Low-temperature polycrystalline silicon waveguides for low loss transmission in the near-to-mid-infrared region.

Low-temperature deposited polycrystalline silicon waveguides are emerging as a flexible platform that allows for dense optoelectronic integration. Here, the optical transmission properties of poly-silicon waveguides have been characterized from the near-to-mid-infrared wavelength regime, extending the optical transmission well beyond previous reports in the telecom band. The poly-Si waveguides with a dimension of 3 µm × âˆ¼0.6 µm have been produced from pre-patterned amorphous silicon waveguides that are post-processed through laser melting, reflowing, and crystallization using a highly localized laser induced heat treatment at a wavelength of 532 nm. Low optical transmission losses (<3 dB cm-1) have been observed at 1.55 µm as well as across the wavelength range of 2-2.25 µm, aided by the relatively large waveguide heights that are enabled by the deposition process. The results demonstrate the suitability of low-temperature poly-silicon waveguides to find wide ranging applications within integrated mid-infrared systems.

Cross-phase modulation instability in PM ANDi fiber-based supercontinuum generation.

We demonstrate broadband supercontinuum generation in an all-normal dispersion polarization-maintaining photonic crystal fiber and report the observation of a cross-phase modulation instability sideband generated outside of the supercontinuum bandwidth. We demonstrate that this sideband is polarized on the slow axis and can be suppressed by pumping on the fiber's fast axis. We theoretically confirm and model this nonlinear process using phase-matching conditions and numerical simulations, obtaining good agreement with the measured data.

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.

Nanoimprinting and tapering of chalcogenide photonic crystal fibers for cascaded supercontinuum generation.

Improved long-wavelength transmission and supercontinuum (SC) generation is demonstrated by antireflective (AR) nanoimprinting and tapering of chalcogenide photonic crystal fibers (PCFs). Using a SC source input spanning from 1 to 4.2 µm, the total transmission of a 15 µm core diameter PCF was improved from ∼53% to ∼74% by nanoimprinting of AR structures on both input and output facets of the fiber. Through a combined effect of reduced reflection and redshifting of the spectrum to 5 µm, the relative transmission of light >3.5 µm in the same fiber was increased by 60.2%. Further extension of the spectrum to 8 µm was achieved using tapered fibers. The spectral broadening dynamics and output power were investigated using different taper parameters and pulse repetition rates.

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.

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.

Preliminary Characterization of El Tor Vibriophage M4.

AIMS: The aim of the present study is the preliminary characterization of an El Tor vibriophage M4 (ATCC 51352-B4). METHODS: We studied the growth characteristics and sustainability of this phage under various stresses like temperature, pH, and UV. The phage morphology and phage genome were also examined using electron microscopy. RESULTS: Sustainability studies showed that the phage is more stable in acidic conditions, which is very uncommon among vibriophages. Studies also showed that M4 is a thermostable phage and it is inactivated by temperatures above 60°C but, like other phages, UV has a high inactivating effect on it. Morphological and genomic studies by electron microscopy showed that this phage has a long contractile tail and a big head. The genome is linear and about 120 kb in length. The genome also has a high packaging density as the value of Vm (the volume occupied per Da of biological macromolecule) is low for this phage. The phage-bacterial interaction was studied by negative staining as well as ultrathin sectioning methods. CONCLUSIONS: M4 belongs to the Myoviridae family and it is generally thermostable. It is prone to destruction by alkali and UV. It also has a large DNA which is densely packed inside of a big capsid.

The ß-prism lectin domain of Vibrio cholerae hemolysin promotes self-assembly of the ß-pore-forming toxin by a carbohydrate-independent mechanism.

