A spectroscopy-based proof-of-concept (POC) for developing loading of pathogen analyzer (LOPA) for dairy products.

Detection of bacterial contamination in dairy products of daily use is a challenge worldwide. We have utilized Methylene Blue Reduction Test (MBRT) for quantification of the microbial count in dairy products (milk) and developed a proof-of-concept (POC) based on this for in-filed applications. In this study, we have used pasteurized milk contaminated with model bacteria Escherichia coli, and Staphylococcus aureus for the calibration and validation of the developed POC. The conversion of MB to Leuco-MB i.e., the colorimetric change due to the reduction of MB to Leuco-MB in presence of microbes has been utilized as the tool to detect presence of microbes in milk. The absorbance peak for methylene blue (MB) at 664 nm decreases significantly in presence of microbes and the blue color becomes faded. In our study, we have employed methylene blue (MB) discolouration phenomenon to estimate the microbial count in milk samples using our developed spectroscopy based POC. The limit of detection (LOD) and the limit of quantitation (LOQ) of the POC were found to be 0.32 CFU/mL and 0.97 CFU/mL. The end users of the developed POC are primarily those involved in the production, processing, testing, regulation, and research of dairy products to ensure they meet safety standards and protect public health. These include retailers, dairy farmers, dairy processors, quality control laboratories, regulatory agencies and research institutions. In our experiment, we have observed a significant change in MB absorption in the milk contaminated with microbes. The indigenously developed sensor strips designed for the working of the POC turn to colorless Leuco-MB compared to milk without the microbes. The analysis of the strips has been measured in the developed device.

A study of scarless wound healing through programmed inflammation, proliferation and maturation using a redox balancing nanogel.

In the study, we have shown the efficacy of an indigenously developed redox balancing chitosan gel with impregnated citrate capped Mn3O4 nanoparticles (nanogel). Application of the nanogel on a wound of preclinical mice model shows role of various signaling molecules and growth factors, and involvement of reactive oxygen species (ROS) at every stage, namely hemostasis, inflammation, and proliferation leading to complete maturation for the scarless wound healing. While in vitro characterization of nanogel using SEM, EDAX, and optical spectroscopy reveals pH regulated redox buffering capacity, in vivo preclinical studies on Swiss albino involving IL-12, IFN-γ, and α-SMA signaling molecules and detailed histopathological investigation and angiogenesis on every stage elucidate role of redox buffering for the complete wound healing process.

Targeted Redox Balancing through Pulmonary Nanomedicine Delivery Reverses Oxidative Stress Induced Lung Inflammation.

Non-invasive delivery of drugs is important for the reversal of respiratory diseases essentially by-passing metabolic pathways and targeting large surface area of drug absorption. Here, we study the inhalation of a redox nano medicine namely citrate functionalized Mn3O4 (C-Mn3O4) duly encapsulated in droplet evaporated aerosols for the balancing of oxidative stress generated by the exposure of Chromium (VI) ion, a potential lung carcinogenic agent. Our optical spectroscopic in-vitro experiments demonstrates the efficacy of redox balancing of the encapsulated nanoparticles (NP) for the maintenance of a homeostatic condition. The formation of Cr-NP complex as an excretion of the heavy metal is also demonstrated through optical spectroscopic and high resolution transmission optical microscopy (HRTEM). Our studies confirm the oxidative stress mitigation activity of the Cr-NP complex. A detailed immunological assay followed by histopathological studies and assessment of mitochondrial parameters in pre-clinical mice model with chromium (Cr) induced lung inflammation establishes the mechanism of drug action to be redox-buffering. Thus, localised delivery of C-Mn3O4 NPs in the respiratory tract via aerosols can act as an effective nanotherapeutic agent against oxidative stress induced lung inflammation.

Structurally Dynamic Monocyte-Liposome Hybrid Vesicles as an Anticancer Drug Delivery Vehicle: A Crucial Correlation of Microscopic Elasticity and Ultrafast Dynamics.

