state, even without any noticeable poikilocytic transformation in micrometric domain.
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Membrana Eritrocítica , Citometría de Flujo , Fractales , Humanos , Microscopía de Fuerza Atómica , Microscopía Confocal , Redes Neurales de la Computación , Propiedades de SuperficieRESUMEN
BACKGROUND: Sex differences in the incidence and prognosis of respiratory diseases have been reported. Studies have shown that women are at increased risk of adverse health outcomes from air pollution than men, but sex-specific immune gene expression patterns and regulatory networks have not been well studied in the lung. MicroRNAs (miRNAs) are environmentally sensitive posttranscriptional regulators of gene expression that may mediate the damaging effects of inhaled pollutants in the lung, by altering the expression of innate immunity molecules. METHODS: Male and female mice of the C57BL/6 background were exposed to 2 ppm of ozone or filtered air (control) for 3 h. Female mice were also exposed at different stages of the estrous cycle. Following exposure, lungs were harvested and total RNA was extracted. We used PCR arrays to study sex differences in the expression of 84 miRNAs predicted to target inflammatory and immune genes. RESULTS: We identified differentially expressed miRNA signatures in the lungs of male vs. female exposed to ozone. In silico pathway analyses identified sex-specific biological networks affected by exposure to ozone that ranged from direct predicted gene targeting to complex interactions with multiple intermediates. We also identified differences in miRNA expression and predicted regulatory networks in females exposed to ozone at different estrous cycle stages. CONCLUSION: Our results indicate that both sex and hormonal status can influence lung miRNA expression in response to ozone exposure, indicating that sex-specific miRNA regulation of inflammatory gene expression could mediate differential pollution-induced health outcomes in men and women.
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Contaminantes Atmosféricos/efectos adversos , Inflamación/inducido químicamente , Pulmón/efectos de los fármacos , MicroARNs , Ozono/efectos adversos , Caracteres Sexuales , Animales , Modelos Animales de Enfermedad , Estradiol/sangre , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Inflamación/sangre , Inflamación/genética , Pulmón/metabolismo , Hormona Luteinizante/sangre , Masculino , Ratones Endogámicos C57BL , Progesterona/sangreRESUMEN
Fluorescence Resonance Energy Transfer between two dyes Fluorescein and Rhodamine 6G were investigated in solution in the presence and absence of Zinc oxide nanoparticle. Zinc oxide nanostructure is used as the fluorescence enhancing agent for the present study since donor (Fluorescein) fluorescence increase significantly in presence of nanoparticle. Accordingly, the energy transfer efficiency in the presence of nanoparticle increases. The maximum efficiency was 69% for acceptor (Rhodamine 6G) concentration of 0.75×10-5M. The energy transfer efficiency was found to be pH sensitive and it varies from 4.15% to 90.00% in mixed dye solution for a change in pH from 1.5 to 10.0. With proper calibration it is possible to use the present system under investigation to sense pH which is better with respect to our previous reported results [Spectrochim. Acta Part A. 149 (2015) 143-149] as it can sense a wide range of pH and with better sensitivity.
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Fluorescence Resonance Energy Transfer between two organic dyes Fluorescein and Rhodamine 6G was investigated in aqueous solution in presence and absence of synthetic clay laponite. Spectroscopic studies suggest that both the dyes were present mainly as monomer in solution. Fluorescence Resonance Energy Transfer occurred from Fluorescein to Rhodamine 6G in solutions. Energy transfer efficiency increases in presence of laponite and the maximum efficiency was 72.00% in aqueous laponite dispersion. Energy transfer efficiency was found to be pH sensitive. It has been demonstrated that with proper calibration it is possible to use the present system under investigation to sense pH over a wide range from 1.5 to 8.0.
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Fluoresceína/química , Colorantes Fluorescentes/química , Rodaminas/química , Transferencia Resonante de Energía de Fluorescencia , Silicatos/química , Agua/químicaRESUMEN
This communication reports our investigations on the Fluorescence Resonance Energy Transfer (FRET) between two laser dyes Acriflavine and Rhodamine B in absence and presence of DNA at different pH. It has been observed that energy transfer efficiency is largely affected by the presence of DNA as well as the pH of the system. It is well known that with increase in pH, DNA conformation changes from double stranded to single stranded (denaturation) and finally form random coil. Based on our experimental results two different types of molecular logic gates namely, XOR and OR logic have been demonstrated which can be used to have an idea about DNA conformation in solution.