Virus Removal and Inactivation Mechanisms during Iron Electrocoagulation: Capsid and Genome Damages and Electro-Fenton Reactions.

Virus destabilization and inactivation are critical considerations in providing safe drinking water. We demonstrate that iron electrocoagulation simultaneously removed (via sweep flocculation) and inactivated a non-enveloped virus surrogate (MS2 bacteriophage) under slightly acidic conditions, resulting in highly effective virus control (e.g., 5-logs at 20 mg Fe/L and pH 6.4 in 30 min). Electrocoagulation simultaneously generated H2O2 and Fe(II) that can potentially trigger electro-Fenton reactions to produce reactive oxygen species such as •OH and high valent oxoiron(IV) that are capable of inactivating viruses. To date, viral attenuation during water treatment has been largely probed by evaluating infective virions (as plaque forming units) or genomic damage (via the quantitative polymerase chain reaction). In addition to these existing means of assessing virus attenuation, a novel technique of correlating transmission electron micrographs of electrocoagulated MS2 with their computationally altered three-dimensional electron density maps was developed to provide direct visual evidence of capsid morphological damages during electrocoagulation. The majority of coliphages lost at least 10-60% of the capsid protein missing a minimum of one of the 5-fold and two of 3- and 2-fold regions upon electrocoagulation, revealing substantial localized capsid deformation. Attenuated total reflectance-Fourier transform infrared spectroscopy revealed potential oxidation of viral coat proteins and modification of their secondary structures that were attributed to reactive oxygen species. Iron electrocoagulation simultaneously disinfects and coagulates non-enveloped viruses (unlike conventional coagulation), adding to the robustness of multiple barriers necessary for public health protection and appears to be a promising technology for small-scale distributed water treatment.

Removal and Inactivation of an Enveloped Virus Surrogate by Iron Conventional Coagulation and Electrocoagulation.

It is imperative to understand the behavior of enveloped viruses during water treatment to better protect public health, especially in the light of evidence of detection of coronaviruses in wastewater. We report bench-scale experiments evaluating the extent and mechanisms of removal and/or inactivation of a coronavirus surrogate (ϕ6 bacteriophage) in water by conventional FeCl3 coagulation and Fe(0) electrocoagulation. Both coagulation methods achieved ∼5-log removal/inactivation of ϕ6 in 20 min. Enhanced removal was attributed to the high hydrophobicity of ϕ6 imparted by its characteristic phospholipid envelope. ϕ6 adhesion to freshly precipitated iron (hydr)oxide also led to envelope damage causing inactivation in both coagulation techniques. Fourier transform infrared spectroscopy revealed oxidative damages to ϕ6 lipids only for electrocoagulation consistent with electro-Fenton reactions. Monitoring ϕ6 dsRNA by a novel reverse transcription quantitative polymerase chain reaction (RT-qPCR) method quantified significantly lower viral removal/inactivation in water compared with the plaque assay demonstrating that relying solely on RT-qPCR assays may overstate human health risks arising from viruses. Transmission electron microscopy and computationally generated electron density maps of ϕ6 showed severe morphological damages to virus' envelope and loss of capsid volume accompanying coagulation. Both conventional and electro- coagulation appear to be highly effective in controlling enveloped viruses during surface water treatment.

Computational image analysis of the baseplate-tail complex of O1 ElTor vibriophage M4.

We performed an in-depth computational image analysis of the baseplate-tail complex of the M4 vibriophage and identified seven major densities in its baseplate, which notably share structural similarities with baseplate modules of a number of other bacteriophages belonging to different species. Employing computational analysis, we explained the helical organization of the sheath protein, wrapping the tail tube. Based on the results obtained in this work along with the proteomics information published previously, we are able to decipher the plausible roles assigned to the different components of the M4 baseplate during infection of the host.

Correction to: Structural analysis and proteomics studies on the Myoviridae vibriophage M4.

Unfortunately, the original article was published with an incorrect figure. Figure 11 contains errors and needs to be withdrawn.

Structural analysis and proteomics studies on the Myoviridae vibriophage M4.

