Biomimetic-Membrane-Protected Plasmonic Nanostructures as Dual-Modality Contrast Agents for Correlated Surface-Enhanced Raman Scattering and Photoacoustic Detection of Hidden Tumor Lesions.

Optical imaging and spectroscopic modalities are of considerable current interest for in vivo cancer detection and image-guided surgery, but the turbid or scattering nature of biomedical tissues has severely limited their abilities to detect buried or occluded tumor lesions. Here we report the development of a dual-modality plasmonic nanostructure based on colloidal gold nanostars (AuNSs) for simultaneous surface-enhanced Raman scattering (SERS) and photoacoustic (PA) detection of tumor phantoms embedded (hidden) in ex vivo animal tissues. By using red blood cell membranes as a naturally derived biomimetic coating, we show that this class of dual-modality contrast agents can provide both Raman spectroscopic and PA signals for the detection and differentiation of hidden solid tumors with greatly improved depths of tissue penetration. Compared to previous polymer-coated AuNSs, the biomimetic coatings are also able to minimize protein adsorption and cellular uptake when exposed to human plasma without compromising their SERS or PA signals. We further show that tumor-targeting peptides (such as cyclic RGD) can be noncovalently inserted for targeting the ανß3-integrin receptors expressed on metastatic cancer cells and tracked via both SERS and PA imaging (PAI). Finally, we demonstrate image-guided resections of tumor-mimicking phantoms comprising metastatic tumor cells buried under layers of skin and fat tissues (6 mm in thickness). Specifically, PAI was used to determine the precise tumor location, while SERS spectroscopic signals were used for tumor identification and differentiation. This work opens the possibility of using these biomimetic dual-modality nanoparticles with superior signal and biological stability for intraoperative cancer detection and resection.

Properties of Carbon Dots versus Small Molecules from "Bottom-up" Synthesis.

A major challenge in the "bottom-up" solvothermal synthesis of carbon dots (CDs) is the removal of small-molecule byproducts, noncarbonized polyamides, or other impurities that confound the optical properties. In previously reported benzene diamine-based CDs, the observed fluorescence signal already has been shown to arise from free small molecules, not from nanosized carbonized dots. Here we have unambiguously identified the small-molecule species in the synthesis of CDs starting with several isomers of benzene diamine by directly matching their NMR, mass spectrometry, and optical data with commercially available small organic molecules. By combining dialysis and chromatography, we have sufficiently purified the CD reaction mixtures to measure the CD size by TEM and STM, elemental composition, optical absorption and emission, and single-particle blinking dynamics. The results can be rationalized by electronic structure calculations on small model CDs. Our results conclusively show that the purified benzene diamine-based CDs do not emit red fluorescence, so the quest for full-spectrum fluorescence from isomers of a single precursor molecule remains open.

Biodegradable and switchable near-infrared fluorescent probes for hypoxia detection.

Aims: Among solid tumors, hypoxia is a common characteristic and responsible for chemotherapeutic resistance. Hypoxia-sensitive imaging probes are therefore essential for early tumor detection, growth monitoring and drug-response evaluation. Despite significant efforts, detecting hypoxic oxygen levels remains challenging. Materials & methods: This paper demonstrates the use of an amine-rich carbon dot probe functionalized with an imidazole group that exhibits reversible fluorescence switching in normoxic and hypoxic environments. Results & conclusion: We demonstrate the ability to emit near-infrared light only under hypoxic conditions. The probes are found to be biodegradable in the presence of human digestive enzymes such as lipase. Ex vivo tissue imaging experiments revealed promising near-infrared signals even at a depth of 5 mm for the probe under ex vivo imaging conditions.

Hypoxia is the state where oxygen is not adequately available at the tissue level and is the common cause of resistance toward chemotherapeutics. Hence, probes that can detect hypoxia are important in detecting early tumor progression. Here in this paper, we have developed a fluorescent probe which helps in determining normoxic and hypoxic environments. This probe emits near-infrared light only under hypoxic conditions. The phenomena have been established herein by extensive experiments.

Cell-Membrane Coated Nanoparticles for Tumor Delineation and Qualitative Estimation of Cancer Biomarkers at Single Wavelength Excitation in Murine and Phantom Models.