A biomimetic cell-based carrier system based on monocyte membranes and liposomes has been designed to create a hybrid "Monocyte-LP" which inherits the surface antigens of the monocytes along with the drug encapsulation property of the liposome. Förster resonance energy transfer (FRET) and polarization gated anisotropy measurements show the stiffness of the vesicles obtained from monocyte membranes (Mons), phosphatidylcholine membranes (LP), and Monocyte-LP to follow an increasing order of Mons > Monocyte-LP > LP. The dynamics of interface bound water molecules plays a key role in the elasticity of the vesicles, which in turn imparts higher delivery efficacy to the hybrid Monocyte-LP for a model anticancer drug doxorubicin than the other two vesicles, indicating a critical balance between flexibility and rigidity for an efficient cellular uptake. The present work provides insight on the influence of elasticity of delivery vehicles for enhanced drug delivery.

A Mutagen Acts as a Potent Reducing Agent of Glycated Hemoglobin: a Combined Ultrafast Electron Transfer and Computational Studies.

Glycated hemoglobin (GHb) found in mammals undergoes irreversible damage when exposed to external redox agents, which is much more vulnerable than its normal counterpart hemoglobin (Hb). Besides the oxygen regulation throughout the body, Hb plays a vital role in balancing immunological health and the redox cycle. Photoinduced ultra-fast electron transfer phenomena actively participate in regulation of various kind of homeostasis involved in such biomacromolecules. In the present study we have shown that a well-known mutagen Ethidium Bromide (EtBr) reduces GHb in femtosecond time scale (efficiently) upon photoexcitation after efficient recognition in the biomolecule. We have performed similar experiment by colocalizing EtBr and Iron (Fe(III)) on the micellar surface as Hb mimic in order to study the excited state EtBr dynamics to rationalize the time scale obtained from EtBr in GHb and Hb. While other experimental techniques including Dynamic Light Scattering (DLS), Zeta potential, absorbance and emission spectroscopy have been employed for the confirmation of structural perturbation of GHb compared to Hb, a detailed computational studies involving molecular docking and density functional theory (DFT) have been employed for the explanation of the experimental observations.

Ultrafast spectroscopic studies on the interaction of reactive oxygen species with a probe impregnated in nanoscopic and microscopic matrix formulation.

Reactive oxygen species (ROS) plays important role to maintain homeostasis in living bodies. Here we have studied interaction of ROS generated from hydrogen peroxide (H2O2) with a well-known spectroscopic probe Rose Bengal (RB) encapsulated in nanoscopic sodium dodecyl sulphate (SDS) micelles in aqueous medium and entrapped in microscopic nylon 66 solid matrix generated using electrospinning technique. A detailed spectroscopic characterization of ROS with SDS encapsulated RB (RB-SDS) shows efficient interaction compared to that in bulk medium. The time resolved analysis on the probe based on femtosecond resolved 2D-spectrum time images collected from streak camera reveal the simultaneous existence of an ultrafast electron (∼6 ps) and a hole transfer mechanism (∼93 ps) resulting from generation of hydroxyl radicals through photobleaching of the probe in presence of H2O2. Based on the spectroscopic and time resolved studies of RB in bulk and in restricted (SDS) medium, we have further translated it for the development of an in-field prototype device which utilizes RB as a ROS sensor impregnated in a nylon thin film. The microscopic nylon solid matrix characterized by scanning electron microscope (SEM) shows porous structure for holding sample containing ROS. Our study quantitatively measures the amount of ROS by using RB embedded microfiber membrane. Thus, our developed prototype device based on RB embedded on the nylon matrix would be beneficial for the potential use in quantification of ROS in extracellular fluids and food materials.

Excited-State Dynamics of a Photoacid: A Potential Probe for Recognizing Transition from Lamellar to Nonlamellar Inverted Structures of Liposome based Nanocarriers.