Bacteriophages play a crucial role in tracking the spread of bacterial epidemics. The frequent emergence of antibiotic-resistant bacterial strains throughout the world has motivated studies on bacteriophages that can potentially be used in phage therapy as an alternative to conventional antibiotic treatment. A recent outbreak of cholera in Haiti took many lives due to a rapid development of resistance to the available antibiotics. The properties of vibriophages, bacteriophages that infect Vibrio cholerae, are therefore of practical interest. A detailed understanding of the structure and assembly of a vibriophage is potentially useful in developing phage therapy against cholera as well as for fabricating artificial nanocontainers. Therefore, the aim of the present study was to determine the three-dimensional organization of vibriophage M4 at sub-nanometer resolution by electron microscopy and single-particle analysis techniques to facilitate its use as a therapeutic agent. We found that M4 has a large capsid with T = 13 icosahedral symmetry and a long contractile tail. This double-stranded DNA phage also contains a head-to-tail connector protein complex that joins the capsid to the tail and a prominent baseplate at the end of the tail. This study also provides information regarding the proteome of this phage, which is proteins similar to that of other Myoviridae phages, and most of the encoded proteins are structural proteins that form the exquisite architecture of this bacteriophage.

Quantification of the Healing Effect in Hepatic Fibrosis Induced by Chitosan Nano-Encapsulated Green Tea in Rat Model.

Green tea extract exhibits several beneficial activities for the human body. In the present work, we explored the healing effect of a novel bio-nano particle named 'Chitosan nano-encapsulated green tea extract' at the ultrastructural level by performing experiments on rat hepatocytes. A fixed volume of Chitosan nano-encapsulated green tea extract solution was administered orally to a group of adult rats for 25 days, after being treated with carbon-tetrachloride and ethanol doses for 3 weeks. Images of ultra-thin sections of liver samples from these rats were recorded in a transmission electron microscope. Using computational analysis, we probed the images quantitatively and found that Chitosan nano-encapsulated green tea extract heals nearly 25% of the sub-cellular area infected with hepatic fibrosis, suggesting its high medicinal value. Scanning electron microscopy was performed to study the topographical changes of cell surface as well as the extracellular matrix network between the hepatocytes, which further confirmed the healing effect.

Three-dimensional structure of the toxin-delivery particle antifeeding prophage of Serratia entomophila.

The Serratia entomophila antifeeding prophage (Afp) is a bullet-shaped toxin-delivery apparatus similar to the R-pyocins of Pseudomonas aeruginosa. Morphologically it resembles the sheathed tail of bacteriophages such as T4, including a baseplate at one end. It also shares features with the type VI secretion systems. Cryo-electron micrographs of tilted Afp specimens (up to 60 degrees) were analyzed to determine the correct cyclic symmetry to overcome the limitation imposed by exclusively side views in nominally untilted specimens. An asymmetric reconstruction shows clear 6-fold cyclic symmetry contrary to a previous conclusion of 4-fold symmetry based on analysis of only the preferred side views (Sen, A., Rybakova, D., Hurst, M. R., and Mitra, A. K. (2010) J. Bacteriol. 192, 4522-4525). Electron tomography of negatively stained Afp revealed right-handed helical striations in many of the particles, establishing the correct hand. Higher quality micrographs of untilted specimens were processed to produce a reconstruction at 2.0-nm resolution with imposed 6-fold symmetry. The helical parameters of the sheath were determined to be 8.14 nm for the subunit rise along and 40.5° for the rotation angle around the helix. The sheath is similar to that of the T4 phage tail but with a different arrangement of the subdomain of the polymerizing sheath protein(s). The central tube is similar to the diameter and axial width of the Hcp1 hexamer of P. aeruginosa type VI secretion system. The tube extends through the baseplate into a needle resembling the "puncture device" of the T4 tail. The tube contains density that may be the toxin and/or a length-determining protein.

Role of antifeeding prophage (Afp) protein Afp16 in terminating the length of the Afp tailocin and stabilizing its sheath.