Real-time guidance through fluorescence imaging improves the surgical outcomes of tumor resections, reducing the chances of leaving positive margins behind. As tumors are heterogeneous, it is imperative to interrogate multiple overexpressed cancer biomarkers with high sensitivity and specificity to improve surgical outcomes. However, for accurate tumor delineation and ratiometric detection of tumor biomarkers, current methods require multiple excitation wavelengths to image multiple biomarkers, which is impractical in a clinical setting. Here, we have developed a biomimetic platform comprising near-infrared fluorescent semiconducting polymer nanoparticles (SPNs) with red blood cell membrane (RBC) coating, capable of targeting two representative cell-surface biomarkers (folate, αυß3 integrins) using a single excitation wavelength for tumor delineation during surgical interventions. We evaluate our single excitation ratiometric nanoparticles in in vitro tumor cells, ex vivo tumor-mimicking phantoms, and in vivo mouse xenograft tumor models. Favorable biological properties (improved biocompatibility, prolonged blood circulation, reduced liver uptake) are complemented by superior spectral features: (i) specific fluorescence enhancement in tumor regions with high tumor-to-normal tissue (T/NT) ratios in ex vivo samples and (ii) estimation of cell-surface tumor biomarkers with single wavelength excitation providing insights about cancer progression (metastases). Our single excitation, dual output approach has the potential to differentiate between the tumor and healthy regions and simultaneously provide a qualitative indicator of cancer progression, thereby guiding surgeons in the operating room with the resection process.

1 H, 15 N and 13 C chemical shift assignments of the N-terminal domain of the two isoforms of the human apolipoprotein E.

Apolipoprotein E (ApoE) is one of the major lipid transporters in humans. It is also implicated in pathological conditions like Alzheimer's and cardiovascular diseases. The N-terminal domain of ApoE binds low-density lipoprotein receptors (LDLR) while the C-terminal domain binds to the lipid. I report the backbone and aliphatic side-chain NMR chemical shifts of the N-terminal domain of two isoforms of ApoE, namely ApoE3 NTD (BMRB No. 51,122) and ApoE4 NTD (BMRB No. 51,123) at pH 3.5 (20 °C).

Non invasive assessment of human oocytes and embryos in assisted reproduction: Review on present practices and future trends.

Oocyte and embryo grading is one of the most important steps in assisted reproductive technology to identify the best among cultured embryos for transfer or vitrification. The most commonly used non-invasive method is morphological assessment of gametes and embryos using a microscope. This method despite being simple and cost-effective has interobserver and intraobserver variability and subjectivity and has little to offer about the physiological state of embryos. This review sourced research articles and reviews pertaining to other non-invasive assessment methodologies from Medline and PubMed to collate latest technologies in vogue and identify novel methodologies of the future. The review assesses the current understanding in oocyte and embryo grading and touches upon novel non-invasive techniques and potential biomarkers to identify the best embryo. The latest available literature on time-lapse imaging, hierarchical algorithms, omics (consisting of proteomics and secretomes), miRNAs, mitochondrial RNAs and artificial intelligence has been accessed to summarize the enormous information available, to identify gaps in current interpretations, to identify emerging technologies and to provide direction for future research. This review will greatly benefit anyone practicing assisted reproduction and clinical embryology.

Rational Design of Surface-State Controlled Multicolor Cross-Linked Carbon Dots with Distinct Photoluminescence and Cellular Uptake Properties.

We disclose for the first time a facile synthetic methodology for the preparation of multicolor carbon dots (CDs) from a single source barring any chromatographic separations. This was achieved via sequential intraparticle cross-linking of surface abundant carboxylic acid groups on the CDs synthesized from a precursor to control their photoluminescence (PL) spectra as well as affect their degree of cellular internalization in cancer cells. The change in PL spectra with sequential cross-linking was projected by theoretical density functional theory (DFT) studies and validated by multiple characterization tools such as X-ray photoelectron spectroscopy (XPS), PL spectroscopy, ninhydrin assay, etc. The variation in cellular internalization of these cross-linked CDs was demonstrated using inhibitor assays, confocal microscopy, and flow cytometry. We supplemented our findings with high-resolution dark-field imaging to visualize and confirm the colocalization of these CDs into distinct intracellular compartments. Finally, to prove the surface-state controlled PL mechanisms of these cross-linked CDs, we fabricated a triple-channel sensor array for the identification of different analytes including metal ions and biologically relevant proteins.

Machine Learning-Assisted Array-Based Detection of Proteins in Serum Using Functionalized MoS2 Nanosheets and Green Fluorescent Protein Conjugates.

