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.

Host-Assisted Delivery of a Model Drug to Genomic DNA: Key Information from Ultrafast Spectroscopy and in silico Study.

Drug delivery to a target without adverse effects is one of the major criteria for clinical use. Herein, we have made an attempt to explore the delivery efficacy of SDS surfactant in a monomer and micellar stage during the delivery of the model drug, Toluidine Blue (TB) from the micellar cavity to DNA. Molecular recognition of pre-micellar SDS encapsulated TB with DNA occurs at a rate constant of k1 ∼652 s-1 . However, no significant release of encapsulated TB at micellar concentration was observed within the experimental time frame. This originated from the higher binding affinity of TB towards the nano-cavity of SDS at micellar concentration which does not allow the delivery of TB from the nano-cavity of SDS micelles to DNA. Thus, molecular recognition controls the extent of DNA recognition by TB which in turn modulates the rate of delivery of TB from SDS in a concentration-dependent manner.

Picosecond-resolved fluorescence resonance energy transfer (FRET) in diffuse reflectance spectroscopy explores biologically relevant hidden molecular contacts in a non-invasive way.

The potentiality of Förster resonance energy transfer (FRET) for studying molecular interactions inside biological tissues with improved spatial (Angström) and temporal (picosecond) resolution is well established. On the other hand, the efficacy of diffuse reflectance spectroscopy (DRS) that uses optical radiation in order to determine physiological parameters including haemoglobin, and oxygen saturation is well known. Here we have made an attempt to combine diffuse reflectance spectroscopy (DRS) with picosecond-resolved FRET in order to show improvement in the exploration of molecular contacts in biological tissue models. We define the technique as ultrafast time-resolved diffuse reflectance spectroscopy (UTRDRS). The illuminated photon of the fluorophore from the surface of the tissue-mimicking layers carries the hidden information of the molecular contact. In order to investigate the validation of the Kubelka-Munk (KM) formulism for the developed UTRDRS technique in tissue phantoms, we have studied the propagation of incandescent and picosecond-laser light through several layers of cellulose membranes. While picosecond-resolved FRET in the diffuse reflected light confirms the hidden nano-contact (4.6 nm) of two different dye layers (8-anilino-1-naphthalenesulfonic acid and Nile blue), high-resolution optical microscopy on the cross-section of the layers reveals the proximity and contacts of the layers with limited spatial resolution (∼300 nm). We have also investigated two biologically relevant molecules, namely carboxyfluorescein and haemoglobin, in tissue phantom layers in order to show the efficacy of the UTRDRS technique. Overall, our studies based on UTRDRS in tissue mimicking layers may have potential applications in non-invasive biomedical diagnosis for patients suffering from skin diseases.

Deciphering the response of asymmetry in the hydrophobic chains of novel cationic lipids towards biological function.

Cationic liposomes, a type of non-viral vectors, often play the important biological function of delivering nucleic acids during cell transfection. Variations in the molecular architecture of di-alkyl dihydroxy ethyl ammonium chloride-based cationic lipids involving hydrophobic tails have been found to influence their biological function in terms of cell transfection efficiency. For example, liposomes based on a cationic lipid (Lip1814) with asymmetry in the hydrophobic chains were found to display higher transfection efficacy in cultured mammalian cell lines than those comprising of symmetric Lip1818 or asymmetric Lip1810. The effect of variations in the molecular architecture of the cationic lipids on the biological activity of liposomes has been explored here via the photophysical studies of 8-anilino-1-naphthalenesulphonate (ANS) and Nile Red (NR) in three cationic liposomes, namely Lip1810, Lip1814 and Lip1818. Time-resolved fluorescence of ANS revealed reduced hydration at the lipid-water interface and enhanced relaxation dynamics of surface water (lipid headgroup bound water molecules) in Lip1810- and Lip1814-based liposomes in the presence of cholesterol. As the probe ANS failed to be incorporated into the lipid-water interface of Lip1818 due to the significantly high rigidity of these liposomes, no information concerning the extent of hydration of the lipid-water interface or the interfacial water dynamics could be obtained. Time-resolved polarization-gated anisotropy measurements of NR in the presence of cholesterol revealed the rigidity of the cationic liposomes to be increasing in the order of Lip1810 < Lip1814 < Lip1818. In the presence of cholesterol, moderately higher rigidity, reduced membrane hydration and enhanced relaxation dynamics of the interfacial water molecules gave rise to the superior cell transfection efficacy of Lip1814-based cationic liposomes than those of the highly flexible Lip1810 or the highly rigid Lip1818.