Vibrio cholerae cytolysin/hemolysin (VCC) is an amphipathic 65-kDa ß-pore-forming toxin with a C-terminal ß-prism lectin domain. Because deletion or point mutation of the lectin domain seriously compromises hemolytic activity, it is thought that carbohydrate-dependent interactions play a critical role in membrane targeting of VCC. To delineate the contributions of the cytolysin and lectin domains in pore formation, we used wild-type VCC, 50-kDa VCC (VCC(50)) without the lectin domain, and mutant VCC(D617A) with no carbohydrate-binding activity. VCC and its two variants with no carbohydrate-binding activity moved to the erythrocyte stroma with apparent association constants on the order of 10(7) M(-1). However, loss of the lectin domain severely reduced the efficiency of self-association of the VCC monomer with the ß-barrel heptamer in the synthetic lipid bilayer from ∼83 to 27%. Notably, inactivation of the carbohydrate-binding activity by the D617A mutation marginally reduced oligomerization to ∼77%. Oligomerization of VCC(50) was temperature-insensitive; by contrast, VCC self-assembly increased with increasing temperature, suggesting that the process is driven by entropy and opposed by enthalpy. Asialofetuin, the ß1-galactosyl-terminated glycoprotein inhibitor of VCC-induced hemolysis, promoted oligomerization of 65-kDa VCC to a species that resembled the membrane-inserted heptamer in stoichiometry and morphology but had reduced global amphipathicity. In conclusion, we propose (i) that the ß-prism lectin domain facilitated toxin assembly by producing entropy during relocation in the heptamer and (ii) that glycoconjugates inhibited VCC by promoting its assembly to a water-soluble, less amphipathic oligomer variant with reduced ability to penetrate the bilayer.

An inducible and secreted eukaryote-like serine/threonine kinase of Salmonella enterica serovar Typhi promotes intracellular survival and pathogenesis.

Eukaryote-like serine/threonine kinases (eSTKs) constitute an important family of bacterial virulence factors. Genome analysis had predicted putative eSTKs in Salmonella enterica serovar Typhi, although their functional characterization and the elucidation of their role in pathogenesis are still awaited. We show here that the primary sequence and secondary structure of the t4519 locus of Salmonella Typhi Ty2 have all the signatures of eukaryotic superfamily kinases. t4519 encodes a ∼39-kDa protein (T4519), which shows serine/threonine kinase activities in vitro. Recombinant T4519 (rT4519) is autophosphorylated and phosphorylates the universal substrate myelin basic protein. Infection of macrophages results in decreased viability of the mutant (Ty2Δt4519) strain, which is reversed by gene complementation. Moreover, reactive oxygen species produced by the macrophages signal to the bacteria to induce T4519, which is translocated to the host cell cytoplasm. That T4519 may target a host substrate(s) is further supported by the activation of host cellular signaling pathways and the induction of cytokines/chemokines. Finally, the role of T4519 in the pathogenesis of Salmonella Typhi is underscored by the significantly decreased mortality of mice infected with the Ty2Δt4519 strain and the fact that the competitive index of this strain for causing systemic infection is 0.25% that of the wild-type strain. This study characterizes the first eSTK of Salmonella Typhi and demonstrates its role in promoting phagosomal survival of the bacteria within macrophages, which is a key determinant of pathogenesis. This, to the best of our knowledge, is the first study to describe the essential role of eSTKs in the in vivo pathogenesis of Salmonella spp.

Antibacterial activities of polyethylene glycol, tween 80 and sodium dodecyl sulphate coated silver nanoparticles in normal and multi-drug resistant bacteria.

Antibacterial activity of silver nanoparticles coated with different functionalizing agents i.e., polyethylene glycol, tween 80 and sodium dodecyl sulphate were evaluated on both normal and multi-drug resistant strains of bacteria. Under the same reaction conditions, these functionalizing agents were added separately to coat silver nanoparticles. Among these, polyethylene glycol coated nanoparticles were most effective in killing all the bacterial strains which includes Escherichia coli DH5a, Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus and multi-drug resistant clinical isolates of Shigella spp. (flexneri, boydii, sohnea) and Vibrio cholerae. The minimum inhibitory concentration of polyethylene glycol coated silver nanoparticles was also less compared to the other two sets of nanoparticles. Consistence with that polyethylene glycol coated nanoparticles produced more intracellular reactive oxygen species in bacteria. Moreover, when human cell lines MCF7 and Chang Liver were incubated in presence of these nanoparticles for 18 h with same concentrations as used for bacteria, no toxicity was observed. But significant increase in cell killing was observed with longer incubation time. Thus our present investigation implicates the potential therapeutic use of silver nanoparticles as antibacterial agent particularly the polyethylene glycol coated one.