Abnormal concentrations of a specific protein or the presence of some biomarker proteins may indicate life-threatening diseases. Pattern-based detection of specific analytes using affinity-regulated receptors is one of the potential alternatives to specific antigen-antibody-based detection. In this report, we have schemed a sensor array by using various functionalized two-dimensional (2D)-MoS2 nanosheets and green fluorescent protein (GFP) as the receptor and the signal transducer, respectively. Two-dimensional MoS2 has been used as a promising candidate for recognition of the bioanalytes because of its high surface-to-volume ratio compared to those of other nanomaterials. Easy surface tunability of this material provides additional advantages to analyze the target of interest. The optimized 2D-MoS2-GFP conjugates are able to discriminate 15 different proteins at 50 nM concentration with a detection limit of 1 nM. Moreover, proteins in the binary mixture and in the presence of serum were discriminated successfully. Ten different proteins in serum media at relevant concentrations were classified successfully with 100% jackknifed classification accuracy, which proves the potentiality of the above system. We have also implemented and discussed the implication of using different machine learning models on the pattern recognition problem associated with array-based sensing.

A descriptive study of community-based profile and attitudes of body donors of an urban conglomeration in Western India.

BACKGROUND: Body donation is considered an altruistic act done by people for advancement of science. There has been a significant depreciation in the availability of cadavers for teaching and research all over the world. Unlike India, many countries have studied the profile and attitudes of potential body donors to improve body donation. With a huge health sector, this Indian study studied the profile and attitudes of body donors and their role in aiding science through body donation. METHODS: Four hundred thirty-one survey forms with informed consent were sent to registered body donors. The questionnaire focused on demographic parameters and their perception on body donation. The data were collated, compared and interpreted with similar studies done worldwide. RESULTS: The study shows an interesting pattern among the Indian donors in comparison to the West. An Indian donor is a male individual in 70s, highly educated and religious with good social background. Despite many similarities, there are important distinguishing features unique to our country and many myths surrounding body donation. CONCLUSION: The Indian donor is highly educated male and charitable with strong belief in God. He believes in aiding medical science through body donation.

Machine Learning-Assisted Array-Based Biomolecular Sensing Using Surface-Functionalized Carbon Dots.

Fluorescent array-based sensing is an emerging differential sensing platform for sensitive detection of analytes in a complex environment without involving a conventional "lock and key" type-specific interaction. These sensing techniques mainly rely on different optical pattern generation from a sensor array and their pattern recognition to differentiate analytes. Currently emerging, compelling pattern-recognition method, Machine Learning (ML), enables a machine to "learn" a pattern by training without having the recognition method explicitly programmed into it. Thus, ML has an enormous potential to analyze these sensing data better than widely used statistical pattern-recognition methods. Here, an array-based sensor using easy-to-synthesize carbon dots with varied surface functionality is reported, which can differentiate between eight different proteins at 100 nM concentration. The utility of using machine learning algorithms in pattern recognition of fluorescence signals from the array has also been demonstrated. In analyzing the array-based sensing data, Machine Learning algorithms like "Gradient-Boosted Trees" have achieved a 100% prediction efficiency compared to inferior-performing classical statistical method "Linear Discriminant Analysis".

A crossover comparative study to assess efficacy of competency based medical education (CBME) and the traditional structured (TS) method in selected competencies of living anatomy of first year MBBS curriculum: A pilot study.

BACKGROUND: Competency based medical education (CBME) is outcome based teaching methodology where a student learns a set of measurable competencies for early clinical exposure. Inspite of ample resources on CBME, there are limited studies on its implementation. This study will try to demonstrate improvement in the performance of students using CBME as a teaching tool over the traditional structured method (TS). METHODS: Forty student volunteers were chosen and divided into two groups. The crossover design exposed the group of students to CBME and TS spread over two periods with a wash out period in between. The intervention group was exposed to selected list of competencies in living Anatomy with feedbacks and formative assessments. The summative assessments were held at the end of each period. RESULTS: The mean scores of CBME and TS in group 1 is 130.625 and 113.65 while in group 2 is 139.425 and 112.075 respectively. The treatment and period effect is significant. Estimate of treatment effect is 22.1625. The average improvement in treatment scores is by 11%. Two tailed paired sample T test reveals significant improvement in the scores post intervention. CONCLUSION: CBME method produces better performance of the students in the competencies of living anatomy.

Exchangeable deuterons introduce artifacts in amide 15N CEST experiments used to study protein conformational exchange.

Protein molecules sample different conformations in solution and characterizing these conformations is crucial to understanding protein function. 15N CEST experiments are now routinely used to study slow conformational exchange of protein molecules between a 'visible' major state and 'invisible' minor states. These experiments have also been adapted to measure the solvent exchange rates of amide protons by exploiting the one bond deuterium isotope effect on the amide 15N chemical shifts. However at moderately high temperatures (~ 50 °C) that are sometimes required to populate protein minor conformers to levels (~ 1%) that can be detected by CEST experiments solvent H/D exchange can lead to 'dips' in low B115N CEST profiles that can be wrongly assigned to the conformational exchange process being characterized. This is demonstrated in the case of ~ 18 kDa T4 Lysozyme (T4L) at 50 °C and the ~ 11 kDa E. coli hibernation promoting factor (HPF) at 52 °C. This problem is trivially solved by eliminating the exchangeable deuterons in the solvent by using either an external D2O lock or by using a small amount (~ 1-3%) of a molecule like d6-DMSO that does not contain exchangeable deuterons to lock the spectrometer.