Liposomes of a cationic lipid dioctadecyldimethylammonium bromide (DODAB) are efficient nanocarriers of nucleic acids. Incorporation of a neutral lipid monoolein (MO) in excess (xMO >0.5) changes the lamellar organization of DODAB liposomes into non-lamellar inverted structures of DODAB/MO liposomes facilitating nucleic acid delivery to cells. Photoexcitation of 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS), a photoacid, initiates an excited state proton transfer (ESPT) reaction in its protonated form (ROH*) generating the deprotonated anionic form (RO- *). The fluorescence intensity ratio (IROH* /IRO-* ) of these two forms is governed by the ESPT dynamics, and increases with increasing MO content (xMO ) in the cationic liposomes of DODAB. Transition from lamellar organization of DODAB liposomes into non-lamellar inverted structures of DODAB/MO liposomes, due to incorporation of MO (xMO ~0.7), is manifested by a significant increase of ESPT time (τPT ) and the time constant of wobbling motion (τW ) of HPTS. Thus, the lamellar organizations of DODAB or DODAB-rich (xMO 0.2) liposomes and the non-lamellar organizations of MO-rich (xMO ~0.7) liposomes are recognized by significantly different excited state dynamics of the photoacid.

A spectroscopy based prototype for the noninvasive detection of diabetes from human saliva using nanohybrids acting as nanozyme.

The recent prediction of diabetes to be a global pandemic invites a detection strategy preferably non-invasive, and bloodless to manage the disease and the associated complications. Here, we have synthesized chitosan polymer functionalized, organic-inorganic bio-compatible nano-hybrids of Mn3O4 nanoparticles, and characterized it by utilizing several optical methodologies for the structural characterization which shows the Michaelis Menten (MM) kinetics for glucose and alpha-amylase protein (well-known diabetes biomarkers). We have also studied the potentiality for the detection of alpha-amylase in human salivary secretion which is reported to be strongly correlated with uncontrolled hyperglycemia. Finally, we have developed a prototype for the measurement of glucose (LOD of 0.38 mg/dL, LOQ of 1.15 mg/dL) and HbA1c (LOD of 0.15% and LOQ of 0.45%) utilizing the basic knowledge in the study for the detection of uncontrolled hyperglycemia at the point-of-care. With the limited number of clinical trials, we have explored the potential of our work in combating the diabetic pandemic across the globe in near future.

Spectroscopic studies on a natural biomarker for the identification of origin and quality of tea extracts for the development of a portable and field deployable prototype.

Even in the era of smart technologies and IoT enabled devices, tea testing technique continues to be a person specific subjective task. In this study, we have employed optical spectroscopy-based detection technique for the quantitative validation of tea quality. In this regard, we have employed the external quantum yield of quercetin at 450 nm (λex = 360 nm), which is an enzymatic product generated by the activity of ß-glucosidase on rutin, a naturally occurring metabolite responsible for tea-flavour (quality). We have found that a specific point in a graph representing Optical Density and external Quantum Yield as independent and dependent variables respectively of an aqueous tea extract objectively indicates a specific variety of the tea. A variety of tea samples from various geographical origin have been analysed with the developed technique and found to be useful for the tea quality assessment. The principal component analysis distinctly showed the tea samples originated from Nepal and Darjeeling having similar external quantum yield, while the tea samples from Assam region had a lower external quantum yield. Furthermore, we have employed experimental and computational biology techniques for the detection of adulteration and health benefit of the tea extracts. In order to assure the portability/field use, we have also developed a prototype which confirms the results obtained in the laboratory. We are of the opinion that the simple user interface and almost zero maintenance cost of the device will make it useful and attractive with minimally trained manpower at low resource setting.

Unraveling an Ultrafast Electron Transport Mechanism in a Photocatalytic "Micromachine" for Their Potential Light Harvesting Applications.