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.

The role of C-terminus carbohydrate-binding domain of Vibrio cholerae haemolysin/cytolysin in the conversion of the pre-pore ß-barrel oligomer to a functional diffusion channel.

BACKGROUND & OBJECTIVES: Vibrio cholerae cytolysin/hemolysin (VCC) is a 65 kDa pore-forming toxin (PFT) secreted by O1 El Tor and non-O1 strains. The purified toxin, which contains two C-terminus carbohydrate-binding domains in addition to the cytolytic domain at the core, causes lysis of a wide spectrum of eukaryotic cells at picomolar concentrations, apoptogenesis of intestinal and immune cells and accumulation of fluid in rabbit ligated ileal loop. Therefore, it may potentially complement the action of cholera toxin (CT) in diarrheagenic strains that do not produce CT. We showed earlier that ß1-galactosyl-terminated glycoconjugates are strong inhibitors of its pore-forming activity, though carbohydrates are not functional receptors of VCC. Here, we investigate how the 15 kDa C-terminus ß-prism lectin domain contributed to pore formation in erthrocytes. METHODS: VCC was isolated from the culture supernatant of late log phase grown bacteria and purified to homogeneity by chromatography. The 50 kDa truncated variant was generated by restricted proteolysis. Liposome was prepared by sonication of a suspension of phospholipids and calceine release assay was done by spectrofluorometric monitoring of the released dye trapped in liposome. Formation of ß-barrel oligomers in erythrocyte stroma was monitored by scanning electron microscopy. RESULTS: Proteolytic truncation of the C-terminus ß-prism lectin domain decreased hemolytic activity of the toxin by ~800-fold without causing a significant change in pore-forming activity toward synthetic lipid vesicles devoid of incorporated glycoproteins/glycolipids. Truncation at the C-terminus did not impair membrane-binding or assembly to the oligomeric pore. INTERPRETATION & CONCLUSIONS: Our data indicated that the C-terminus domain played a critical role in translocation of the pre-pore oligomeric assembly from the cell surface or lipid-water interface to the hydrocarbon core of the membrane bilayer, signaling the formation of functional diffusion channels.

Three-dimensional structure of different functional forms of the Vibrio cholerae hemolysin oligomer: a cryo-electron microscopic study.

Vibrio cholerae hemolysin (HlyA) is a 65-kDa water-soluble pore-forming toxin that causes lysis of eukaryotic cells by destroying selective permeability of the plasma membrane bilayer. The HlyA monomer self-assembles on the target cell surface to the more stable beta-barrel amphipathic heptamer, which inserts into the membrane bilayer to form a diffusion channel. Deletion of the 15-kDa beta-prism lectin domain at the C terminus generates a 50-kDa hemolysin variant (HlyA50) with an approximately 1,000-fold decrease in hemolytic activity. Because functional differences are eventually dictated by structural differences, we determined three-dimensional structures of 65- and 50-kDa HlyA oligomers, using cryo-electron microscopy and single-particle methods. Our study clearly shows that the HlyA oligomer has sevenfold symmetry but that the HlyA50 oligomer is an asymmetric molecule. The HlyA oligomer has bowl-like, arm-like, and ring-like domains. The bowl-like domain is coupled with the ring-like domain, and seven side openings are present just beneath the ring-like domain. Although a central channel is present in both HlyA and HlyA50 oligomers, they differ in pore size as well as in shape of the molecules and channel. These structural differences may be relevant to the striking difference in efficiencies of functional channel formation by the two toxin forms.