Modulation of Kinetic Pathways of Enzyme-Substrate Interaction in a Microfluidic Channel: Nanoscopic Water Dynamics as a Switch.

Enzyme-mediated catalysis is attributed to enzyme-substrate interactions, with models such as "induced fit" and "conformational selection" emphasizing the role of protein conformational transitions. The dynamic nature of the protein structure, thus, plays a crucial role in molecular recognition and substrate binding. As large-scale protein motions are coupled to water motions, hydration dynamics play a key role in protein dynamics, and hence, in enzyme catalysis. Here, microfluidic techniques and time-dependent fluorescence Stokes shift (TDFSS) measurements are employed to elucidate the role of nanoscopic water dynamics in the interaction of an enzyme, α-Chymotrypsin (CHT), with a substrate, Ala-Ala-Phe-7-amido-4-methylcoumarin (AMC) in the cationic reverse micelles of benzylhexadecyldimethylammonium chloride (BHDC/benzene) and anionic reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT/benzene). The kinetic pathways unraveled from the microfluidic setup are consistent with the "conformational selection" fit for the interaction of CHT with AMC in the cationic reverse micelles, whereas an "induced fit" mechanism is indicated for the anionic reverse micelles. In the cationic reverse micelles of BHDC, faster hydration dynamics (≈550 ps) aid the pathway of "conformational selection", whereas in the anionic reverse micelles of AOT, the significantly slower dynamics of hydration (≈1600 ps) facilitate an "induced fit" mechanism for the formation of the final enzyme-substrate complex. The role of water dynamics in dictating the mechanism of enzyme-substrate interaction becomes further manifest in the neutral reverse micelles of Brij-30 and Triton X-100. In the former, the faster water dynamics aid the "conformational selection" pathway, whereas the significantly slower dynamics of water molecules in the latter are conducive to the "induced fit" mechanism in the enzyme-substrate interaction. Thus, nanoscopic water dynamics act as a switch in modulating the pathway of recognition of an enzyme (CHT) by the substrate (AMC) in reverse micelles.

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.

Label-Free Physical-Analytical Techniques Reveal Epigenetic Modifications of Breast Cancer Chromosomes.

Epigenetic dysregulation including DNA methylation and histone modifications is being increasingly recognized as a promising biomarker for the diagnosis and prognosis of cancer. Herein, we devised a label-free analytical toolbox comprising IR, UV-vis, CD spectroscopy, and cyclic voltammetry, which is capable to differentiate significantly hyper-methylated breast cancer chromosomes from the normal breast epithelial counterparts.

Synthesis and characterization of a nano-formulation for long lasting sterilization effect.

The current COVID-19 pandemic has increased the use of alcohol based hand sanitisers globally. These available alcohol based sanitisers cannot provide an antibacterial effect for an extended period of time, after the evaporation of ethanol. Hence, the need for a sanitiser with an anti-microbial activity combined with a long lasting effect is the need of the hour. In this study, we report the synthesis of a long lasting sanitiser from ozonated omega 9 fatty acid esters in an ethanolic medium. The formed vesicles made of the fatty acids have been characterized by DLS, Zeta potential, and time resolved fluorescence anisotropy studies. Ethanol although, provides an antibacterial effect, the effect is more pronounced in our prepared formulation owing to its high peroxide value that generates additional oxidative stress. Finally, this additional antimicrobial effect will have relevance in the current COVID-19 scenario in providing a long lasting hand sanitiser.

A novel molecular reporter for probing protein DNA recognition: An optical spectroscopic and molecular modeling study.