Following the seminal discovery of Richard Feynman, several micromachines have been made that are capable of several applications, such as solar energy harvesting, remediation of environmental pollution, etc. Here we have synthesized a nanohybrid combining TiO2 nanoparticle and light harvesting robust organic molecule RK1 (2-cyano-3-(4-(7-(5-(4-(diphenylamino)phenyl)-4-octylthiophen-2-yl)benzo[c][1,2,5] thiadiazol-4-yl)phenyl) acrylic acid) as a model micromachine having solar light harvesting ability potential for application in photocatalysis, preparation of solar active devices, etc. Detailed structural characterization, including High Resolution Transmission Electronic Microscopy (HRTEM) and Fourier-transform infrared spectroscopy (FTIR), has been performed on the nanohybrid. We have studied the excited-state ultrafast dynamics of the efficient push-pull dye RK1 in solution, on mesoporous semiconductor nanoparticles, and in insulator nanoparticles by streak camera (resolution of the order of 500 fs). The dynamics of such photosensitizers in polar solvents have been reported, and it has been observed that completely different dynamics occur when they are attached to the surface of the semiconductor/insulator nanosurface. A femtosecond-resolved fast electron transfer has been reported when photosensitizer RK1 has been attached to the surface of the semiconductor nanoparticle, which in turn plays a crucial role in the development of an efficient light harvesting material. The generation of reactive oxygen species as a result of femtosecond-resolved photoinduced electron injection in the aqueous medium is also investigated in order to explore the possibility of redox-active micromachines, which are found to be crucial for efficient and enhanced photocatalysis.

Sensing Bioavailable Water Content of Granulated Matrices: A Combined Experimental and Computational Study.

This paper represents the synthesis, characterization and validation of a cobalt chloride functionalised nano-porous cellulose membrane, a unique sensor for non-contact measurement of water potential in various biomedical and environmentally important matrices. The developed nano sensor, along with associated electronic components, is assembled as a prototype device called "MEGH" (Measuring Essential Good Hydration) to measure essential hydration of matrices of both environmental and biomedical importance, including soil and human skin. The relative humidity above the soil surface in equilibrium with the soil moisture has been studied for both hydrophobic and hydrophilic soil types. Our studies confirm that the percentage of water available to plants is greater in hydrophobic soil rather than in hydrophilic soil, which has also been corroborated using simulation studies. Furthermore, the requirement of hydration in human skin has also been evaluated by measuring the water potential of both dry and moist skin.

Non-invasive estimation of hemoglobin, bilirubin and oxygen saturation of neonates simultaneously using whole optical spectrum analysis at point of care.

The study was aimed to evaluate the performance of a newly developed spectroscopy-based non-invasive and noncontact device (SAMIRA) for the simultaneous measurement of hemoglobin, bilirubin and oxygen saturation as an alternative to the invasive biochemical method of blood sampling. The accuracy of the device was assessed in 4318 neonates having incidences of either anemia, jaundice, or hypoxia. Transcutaneous bilirubin, hemoglobin and blood saturation values were obtained by the newly developed instrument which was corroborated with the biochemical blood tests by expert clinicians. The instrument is trained using Artificial Neural Network Analysis to increase the acceptability of the data. The artificial intelligence incorporated within the instrument determines the disease condition of the neonate. The Pearson's correlation coefficient, r was found to be 0.987 for hemoglobin estimation and 0.988 for bilirubin and blood gas saturation respectively. The bias and the limits of agreement for the measurement of all the three parameters were within the clinically acceptance limit.

A portable spectroscopic instrument for multiplexed monitoring of acute water toxicity: Design, testing, and evaluation.

The deteriorating water environment worldwide, mainly due to population explosion and uncontrolled direct disposal of harmful industrial and farming wastes, earnestly demands new approaches and accurate technologies to monitor water quality before consumption overcoming the shortcomings of the current methodologies. A spectroscopic water quality monitoring and early-warning instrument for evaluating acute water toxicity are the need of the hour. In this study, we have developed a prototype capable of the quantification of dissolved organic matter, dissolved chemicals, and suspended particulate matter in trace amounts dissolved in the water. The prototype estimates the water quality of the samples by measuring the absorbance, fluorescence, and scattering of the impurities simultaneously. The performance of the instrument was evaluated by detecting common water pollutants such as Benzopyrene, Crystal Violet, and Titanium di-oxide. The limit of detection values was found to be 0.50, 23.9, and 23.2 ppb (0.29 µM), respectively.

Understanding on CRISPR/Cas9 mediated cutting-edge approaches for cancer therapeutics.