Simultaneous Exfoliation and Functionalization of 2H-MoS2 by Thiolated Surfactants: Applications in Enhanced Antibacterial Activity.

Two-dimensional transition metal dichalcogenides (TMDs), such as MoS2, generally exist in two different polymorphic structures, metallic (1T phase) and semiconducting (2H phase). In context of their wide spectrum of applications ranging from electronic to biomedicine, the aspects of ligand conjugation and solution processability are highly significant. In addition, the assessment of their antibacterial property and biocompatibility is equally important to explore their biomedical applications. Here we report a new method for the exfoliation and direct functionalization of 2H-MoS2 using surfactant molecules with thiol functionality. We found that the exfoliated MoS2 using thiolated ligands are functionalized with desired functionality and the processing scheme can be extended to other TMDs. Functionalized 2H-MoS2 exhibits highly enhanced antibacterial efficiency compared to similarly functionalized metallic 1T-MoS2 against pathogenic bacteria. The newly synthesized functionalized 2H-MoS2 exhibits better hemocompatibility, which makes it suitable for in vivo applications. This convenient functionalization method opens the door for many other applications of functionalized semiconducting 2H-MoS2 and other TMDs.

Atomic resolution mechanism of ligand binding to a solvent inaccessible cavity in T4 lysozyme.

Ligand binding sites in proteins are often localized to deeply buried cavities, inaccessible to bulk solvent. Yet, in many cases binding of cognate ligands occurs rapidly. An intriguing system is presented by the L99A cavity mutant of T4 Lysozyme (T4L L99A) that rapidly binds benzene (~106 M-1s-1). Although the protein has long served as a model system for protein thermodynamics and crystal structures of both free and benzene-bound T4L L99A are available, the kinetic pathways by which benzene reaches its solvent-inaccessible binding cavity remain elusive. The current work, using extensive molecular dynamics simulation, achieves this by capturing the complete process of spontaneous recognition of benzene by T4L L99A at atomistic resolution. A series of multi-microsecond unbiased molecular dynamics simulation trajectories unequivocally reveal how benzene, starting in bulk solvent, diffuses to the protein and spontaneously reaches the solvent inaccessible cavity of T4L L99A. The simulated and high-resolution X-ray derived bound structures are in excellent agreement. A robust four-state Markov model, developed using cumulative 60 µs trajectories, identifies and quantifies multiple ligand binding pathways with low activation barriers. Interestingly, none of these identified binding pathways required large conformational changes for ligand access to the buried cavity. Rather, these involve transient but crucial opening of a channel to the cavity via subtle displacements in the positions of key helices (helix4/helix6, helix7/helix9) leading to rapid binding. Free energy simulations further elucidate that these channel-opening events would have been unfavorable in wild type T4L. Taken together and via integrating with results from experiments, these simulations provide unprecedented mechanistic insights into the complete ligand recognition process in a buried cavity. By illustrating the power of subtle helix movements in opening up multiple pathways for ligand access, this work offers an alternate view of ligand recognition in a solvent-inaccessible cavity, contrary to the common perception of a single dominant pathway for ligand binding.

2D-MoS2-Based ß-Lactamase Inhibitor for Combination Therapy against Drug-Resistant Bacteria.

Despite the remarkable improvement in modern medicine, the ever-increasing abundance of antibiotic-resistant microorganisms remains a catastrophic threat to global health care. ß-Lactamase is playing one of the major roles in antibiotic resistance by making the conventional antibacterial agents abortive by destroying their lactam ring. The combination therapy of traditional antibiotics along with ß-lactamase inhibitors is a potential solution to this problem. In this work, we have screened various functionalized two-dimensional molybdenum disulfide (2D-MoS2) nanomaterials as enzyme inhibitors that effectively bind with ß-lactamase enzyme and reveal competitive inhibition. Among these, carboxylate-functionalized negatively charged 2D-MoS2 is the most potent inhibitor, and in vitro combinatorial application of this with conventional antibiotics has been able to remarkably suppress relevant drug-resistant bacterial growth rate. This study will help to further explore different surface-functionalized 2D nanomaterials with improved ß-lactamase inhibition to fight against multidrug-resistant bacterial infections.