The Serratia entomophila antifeeding prophage Afp, forms a phage-tail-like particle that acts on the New Zealand grass grub, Costelytra zealandica with a 3-day LD50 of approximately 500 Afp particles per larva. Genes (afp1-18) encoding components of Afp were expressed and their products purified allowing morphological assessment of the products by transmission electron microscopy (TEM). Expression of afp1-15 resulted in the formation of a non-sheathed structure termed the tube-baseplate complex or TBC, composed of an irregular-length tube attached to a baseplate with associated tail fibres. Expression of afp1-16 produced mature, normal-length Afp particles, whereas coexpression of afp16 with afp1-15 in trans resulted in the formation of aberrant Afp particles of variable lengths. A C-terminally truncated Afp16 mutant resulted in a phenotype intermediate between mature Afp and TBC. The addition of purified Afp16 to Afp unravelled by acidic treatment resulted in the formation of shorter tubes when specimen pH was adjusted to 7 than those formed in the absence of Afp16. Analysis of TEM images of purified Afp16 revealed a hexameric ring-like structure similar to that formed by gp3 of phage T4 and gpU of phage λ. Our results suggest that Afp16 terminates tube elongation and is involved in sheath formation.

Hetero-Multivalent Targeted Liposomal Drug Delivery to Treat Pseudomonas aeruginosa Infections.

Pseudomonas aeruginosa is the leading nosocomial and community-acquired pathogen causing a plethora of acute and chronic infections. The Centers for Disease Control and Prevention has designated multidrug-resistant isolates of P. aeruginosa as a serious threat. A novel delivery vehicle capable of specifically targeting  P. aeruginosa, and encapsulating antimicrobials, may address the challenges associated with these infections. We have developed hetero-multivalent targeted liposomes functionalized with host cell glycans to increase the delivery of antibiotics to the site of infection. Previously, we have demonstrated that compared with monovalent liposomes, these hetero-multivalent liposomes bind with higher affinity to P. aeruginosa. Here, compared with nontargeted liposomes, we have shown that greater numbers of targeted liposomes are found in the circulation, as well as at the site of P. aeruginosa (PAO1) infection in the thighs of CD-1 mice. No significant difference was found in the uptake of targeted, nontargeted, and PEGylated liposomes by J774.A1 macrophages. Ciprofloxacin-loaded liposomes were formulated and characterized for size, encapsulation, loading, and drug release. In vitro antimicrobial efficacy was assessed using CLSI broth microdilution assays and time-kill kinetics. Lastly, PAO1-inoculated mice treated with ciprofloxacin-loaded, hetero-multivalent targeted liposomes survived longer than mice treated with ciprofloxacin-loaded, monovalent targeted, or nontargeted liposomes and free ciprofloxacin. Thus, liposomes functionalized with host cell glycans target P. aeruginosa resulting in increased retention of the liposomes in the circulation, accumulation at the site of infection, and increased survival time in a mouse surgical site infection model. Consequently, this formulation strategy may improve outcomes in patients infected with P. aeruginosa.

Structure of vibriophage D10 tail sheath revealed by electron microscopy and computational image processing.

Vibriophage D10, a member of the Vibrio cholerae O-1El-Tor phage typing scheme, is used to detect the spread of the cholera epidemic and belongs to the Myoviridae family. The outer sheath of the tail of vibriophage is highly contractile in nature. We have used electron microscopy and computational image-processing techniques to determine the structure of this contractile tail sheath. The three-dimensional density map of the tail sheath reveals the presence of ∼35 Å long and ∼25 Å wide protrusions, extending out of the tail structure. The electron micrographs revealed that the tail sheaths of a considerable number of D10 phage particles undergo axial compression up to 51% at almost neutral pH (7.2) and at room temperature (20°). We find that the genome of the phage particles is ejected out when the tail sheath of the phage particles are deliberately made to contract by subjecting them to a surrounding environment of pH 10.5. We infer that the contraction of the tail sheath is responsible for the loss of the phage genome even at neutral pH and room temperature. This may be a plausible reason for the unusual behavior of rapid decline of the phage within a span of 48-72 h of its preparation.