The research focus on CRISPR/Cas9 has gained substantial concentration since the discovery of 'an unusual repeat sequence' reported by Ishino et al. (J Bacteriol 169:5429-5433, 1987) and the journey comprises the recent Nobel Prize award (2020), conferred to Emmanuelle Charpentier and Jennifer Doudna. Cumulatively, the CRISPR has a short, compact, and most discussed success of its application in becoming one of the most versatile and paradigm shifting technologies of Biological Research. Today, the CRISPR/Cas9 genome editing system is almost ubiquitously utilized in many facets of biological research where its tremendous gene manipulation capability has been harnessed to create miracles. From 2012, the CRISPR/Cas 9 system has been showcased in almost 15,000 research articles in the PubMed database, till date. Backed by some strong molecular evidence, the CRISPR system has been utilized in a few clinical trials targeted towards various pathologies. While the area covered by CRISPR is cosmic, this review will focus mostly on the utilization of CRISPR/Cas9 technology in the field of cancer therapy.

Corrigendum: CAR T Cells Targeting the Tumor MUC1 Glycoprotein Reduce Triple-Negative Breast Cancer Growth.

[This corrects the article DOI: 10.3389/fimmu.2019.01149.].

Spectroscopy of an intrinsic fluorophore in animal and plant milk for potential identification of their quality.

Riboflavin (RF), commonly known as vitamin B2, is an essential ingredient in any milk variety of animal origin. The photophysics of the molecule RF, including its interaction with biological macromolecules, are well studied. Here, we have investigated the possibility of the molecule as a potential biomarker of milk quality. We also found omnipresence of this molecule in milk of plant origin (soy milk). Spectroscopic studies on various animal and plant milks of different commercial origins confirmed the potential of RF for use in identifying the quality of the milk varieties. Our developed strategy involved identification or spectroscopic signature of RF by measuring optical density at 365 nm (quality factor 1) and fluorescence intensity around 520 nm (excitation at 365 nm; quality factor 2) on a very small amount of whole milk (10 µL). We also developed a prototype device called Mil-Q-Way to be used in the real field. The required interfacing software in the LabView platform was also developed. A 2-parameter plot (quality factor 1 on the x-axis and quality factor 2 on the y-axis) called the Mil-Q-Way plot clearly differentiates the quality of milks of different commercial origins. The low-cost device based on simple spectroscopy was shown to screen for the presence of harmful adulterants in drinkable milk.

Indomethacin enhances anti-tumor efficacy of a MUC1 peptide vaccine against breast cancer in MUC1 transgenic mice.

In recent years, vaccines against tumor antigens have shown potential for combating invasive cancers, including primary tumors and metastatic lesions. This is particularly pertinent for breast cancer, which is the second-leading cause of cancer-related death in women. MUC1 is a glycoprotein that is normally expressed on glandular epithelium, but is overexpressed and under-glycosylated in most human cancers, including the majority of breast cancers. This under-glycosylation exposes the MUC1 protein core on the tumor-associated form of the protein. We have previously shown that a vaccine consisting of MUC1 core peptides stimulates a tumor-specific immune response. However, this immune response is dampened by the immunosuppressive microenvironment within breast tumors. Thus, in the present study, we investigated the effectiveness of MUC1 vaccination in combination with four different drugs that inhibit different components of the COX pathway: indomethacin (COX-1 and COX-2 inhibitor), celecoxib (COX-2 inhibitor), 1-methyl tryptophan (indoleamine 2,3 dioxygenase inhibitor), and AH6809 (prostaglandin E2 receptor antagonist). These treatment regimens were explored for the treatment of orthotopic MUC1-expressing breast tumors in mice transgenic for human MUC1. We found that the combination of vaccine and indomethacin resulted in a significant reduction in tumor burden. Indomethacin did not increase tumor-specific immune responses over vaccine alone, but rather appeared to reduce the proliferation and increase apoptosis of tumor cells, thus rendering them susceptible to immune cell killing.

CAR T Cells Targeting the Tumor MUC1 Glycoprotein Reduce Triple-Negative Breast Cancer Growth.