Graphene Oxide as a Carbocatalyst for a Diels-Alder Reaction in an Aqueous Medium.

The Diels-Alder (DA) reaction, a [4+2] cycloaddition reaction, is highly important in synthetic organic chemistry and is frequently used in the synthesis of natural products containing six-membered rings. Herein, we report an efficient protocol for the DA reaction between 9-hydroxymethylanthracene and N-substituted maleimides using two-dimensional graphene oxide (GO) as a heterogeneous carbocatalyst in an aqueous medium at room temperature. High yields, a wide substrate scope, low temperature, excellent functional group tolerance, atom economy, and water as a green solvent are noteworthy features of this protocol. The heterogeneous GO catalyst can be easily recovered and used multiple times without any significant loss in catalytic activity.

High Antibacterial Activity of Functionalized Chemically Exfoliated MoS2.

In view of the implications of inherent resistance of pathogenic bacteria, especially ESKAPE pathogens toward most of the commercially available antibiotics and the importance of these bacteria-induced biofilm formation leading to chronic infection, it is important to develop new-generation synthetic materials with greater efficacy toward antibacterial property. In addressing this issue, this paper reports a proof-of-principle study to evaluate the potential of functionalized two-dimensional chemically exfoliated MoS2 (ce-MoS2) toward inhibitory and bactericidal property against two representative ESKAPE pathogenic strain-a Gram-positive Staphylococcus aureus (MRSA) and a Gram-negative Pseudomonas aeruginosa. More significantly, the mechanistic study establishes a different extent of oxidative stress together with rapid membrane depolarization in contact with ce-MoS2 having ligands of varied charge and hydrophobicity. The implication of our results is discussed in the light of the lack of survivability of planktonic bacteria and biofilm destruction in vitro. A comparison with widely used small molecules and other nanomaterial-based therapeutics conclusively establishes a better efficacy of 2D ce-MoS2 as a new class of antibiotics.

Gross and microscopic study of insertion of levator palpebrae superioris and its anatomical correlation in superior palpebral crease formation and its clinical relevance.

BACKGROUND: To study insertion of LPS and correlate anatomically the formation of superior palpebral crease and its clinical relevance in section of Indian population. METHODS: Twenty-five human eyelids from cadavers ranging from 15-80 years were studied by dissection and histology. RESULTS: The levator aponeurosis traverses through interfascicular space of orbicularis oculi with twenty four inserting into subcutaneous tissue. Low septo aponeurotic sling or preaponeurotic fat was not observed. Lower one third tarsal insertion seen in twenty four specimens. Fibres become denser as it reaches the connective tissue anterior to tarsal plate. The crease was at different levels in relation to the tarsal insertion. CONCLUSION: The aponeurosis insertion is either to the skin, the subcutaneous tissue or into the interfascicular space of the orbicularis. The present study reaffirms the insertion of LPS to the subcutaneous tissue of the eyelid with an extension reaching lower one third of the tarsal plate. The tissues in the anterior tarsal plate are closely packed but to act as a single complex to form a dynamic crease has not been confirmed in the study. No gross or histological difference was observed in the skin of the eyelid in the vicinity of the crease. The pattern of insertion of LPS aponeurosis plays a direct role in the formation of superior palpebral crease an important role in surgeries for Ptosis. The eyelids studied were of Indian origin with crease, and further comparative studies needs to be done for the precise anatomical explanation of single fold eyelids.

Comparative study of anatomical specimens using plastination by araldite HY103, polypropylene resin, 6170H19 Orthocryl and silicone - A qualitative study.

BACKGROUND: Most of the organs and tissues are preserved in formalin with its own set of disadvantages. Plastination is a unique method of permanently preserving tissue in a life like state. Plastination developed by western authorities is a labour and equipment intensive affair. Most common polymer used is S10, however this study uses easily available alternative polymers for plastination. METHOD: Various polymers like Epoxy resins, Polypropylene resins, Orthocryl and silicone were used in plastinating the anatomical specimens. Specific methods were used for solid, hollow organs and brain specimens. The specimens were made to undergo stages of fixation, dehydration, impregnation and curing. The results were studied and interpreted under various parameters. RESULTS: The results were interpreted under various parameters like shrinkage, retention of colour, odour, pliability and retention of gross anatomy. The study concluded that Orthocryl and Epoxy resins retained maximum colour with minimal shrinkage while maximum discolouration was with polypropylene plastinates. Brain sections were best preserved in Orthocryl. CONCLUSION: The study concluded that indigenous methods and materials can produce quality plastinates which can be an important adjunct to traditional methods of teaching however more studies need to be done for refinement.