The cyclic AMP (cAMP)-cAMP receptor protein signaling system mediates resistance of Vibrio cholerae O1 strains to multiple environmental bacteriophages.

Toxigenic Vibrio cholerae, the causative agent of the epidemic diarrheal disease cholera, interacts with diverse environmental bacteriophages. These interactions promote genetic diversity or cause selective enrichment of phage-resistant bacterial clones. To identify bacterial genes involved in mediating the phage-resistant phenotype, we screened a transposon insertion library of V. cholerae O1 El Tor biotype strain C6706 to identify mutants showing altered susceptibility to a panel of phages isolated from surface waters in Bangladesh. Mutants with insertion in cyaA or crp genes encoding adenylate cyclase or cyclic AMP (cAMP) receptor protein (CRP), respectively, were susceptible to a phage designated JSF9 to which the parent strain was completely resistant. Application of the cyaA mutant as an indicator strain in environmental phage monitoring enhanced phage detection, and we identified 3 additional phages to which the parent strain was resistant. Incorporation of the cyaA or crp mutations into other V. cholerae O1 strains caused similar alterations in their phage susceptibility patterns, and the susceptibility correlated with the ability of the bacteria to adsorb these phages. Our results suggest that cAMP-CRP-mediated downregulation of phage adsorption may contribute to a mechanism for the V. cholerae O1 strains to survive predation by multiple environmental phages. Furthermore, the cyaA or crp mutant strains may be used as suitable indicators in monitoring cholera phages in the water.

Prevalence of Shigella boydii in Bangladesh: Isolation and Characterization of a Rare Phage MK-13 That Can Robustly Identify Shigellosis Caused by Shigella boydii Type 1.

Shigellosis, caused by Shigella boydii type 1, is understudied and underreported. For 3 years, GEMS study identified 5.4% of all Shigella as S. boydii. We showed the prevalent serotypes of S. boydii in Bangladesh and phage-based diagnosis of S. boydii type 1, a rapid and low-cost approach. Previously typed 793 clinical S. boydii strains were used for serotype distribution. Twenty-eight environmental water samples were collected for isolation of Shigella phages. Forty-eight serotypes of Shigella and other enteric bacteria were used for testing the susceptibility to phage MK-13. Electron microscopy, restriction enzyme analysis, whole genome sequencing (WGS), and annotation were performed for extensive characterization. S. boydii type 1 is the second most prevalent serotype among 20 serotypes of S. boydii in Bangladesh. We isolated a novel phage, MK-13, which specifically lyses S. boydii type 1, but doesn't lyse other 47 serotypes of Shigella or other enteric bacteria tested. The phage belongs to the Myoviridae family and distinct from other phages indicated by electron microscopy and restriction enzyme analysis, respectively. MK-13 genome consists of 158 kbp of circularly permuted double-stranded DNA with G + C content of 49.45%, and encodes 211 open reading frames including four tRNA-coding regions. The genome has 98% identity with previously reported phage, ΦSboM-AG3, reported to have a broader host range infecting most of the S. boydii and other species of Shigella tested. To our knowledge, MK-13 is the first phage reported to be used as a diagnostic marker to detect S. boydii type 1, especially in remote settings with limited laboratory infrastructure.

Correction: Phenotypic and genomic analyses of bacteriophages targeting environmental and clinical CS3-expressing enterotoxigenic Escherichia coli (ETEC) strains.

[This corrects the article DOI: 10.1371/journal.pone.0209357.].

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.

Phenotypic and genomic analyses of bacteriophages targeting environmental and clinical CS3-expressing enterotoxigenic Escherichia coli (ETEC) strains.

