Inactivation of multidrug-resistant bacteria and bacterial spores and generation of high-potency bacterial vaccines using ultrashort pulsed lasers.

Multidrug-resistant organisms (MDROs) represent a continuing healthcare crisis with no definitive solution to date. An alternative to antibiotics is the development of therapies and vaccines using biocompatible physical methods such as ultrashort pulsed (USP) lasers, which have previously been shown to inactivate pathogens while minimizing collateral damage to human cells, blood proteins, and vaccine antigens. Here we demonstrate that visible USP laser treatment results in bactericidal effect (≥3-log load reduction) against clinically significant MDROs, including methicillin-resistant Staphylococcus aureus and extended spectrum beta-lactamase-producing Escherichia coli.

Selective photonic disinfection of cell culture using a visible ultrashort pulsed laser.

Microbial contamination of cell culture is a major problem encountered both in academic labs and in the biotechnology/pharmaceutical industries. A broad spectrum of microbes including mycoplasma, bacteria, fungi, and viruses are the causative agents of cell culture contamination. Unfortunately, the existing disinfection techniques lack selectivity and/or lead to the development of drug-resistance, and more importantly there is no universal method to address all microbes.

Chemical-free inactivated whole influenza virus vaccine prepared by ultrashort pulsed laser treatment.

There is an urgent need for rapid methods to develop vaccines in response to emerging viral pathogens. Whole inactivated virus (WIV) vaccines represent an ideal strategy for this purpose; however, a universal method for producing safe and immunogenic inactivated vaccines is lacking. Conventional pathogen inactivation methods such as formalin, heat, ultraviolet light, and gamma rays cause structural alterations in vaccines that lead to reduced neutralizing antibody specificity, and in some cases, disastrous T helper type 2-mediated immune pathology.

Pathogen reduction in human plasma using an ultrashort pulsed laser.

Pathogen reduction is a viable approach to ensure the continued safety of the blood supply against emerging pathogens. However, the currently licensed pathogen reduction techniques are ineffective against non-enveloped viruses such as hepatitis A virus, and they introduce chemicals with concerns of side effects which prevent their widespread use. In this report, we demonstrate the inactivation of both enveloped and non-enveloped viruses in human plasma using a novel chemical-free method, a visible ultrashort pulsed laser.

Ultrashort pulsed laser treatment inactivates viruses by inhibiting viral replication and transcription in the host nucleus.

Ultrashort pulsed laser irradiation is a new method for virus reduction in pharmaceuticals and blood products. Current evidence suggests that ultrashort pulsed laser irradiation inactivates viruses through an impulsive stimulated Raman scattering process, resulting in aggregation of viral capsid proteins. However, the specific functional defect(s) in viruses inactivated in this manner have not been demonstrated.

Studies of inactivation mechanism of non-enveloped icosahedral virus by a visible ultrashort pulsed laser.

Background: Low-power ultrashort pulsed (USP) lasers operating at wavelengths of 425 nm and near infrared region have been shown to effectively inactivate viruses such as human immunodeficiency virus (HIV), M13 bacteriophage, and murine cytomegalovirus (MCMV). It was shown previously that non-enveloped, helical viruses such as M13 bacteriophage, were inactivated by a USP laser through an impulsive stimulated Raman scattering (ISRS) process. Recently, enveloped virus like MCMV has been shown to be inactivated by a USP laser via protein aggregation induced by an ISRS process.

Inactivation of enveloped virus by laser-driven protein aggregation.

Ultrafast lasers in the visible and near-infrared range have emerged as a potential new method for pathogen reduction of blood products and pharmaceuticals. However, the mechanism of enveloped virus inactivation by this method is unknown. We report the inactivation as well as the molecular and structural effects caused by visible (425 nm) femtosecond laser irradiation on murine cytomegalovirus (MCMV), an enveloped, double-stranded DNA virus.

Prospects for a novel ultrashort pulsed laser technology for pathogen inactivation.

The threat of emerging pathogens and microbial drug resistance has spurred tremendous efforts to develop new and more effective antimicrobial strategies. Recently, a novel ultrashort pulsed (USP) laser technology has been developed that enables efficient and chemical-free inactivation of a wide spectrum of viral and bacterial pathogens. Such a technology circumvents the need to introduce potentially toxic chemicals and could permit safe and environmentally friendly pathogen reduction, with a multitude of possible applications including the sterilization of pharmaceuticals and blood products, and the generation of attenuated or inactivated vaccines.

Studies of inactivation of encephalomyocarditis virus, M13 bacteriophage, and Salmonella typhimurium by using a visible femtosecond laser: insight into the possible inactivation mechanisms.

We report experimental results on the inactivation of encephalomyocarditis virus, M13 bacteriophage, and Salmonella typhimurium by a visible femtosecond laser. Our results suggest that inactivation of virus and bacterium by a visible femtosecond laser involves completely different mechanisms. Inactivation of viruses by a visible femtosecond laser involves the breaking of hydrogen∕hydrophobic bonds or the separation of the weak protein links in the protein shell of a viral particle.

Photonic approach to the selective inactivation of viruses with a near-infrared subpicosecond fiber laser.