A novel benzo[a]phenoxazine-based fluorescent dye LV2 has been employed as a molecular reporter to probe recognition of a linker histone protein H1 by calf-thymus DNA (DNA). Fluorescence lifetime of LV2 buried in the globular domain of H1 (∼2.1 ns) or in the minor groove of DNA (∼0.93 ns) increases significantly to 2.65 ns upon interaction of the cationic protein with DNA indicating formation of the H1-DNA complex. The rotational relaxation time of the fluorophore buried in the globular domain of H1 increases significantly from 2.2 ns to 8.54 ns in the presence of DNA manifesting the recognition of H1 by DNA leading to formation of the H1-DNA complex. Molecular docking and molecular dynamics (MD) simulations have shown that binding of LV2 is energetically most favourable in the interface of the H1-DNA complex than in the globular domain of H1 or in the minor groove of DNA. As a consequence, orientational relaxation of the LV2 is significantly hindered in the protein-DNA interface compared to H1 or DNA giving rise to a much longer rotational relaxation time (8.54 ns) in the H1-DNA complex relative to that in pure H1 (2.2 ns) or DNA (5.7 ns). Thus, via a significant change of fluorescence lifetime and rotational relaxation time, the benzo[a]phenoxazine-based fluorescent dye buried within the globular domain of the cationic protein, or within the minor groove of DNA, reports on recognition of H1 by DNA.

Proactive pedestrian safety evaluation at urban road network level, an experience in Kolkata City, India.

In developing nations, road traffic crashes involving pedestrians have become a foremost worry. Presently, most of the road safety assessment projects and selection of interventions are still restricted to traditional methods that depend on historical crash data. However, in low and middle-income countries such as India, the availability, reliability, and accuracy of crash data are uncertain. Alternatively, Post Encroachment Time (PET) has added attention as a proximal indicator to examine pedestrian-vehicular potential crashes and address pedestrian risk under mixed traffic conditions. Hence, it will be meaningful to examine if the PET is a good substitute for pedestrian-vehicular crashes and if so, what built environment and pedestrian-level factors influence PET. In this background, the present study establishes a mathematical association between the average PET value of the urban road network level and actual crashes. Afterward, multiple linear regression models are developed to study the impact of the built environment, traffic parameters, and pedestrian-level attributes on PET. The outcomes indicate that vehicle speed, lack of enforcement, absence of traffic signal (for traffic as well as pedestrians), land use type, slum population, inadequate sight distance, pedestrian's state of crossing, and pedestrian's risky crossing behaviour substantially affect the average PET at road network-level.

Dual Sensitization via Electron and Energy Harvesting in a Nanohybrid for Improvement of Therapeutic Efficacy.

We demonstrate experimental evidence of the effect of surface plasmon resonance of noble metal nanoparticles (NPs) on the activity of a well-known biomedicinal drug in the proximity of a semiconductor having a wide band gap for enhanced photodynamic therapy (PDT) efficacy. We have chosen riboflavin (Rf) (or vitamin B2) as a model photosensitizer, attached with ZnO NPs and further attached with gold (Au) NP-decorated ZnO to increase the efficiency. The synthesized nanohybrids are characterized with the help of different microscopic, optical spectroscopic, and density functional theory (DFT)-based techniques. The DFT and time-dependent DFT-based calculations validate the experimental findings. A detailed ultrafast spectroscopic study has been carried out further to study the excited-state charge dynamics in the interface of the nanohybrids. The occurrence of a Förster resonance energy transfer (FRET) between Rf and Au has been found to be the key reason for the increased efficiency in the Rf-ZnO-Au nanohybrid over the Rf-ZnO one. The dipolar coupling between Au and Rf in the Rf-ZnO-Au nanohybrid further facilitates the generation of reactive oxygen species (ROS) in comparison to Rf-ZnO under blue-light irradiation. The greater efficiency in ROS generation by the Rf-ZnO-Au nanohybrid has been utilized for antimicrobial action against methicillin-resistant S. aureus (MRSA). Overall, the present study highlights the dual sensitization for achieving enhanced electron injection efficiency in the Rf-ZnO-Au nanohybrid in order to use it as an antibacterial agent that could be translated in PDT.