Navigating the Light-Sheet Image Analysis Software Landscape: Concepts for Driving Cohesion From Data Acquisition to Analysis.

From the combined perspective of biologists, microscope instrumentation developers, imaging core facility scientists, and high performance computing experts, we discuss the challenges faced when selecting imaging and analysis tools in the field of light-sheet microscopy. Our goal is to provide a contextual framework of basic computing concepts that cell and developmental biologists can refer to when mapping the peculiarities of different light-sheet data to specific existing computing environments and image analysis pipelines. We provide our perspective on efficient processes for tool selection and review current hardware and software commonly used in light-sheet image analysis, as well as discuss what ideal tools for the future may look like.

Structural study of the Serratia entomophila antifeeding prophage: three-dimensional structure of the helical sheath.

The sheath of the Serratia entomophila antifeeding prophage, which is pathogenic to the New Zealand grass grub Costelytra zealandica, is a 3-fold helix formed by a 4-fold symmetric repeating motif disposed around a helical inner tube. This structure, determined by electron microscopy and image processing, is distinct from that of the other known morphologically similar bacteriophage sheaths.

Advanced microscopic evaluation of parallel type I and type II cell deaths induced by multi-functionalized gold nanocages in breast cancer.

Despite aggressive surgical resections and combinatorial chemoradiations, certain highly malignant populations of tumor cells resurrect and metastasize. Mixed-grade cancer cells fail to respond to standard-of-care therapies by developing intrinsic chemoresistance and subsequently result in tumor relapse. Macroautophagy is a membrane trafficking process that underlies drug resistance and tumorigenesis in most breast cancers. Manipulating cellular homeostasis by a combinatorial nanotherapeutic model, one can evaluate the crosstalk between type I and type II cell death and decipher the fate of cancer therapy. Here, we present a multi-strategic approach in cancer targeting to mitigate the autophagic flux with subcellular toxicity via lysosome permeation, accompanied by mitochondrial perturbation and apoptosis. In this way, a nanoformulation is developed with a unique blend of a lysosomotropic agent, an immunomodulating sulfated-polysaccharide, an adjuvant chemotherapeutic agent, and a monoclonal antibody as a broad-spectrum complex for combinatorial nanotherapy of all breast cancers. To the best of our knowledge, this manuscript illustrates for the first time the applications of advanced microscopic techniques such as electron tomography, three-dimensional rendering and segmentation of subcellular interactions, and fate of the multifunctional therapeutic gold nanocages specifically targeted toward breast cancer cells.

Visualizing a Vibrio cholerae O1 El Tor typing bacteriophage belonging to the Myoviridae group and the packaging of its genomic ends inside the phage capsid.

Phage D10, an O1 El Tor tying vibriophage, has been successfully employed to tract the outspread of cholera epidemic. Using Transmission Electron Microscopy and computational image analysis, we have determined the structures of the capsid, head-to-tail connector, the contractile helical tail, the baseplate and combined them to form the complete three-dimensional (3D) D10 phage structure. Using partial denaturation experiments on the genome and using the computed 3D structure of the phage, we have established the packing of the genome ends inside the capsid together with the release styles during the phage infection, respectively. Finally, using the 3D density maps of the different components of the D10 phage, we have presented a simplified picture of morphogenesis of the D10 vibriophage. Using the complete assembled structure of the D10 phage, we have traced the path of the phage genome during the infection process, all the way from the phage head down the tail tube of the tail to the top of the baseplate. To the best of our knowledge, this is first structural study for a long-tailed vibriophage. We have tabulated the structural features of the different components of the phages belonging to the Myoviridae and Siphoviridae. The comparative study suggested the possibility of a common origin of the bacteriophages, irrespective of belonging to different groups and species.

Physicochemical characterization of vibriophage N5.