We report a photonic approach for selective inactivation of viruses with a near-infrared subpicosecond laser. We demonstrate that this method can selectively inactivate viral particles ranging from nonpathogenic viruses such as the M13 bacteriophage and the tobacco mosaic virus to pathogenic viruses such as the human papillomavirus and the human immunodeficiency virus (HIV). At the same time, sensitive materials such as human Jurkat T cells, human red blood cells, and mouse dendritic cells remain unharmed.

Simulations of impulsive laser scattering of biological protein assemblies: application to M13 bacteriophage.

We develop a theoretical framework, based on a bond-polarizability model, for simulating the impulsive force experienced on a protein or an assembly of proteins from a pulsed light source by coupling the laser electric field to an atomic distortion. The mechanism is impulsive stimulated Raman scattering (ISRS) where mechanical distortions produce variation in the electronic polarization through atomic displacements similar to vibrational Raman scattering. The magnitude of the impulsive force is determined from the empirical two-body bond-polarizability model and the intensity of the incident light.

Studies of electron-phonon and phonon-phonon interactions in InN using ultrafast Raman spectroscopy.

Subpicosecond time-resolved Raman spectroscopy has been employed to investigate electron-phonon interactions and phonon dynamics in InN. The electron-longitudinal optical phonon scattering rate and the decay dynamics of longitudinal optical phonons in InN have been directly measured. Our results indicate that hot-phonon effects can play an important role in the electron relaxation and transport in InN.

Inactivation of viruses by laser-driven coherent excitations via impulsive stimulated Raman scattering process.

The inactivation of viruses such as M13 bacteriophages subject to excitations by a very low power visible femtosecond laser has been studied. Our experimental results show that for a visible femtosecond laser having lambda = 425 nm and a pulse width of 100 fs, the M13 bacteriophages are inactivated when the laser power density is greater than or equal to 49 MW/cm(2). The medium lethal laser power density (LD(50)) is 51.

Raman intensity and spectra predictions for cylindrical viruses.

A theoretical framework for predicting low frequency Raman vibrational spectra of viral capsids is presented and applied to the M13 bacteriophage. The method uses a continuum elastic theory for the vibrational modes and a bond-charge polarizability model of an amorphous material to roughly predict the Raman intensities. Comparison is made to experimental results for the M13 bacteriophage virus.

Inactivation of viruses by coherent excitations with a low power visible femtosecond laser.

Background: Resonant microwave absorption has been proposed in the literature to excite the vibrational states of microorganisms in an attempt to destroy them. But it is extremely difficult to transfer microwave excitation energy to the vibrational energy of microorganisms due to severe absorption of water in this spectral range. We demonstrate for the first time that, by using a visible femtosecond laser, it is effective to inactivate viruses such as bacteriophage M13 through impulsive stimulated Raman scattering.

Geldanamycin inhibits hemorrhage-induced increases in caspase-3 activity: role of inducible nitric oxide synthase.

Hemorrhage has been shown to increase inducible nitric oxide synthase (iNOS) and deplete ATP levels in tissues and geldanamycin limits both processes. Moreover, it is evident that inhibition of iNOS reduces caspase-3 and increases survival. Thus we sought to identify the molecular events responsible for the beneficial effect of geldanamycin.

Probing the low-frequency vibrational modes of viruses with Raman scattering--bacteriophage M13 in water.

Raman spectroscopy is used to study low-wave-number ( View Article and Find Full Text PDF

Biology of hypoxia.

Hypoxia is an often seen problem resulting from conditions such as ischemia, hemorrhage, stroke, premature birth, and other cardiovascular difficulties. To find useful remedies that are capable of ameliorating its casualty is an essential effort. Although the underlying mechanisms of the hypoxia-induce injury and cell death are still not fully understood, it has been shown that hypoxia induces nitric oxide (NO) overproduction and inducible nitric oxide synthase (iNOS) overexpression that play important roles in producing injury including increases in polymorphonuclear neutrophils (PMN) infiltration to injured tissues and leukotriene B4 (LTB4) generation.

Lycopene is more potent than beta carotene in the neutralization of singlet oxygen: role of energy transfer probed by ultrafast Raman spectroscopy.

Energy transfer processes between beta carotene, lycopene, and singlet oxygen ((1)O(2)) have been studied by ultrafast Raman spectroscopy. Our experimental results demonstrate that during the neutralization of singlet oxygen by beta carotene the excitation energy of singlet oxygen is transferred directly to the first excited electronic state S(1) of beta carotene. In contrast, the excitation energy of singlet oxygen is transferred directly to the ground excited vibronic state S(0) of lycopene.

Observation of the low frequency vibrational modes of bacteriophage M13 in water by Raman spectroscopy.

Background: Recently, a technique which departs radically from conventional approaches has been proposed. This novel technique utilizes biological objects such as viruses as nano-templates for the fabrication of nanostructure elements. For example, rod-shaped viruses such as the M13 phage and tobacco mosaic virus have been successfully used as biological templates for the synthesis of semiconductor and metallic nanowires.

Enhancement of band gap emission stimulated by defect loss.

Defect radiation has been always considered as the most important loss for an emitter based on band gap emission. Here, we propose a novel approach which goes against this conventional wisdom. Based on the resonance effect between the surface plasmon of metal nanoparticles and defect emission, it is possible to convert the useless defect radiation to the useful excitonic emission with a giant enhancement factor.