Diarrhea due to infection of enterotoxigenic Escherichia coli (ETEC) is of great concern in several low and middle-income countries. ETEC infection is considered to be the most common cause of diarrhea in Bangladesh and is mainly spread through contaminated water and food. ETEC pathogenesis is mediated by the expression of enterotoxins and colonization factors (CFs) that target the intestinal mucosa. ETEC can survive for extended time periods in water, where they are likely to be attacked by bacteriophages. Antibiotic resistance is common amongst enteric pathogens and therefore is the use of bacteriophages (phage) as a therapeutic tool an interesting approach. This study was designed to identify novel phages that specifically target ETEC virulence factors. In total, 48 phages and 195 ETEC isolates were collected from water sources and stool samples. Amongst the identified ETEC specific phages, an enterobacteria phage T7, designated as IMM-002, showed a significant specificity towards colonization factor CS3-expressing ETEC isolates. Antibody-blocking and phage-neutralization assays revealed that CS3 is used as a host receptor for the IMM-002 phage. The bacterial CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated) defence mechanism can invoke immunity against phages. Genomic analyses coupled with plaque assay experiments indicate that the ETEC CRISPR-Cas system is involved in the resistance against the CS3-specific phage (IMM-002) and the previously identified CS7-specific phage (IMM-001). As environmental water serves as a reservoir for ETEC, it is important to search for new antimicrobial agents such as phages in environmental water as well as the human gut. A better understanding of how the interplay between ETEC-specific phages and ETEC isolates affects the ETEC diversity, both in environmental ecosystems and within the host, is important for the development of new treatments for ETEC infections.

Physicochemical characterization of El Tor Vibriophage S20.

OBJECTIVE: To characterize Vibrio cholerae El Tor typing phage S20 (ATCC No. 51352-B3). METHODS AND RESULTS: The phage has a hexagonal head and a short tail. Cryo-electron microscopy and three-dimensional image reconstruction showed that the phage head has icosahedral symmetry. The phage has two major structural polypeptides of 50 and 42 kDa. Adsorption of the phage to its host followed a biphasic kinetics and its intracellular growth is characterized by a latent period of 12 min and a burst size of around 60 particles per infected cell. The phage was found to be stable at a pH range 5.0-9.0 and moderately thermotolerant and highly UV sensitive. Phage genome comprises a 40.7 +/- 1.5-kb linear DNA molecule with random circular permutation and terminal redundancy. The restriction endonucleases AccI, HpaII, HaeIII, HindIII, EcoRV, HincII, DraI and XmnI cut vibriophage S20 DNA. CONCLUSION: Vibriophage S20, which belongs to Podoviridae, has an icosahedral head and the genome, which is double-stranded linear DNA, has random circular permutation and terminal redundancy.

Environmental bacteriophages active on biofilms and planktonic forms of toxigenic Vibrio cholerae: Potential relevance in cholera epidemiology.

METHODS: Phages isolated from environmental waters in Bangladesh were tested for their host specificity towards V. cholerae O1 and O139, and the ability to disperse V. cholerae biofilms formed in the laboratory. Representative phages were further characterized by electron microscopy and whole genome sequencing. Selected phages were then introduced in various combinations to biofilms of toxigenic V. cholerae added to samples of river water, and the dispersion of biofilms as well as the growth kinetics of V. cholerae and the phages were monitored. RESULTS: A phage cocktail composed of three different phages isolated from surface waters in Bangladesh and designated as JSF7, JSF4, and JSF3 could significantly influence the distribution and concentration of the active planktonic form and biofilm associated form of toxigenic V. cholerae in water. While JSF7 showed a biofilm degrading activity and dispersed cells from both V. cholerae O1 and O139 derived biofilms thus increasing the concentration of planktonic V. cholerae in water, JSF4 and JSF3 showed strong bactericidal activity against V. cholerae O1 and O139 respectively. A mixture of all three phages could effectively reduce both biofilm-associated and planktonic V. cholerae in river water microcosms. SIGNIFICANCE: Besides potential applicability in phage-mediated control of cholera, our results have relevance in appreciating possible intricate role of diverse environmental phages in the epidemiology of the disease, since both biofilms and phages influence the prevalence and infectivity of V. cholerae in a variety of ways.