Antibody-derived chimeric antigen receptor (CAR) T cell therapy has achieved gratifying breakthrough in hematologic malignancies but has shown limited success in solid tumor immunotherapy. Monoclonal antibody, TAB004, specifically recognizes the aberrantly glycosylated tumor form of MUC1 (tMUC1) in all subtypes of breast cancer including 95% of triple-negative breast cancer (TNBC) while sparing recognition of normal tissue MUC1. We transduced human T cells with MUC28z, a chimeric antigen receptor comprising of the scFv of TAB004 coupled to CD28 and CD3ζ. MUC28z was well-expressed on the surface of engineered activated human T cells. MUC28z CAR T cells demonstrated significant target-specific cytotoxicity against a panel of human TNBC cells. Upon recognition of tMUC1 on TNBC cells, MUC28z CAR T cells increased production of Granzyme B, IFN-γ and other Th1 type cytokines and chemokines. A single dose of MUC28z CAR T cells significantly reduced TNBC tumor growth in a xenograft model. Thus, MUC28z CAR T cells have high therapeutic potential against tMUC1-positive TNBC tumors with minimal damage to normal breast epithelial cells.

Exciton dissociation in an NIR-active triohybrid nanocrystal leading to efficient generation of reactive oxygen species.

Lead sulfide (PbS) colloidal quantum dots (QDs) are emerging materials for fundamental studies because of their potential application in near infrared (NIR) light harvesting technologies. However, inefficient electron separation, facile charge recombination and defect state trapping of photoexcited carriers are reported as limitations of the PbS QDs to achieve efficient energy conversion. In the present study, we have synthesized a triohybrid by assembling a semiconductor titanium dioxide (TiO2), an organic oxidizing molecule phenothiazine (PTZ) and PbS QDs. The triohybrid along with PbS_TiO2 and PbS_PTZ hybrids has been characterized and the attachment of different components is verified by spectroscopic and microscopic techniques. The interfacial dynamics of the photoexcited carriers in the PbS_TiO2 and PbS_PTZ hybrids have been investigated separately using steady state and time resolved photoluminescence (TRPL) measurements. The photoinduced electron transfer (PET) from the PbS QD to the conduction band (CB) of TiO2 and photoinduced hole transfer (PHT) from the valence band (VB) of the QD to the highest occupied molecular orbital (HOMO) of PTZ have been observed and correlated mechanistically to the energy level alignments obtained from cyclic voltammetric (CV) analysis. The PTZ molecule is also found to act as a surface defect passivator of the PbS QD. Finally, simultaneous exciton dissociation and reduced back recombination phenomena have been correlated with a higher reactive oxygen species (ROS) generation activity of the triohybrid than the other two, under IR light irradiation. Thus, a detailed investigation of carrier dynamics and the mechanism of higher NIR light activity for a novel nanohybrid is explored and analyzed which could be beneficial for NIR catalysis or antibacterial activities.

A tumor specific antibody to aid breast cancer screening in women with dense breast tissue.

Screening for breast cancer has predominantly been done using mammography. Unfortunately, mammograms miss 50% cancers in women with dense breast tissue. Multi-modal screenings offer the best chance of enhancing breast cancer screening effectiveness. We evaluated the use of TAB004, an antibody that recognizes the tumor form of the glycoprotein MUC1 (tMUC1), to aid early detection of breast cancer. Our experimental approach was to follow tMUC1 from the tissue into circulation. We found that 95% of human breast cancer tissues across all subtypes stained positive for TAB004. In breast cancer cell lines, we showed that the amount of tMUC1 released from tumor cells is proportional to the cell's tMUC1 expression level. Finally, we showed that TAB004 can be used to assess circulating tMUC1 levels, which when monitored in the context of cancer immunoediting, can aid earlier diagnosis of breast cancer regardless of breast tissue density. In a blinded pilot study with banked serial samples, tMUC1 levels increased significantly up to 2 years before diagnosis. Inclusion of tMUC1 monitoring as part of a multi-modal screening strategy may lead to earlier stage diagnosis of women whose cancers are missed by mammography.