Inorganic-organic Synergy in Nano-hybrids Makes a New Class of Drug with Targeted Delivery: Glutamate Functionalization of Iron Nanoparticles for Potential Bone Marrow Delivery and X-ray Dynamic Therapy.

The direct delivery of therapeutic molecules is generally inefficient and has several problems. Hence, nanomedicines with targeted and controlled delivery applications have been an exciting field of research for the past decade. In this regard, the adjustable properties of inorganic nanoparticles like particle size distribution, ability to change the targeting ligand to have a higher affinity towards the pathologic cell, and controlled delivery properties have made them indispensable for targeted drug delivery applications. Changing the ligand on the surface of the inorganic nanoparticle can direct different therapeutic molecules to different organs like the liver, spleen, kidney, bone, and even brain. However, while the other targeted nanomedicines are well-reported, the targeting of therapeutics to bone marrow cells is sparse in the literature. Hence, the administration of therapeutics for bone-related disorders, like bone metastases, leads to several problems, such as severe systemic toxicity and suboptimal efficacy. In this direction, we have shown our successful effort to functionalise a model inorganic nanoparticle (Fe2O3) by glutamate ligand which is reported to have a high affinity towards the NMDA receptors of the bone cells. We have performed spectroscopic studies to characterize the nano-hybrid. We have shown that the cargo or the Fe2O3 nanoparticle possesses the ability to generate photo-induced reactive oxygen species (ROS), thereby leading to a therapeutic opportunity for bone metastases. In addition, the nanoparticle also possesses the ability to generate enhanced ROS on X-ray irradiation, which may provide a new strategy for bone metastases and cancer therapy. Also, this paper reviews the advancement in the drug delivery applications of inorganic nanoparticles and highlights the crosstalk between the inorganic nanoparticles with the conjugated targeting ligand for efficient delivery applications.

Interaction of a Jaundice Marker Molecule with a Redox-Modulatory Nano-Hybrid: A Combined Electrochemical and Spectroscopic Study toward the Development of a Theranostic Tool.

This study explores a combined electrochemical and spectroscopic approach to investigate the degradation of bilirubin, a molecular marker of jaundice in humans using a biocompatible nanohybrid (citrate-functionalized Mn3 O4 nanohybrid; C-Mn3 O4 NH). The approach is aimed at the development of a facile theranostic tool for treatment, detection, and prognosis of jaundice. Linear sweep voltammetry (LSV) studies on bilirubin, C-Mn3 O4 NH, a model carrier protein, and its complex with bilirubin reveal the efficacy of the nanohybrid for both degradation and detection of bilirubin. Furthermore, spectroscopic studies depict that distal electron transfer to be the probable mechanism behind the observed bilirubin degradation in physiological milieu.

A Mechanoelastic Glimpse on Hyaluronan-Coated Extracellular Vesicles.

Cancer cells secrete extracellular vesicles (EVs) covered with a carbohydrate polymer, hyaluronan (HA), linked to tumor malignancy. Herein, we have unravelled the contour lengths of HA on a single cancer cell-derived EV surface using single-molecule force spectroscopy (SMFS), which divulges the presence of low molecular weight HA (LMW-HA < 200 kDa). We also discovered that these LMW-HA-EVs are significantly more elastic than the normal cell-derived EVs. This intrinsic elasticity of cancer EVs could be directly allied to the LMW-HA abundance and associated labile water network on EV surface as revealed by correlative SMFS, hydration dynamics with fluorescence spectroscopy, and molecular dynamics simulations. This method emerges as a molecular biosensor of the cancer microenvironment.

The extent of ionization in simulations of radio-loud AGNs impacting kpc gas discs.