Phage N5 is one of the phages of Vibrio cholerae serovar O1 biotype El Tor (Ghosh, A. N., Ansari, M. Q., and Dutta, G. C. Isolation and morphological characterization of El Tor cholera phages. J. Gen. Virol. 70: 2241-2243, 1989). In the present communication the growth curve, molecular weight and confirmation of the genome, partial denaturation map and restriction endonuclease digestion pattern have been determined. Partial denaturation map indicates that the genome has non-permuted / invariant sequence. Presence of cohesive ends has also been documented.

New Vibrio cholerae O1 biotype ElTor bacteriophages.

We report the presence of three new O1 ElTor vibriophages named AS1, AS2 and AS3, isolated from the sewage and pond waters of the outskirts of Kolkata. A few phages, named AS4, with hexagonal heads and abnormally long tails with typical curly projections were also found in the water samples.

Ion-swimming speed variation of Vibrio cholerae cells.

In the present work we report the variation in swimming speed of Vibrio cholerae with respect to the change in concentration of sodium ions in the medium. We have also studied the variation in swimming speed with respect to temperature. We find that the swimming speed initially shows a linear increase with the increase of the sodium ions in the medium and then plateaus. The range within which the swimming speed attains saturation is approximately the same at different temperatures.

The bipolar filaments formed by herpes simplex virus type 1 SSB/recombination protein (ICP8) suggest a mechanism for DNA annealing.

Herpes simplex virus type 1 encodes a multifunctional protein, ICP8, which serves both as a single-strand binding protein and as a recombinase, catalyzing reactions involved in replication and recombination of the viral genome. In the presence of divalent ions and at low temperature, previous electron microscopic studies showed that ICP8 will form long left-handed helical filaments. Here, electron microscopic image reconstruction reveals that the filaments are bipolar, with an asymmetric unit containing two subunits of ICP8 that constitute a symmetrical dimer. This organization of the filament has been confirmed using scanning transmission electron microscopy. The pitch of the filaments is approximately 250 A, with approximately 6.2 dimers per turn. Docking of a crystal structure of ICP8 into the reconstructed filament shows that the C-terminal domain of ICP8, attached to the body of the subunit by a flexible linker containing approximately 10 residues, is packed into a pocket in the body of a neighboring subunit in the crystal in a similar manner as in the filament. However, the interactions between the large N-terminal domains are quite different in the filament from that observed in the crystal. A previously proposed model for ICP8 binding single-stranded DNA (ssDNA), based upon the crystal structure, leads to a model for a continuous strand of ssDNA near the filament axis. The bipolar nature of the ICP8 filaments means that a second strand of ssDNA would be running through this filament in the opposite orientation, and this provides a potential mechanism for how ICP8 anneals complementary ssDNA into double-stranded DNA, where each strand runs in opposite directions.

Initial location of the RNA-dependent RNA polymerase in the bacteriophage Phi6 procapsid determined by cryo-electron microscopy.

The RNA-dependent RNA polymerases (RdRPs) of Cystoviridae bacteriophages, like those of eukaryotic viruses of the Reoviridae, function inside the inner capsid shell in both replication and transcription. In bacteriophage Phi6, this inner shell is first assembled as an icosahedral procapsid with recessed 5-fold vertices that subsequently undergoes major structural changes during maturation. The tripartite genome is packaged as single-stranded RNA molecules via channels on the 5-fold vertices, and transcripts probably exit the mature capsid by the same route. The RdRP (protein P2) is assembled within the procapsid, and it was thought that it should be located on the 5-fold axes near the RNA entry and exit channels. To determine the initial location of the RdRP inside the procapsid of bacteriophage Phi6, we performed cryo-electron microscopy of wild type and mutant procapsids and complemented these data with biochemical determinations of copy numbers. We observe ring-like densities on the 3-fold axes that are strong in a mutant that has approximately 10 copies of P2 per particle; faint in wild type, reflecting the lower copy number of approximately 3; and completely absent in a P2-null mutant. The dimensions and shapes of these densities match those of the known crystal structure of the P2 monomer. We propose that, during maturation, the P2 molecules rotate to occupy positions closer to adjacent 5-fold vertices where they conduct replication and transcription.