We use the results of relativistic hydrodynamic simulations of jet-interstellar medium (ISM) interactions in a galaxy with a radio-loud AGN to quantify the extent of ionization in the central few kpcs of the gaseous galactic disc. We perform post-process radiative transfer of AGN radiation through the simulated gaseous jet-perturbed disc to estimate the extent of photo-ionization by the AGN with an incident luminosity of 1045 erg s-1. We also map the gas that is collisionally ionized due to shocks driven by the jet. The analysis was carried out for simulations with similar jet power (1045 erg s-1) but different jet orientations with respect to the gas disc. We find that the shocks from the jets can ionize a significant fraction (up to 33 [Formula: see text]) of dense gas ([Formula: see text]) in the disc, and that the jets clear out the central regions of gas for AGN radiation to penetrate to larger distances in the disc. Jets inclined towards the disc plane couple more strongly with the ISM and ionize a larger fraction of gas in the disc as compared to the vertical jet. However, similar to previous studies, we find that the AGN radiation is quickly absorbed by the outer layers of dense clouds in the disc, and is not able to substantially ionize the disc on a global scale. Thus, compared to jet-ISM interactions, we expect that photo-ionization by the AGN radiation only weakly affects the star-formation activity in the central regions of the galactic disc (≲ 1 kpc), although the jet-induced shocks can spread farther out.

Decoding the Kinetic Pathways toward a Lipid/DNA Complex of Alkyl Alcohol Cationic Lipids Formed in a Microfluidic Channel.

Complexes of cationic liposomes with DNA have emerged as promising nonviral vectors for delivering genetic information into cells for gene therapy. Kinetics of the liposome/DNA complex (lipoplex) formation on a millisecond time scale are studied by monitoring time evolution of fluorescence of 8-anilino-1-naphthalene sulfonic acid (ANS) and ethidium bromide (EtBr) in a continuous flow microfluidic channel coupled to a fluorescence microscope. The formation of lipoplexes between calf thymus DNA and liposomes based on two novel cationic lipids (Lip1810 and Lip1814) are found to follow a two-step process with kinetic constants for the Lip1814/DNA complex (k1 = 1120-1383 s-1, k2 = 0.227-1.45 s-1) being significantly different from those (k1 = 68.53-98.5 s-1, k2 = 32.3-60.19 s-1) corresponding to formation of the Lip1810/DNA complex. The kinetic pathway leading to the formation of Lip1814/DNA complex is diffusion-controlled whereas the formation of Lip1810/DNA complex occurs by a conformational rearrangement-controlled pathway. The observed difference in the kinetics of lipoplex formation likely originates from different structures of the lipid/DNA complexes.

"Seeing" invisible volatile organic compound (VOC) marker of urinary bladder cancer: A development from bench to bedside prototype spectroscopic device.

Urinary bladder cancer (UBC) is one of the most common cancers and has notoriously high risk of recurrence and mortality across the globe. Current clinical initial diagnostic approaches are either invasive or lacks sensitivity. In this study, an attempt has been made to invent a cost-effective, novel, portable diagnostic device based on the environmental sensitive fluorophores namely Nile Red (NR), Eosin Y (EY) and Rose Bengal (RB). They act as sensing agents for detecting volatile organic compounds (VOC) exclusively present in the urine sample of UBC patients and differentiate the UBC samples from the healthy control group. Upon exposure with a particular group of VOCs, a significant amount of increment in fluorescence intensities of NR, EY and RB were detected and recorded in our indigenously developed "NABIL" device. To check the performance of NABIL, the data collected from the device was compared with the conventional techniques by arranging a clinical trial with 21 healthy controls and 52 UBC patients. With the assistance of our analysis technique based on LabVIEW platform, very high sensitivity and accuracy from healthy controls have been achieved. For UBC patients, it shows impressive diagnostic results. In addition, depending on the sample processing mechanism, NABIL device can also reveal the grade of UBC and prognosis under treatment. Overall, this study contributes a novel, non-invasive, easy-to-use, inexpensive, real-time, accurate method for selectively UBC diagnosis, which can be useful for personalized care/diagnosis and postoperative surveillance, resulting in saving more lives.

Molecular co-localization of multiple drugs in a nanoscopic delivery vehicle for potential synergistic remediation of multi-drug resistant bacteria.

Anti-microbial resistant infection is predicted to be alarming in upcoming years. In the present study, we proposed co-localization of two model drugs viz., rifampicin and benzothiazole used in anti-tuberculosis and anti-fungal agents respectively in a nanoscopic cationic micelle (cetyl triethyl ammonium bromide) with hydrodynamic diameter of 2.69 nm. Sterilization effect of the co-localized micellar formulation against a model multi-drug resistant bacterial strain viz., Methicillin resistant Staphylococcus aureus was also investigated. 99.88% decrease of bacterial growth in terms of colony forming unit was observed using the developed formulation. While Dynamic Light Scattering and Forsters Resonance Energy Transfer between benzothiazole and rifampicin show co-localization of the drugs in the nanoscopic micellar environment, analysis of time-resolved fluorescence decays by Infelta-Tachiya model and the probability distribution of the donor-acceptor distance fluctuations for 5 µM,10 µM and 15 µM acceptor concentrations confirm efficacy of the co-localization. Energy transfer efficiency and the donor acceptor distance are found to be 46% and 20.9 Å respectively. We have also used a detailed computational biology framework to rationalize the sterilization effect of our indigenous formulation. It has to be noted that the drugs used in our studies are not being used for their conventional indication. Rather the co-localization of the drugs in the micellar environment shows a completely different indication of their use in the remediation of multi-drug resistant bacteria revealing the re-purposing of the drugs for potential use in hospital-born multi-drug resistant bacterial infection.

A combined spectroscopic and molecular modeling Study on structure-function-dynamics under chemical modification: Alpha-chymotrypsin with formalin preservative.

Enzyme function can be altered via modification of its amino acid residues, side chains and large-scale domain modifications. Herein, we have addressed the role of residue modification in catalytic activity and molecular recognition of an enzyme alpha-chymotrypsin (CHT) in presence of a covalent cross-linker formalin. Enzyme assay reveals reduced catalytic activity upon increased formalin concentration. Polarization gated anisotropy studies of a fluorophore 8-Anilino-1-naphthalenesulfonic acid (ANS) in CHT show a dip rise pattern in presence of formalin which is consistent with the generation of multiple ANS binding sites in the enzyme owing to modifications of its local amino acid residues. Molecular docking study on amino acid residue modifications in CHT also indicate towards the formation of multiple ANS binding site. The docking model also predicted no change in binding behavior for the substrate Ala-Ala-Phe-7-amido-4-methylcoumarin (AMC) at the active site upon formalin induced amino acid cross-linking.

Investigating the fatal pedestrian crash occurrence in urban setup in a developing country using multiple-risk source model.

Pedestrian fatalities and injuries are a major public health burden in developing countries. In the safety literature, pedestrian crashes have been modelled predominately using single equation regression models, assuming a single underlying source of risk factors. In contrast, the fatal pedestrian crash counts at a site may be an outcome of multiple sources of risk factors, such as poor road infrastructure, land use type, traffic exposures, and operational parameters, site-specific socio-demographic characteristics, as well as pedestrians' poor risk perception and dangerous crossing behavior, which may be influenced by poor road infrastructure and lack of information, etc. However, these multiple sources are generally overlooked in traditional single equation crash prediction models. In this background, this study postulates, and demonstrates empirically, that the total fatal pedestrian crash counts at the urban road network level may arise from multiple simultaneous and interdependent sources of risk factors, rather than one. Each of these sources may distinctively contribute to the total observed crash count. Intersection-level crash data obtained from the "Kolkata Police", India, is utilized to demonstrate the present modelling methodology. The three-components mixture model and a joint econometric model are developed to predict fatal pedestrian crashes. The study outcomes indicate that the multiple-source risk models perform significantly better than the single equation regression model in terms of prediction ability and goodness-of-fit measures. Moreover, while the single equation model predicts total fatal crash counts for individual sites, the multiple risk source model predicts crash count proportions contributed by each source of risk factors and predicts crashes by a particular source.