scholarly journals Leaky Lactococcus Cultures That Externalize Enzymes and Antigens Independently of Culture Lysis and Secretion and Export Pathways

2001 ◽  
Vol 67 (1) ◽  
pp. 251-259 ◽  
Author(s):  
Shirley A. Walker ◽  
Todd R. Klaenhammer
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Propidium Iodide ◽  
Copy Number ◽  
Critical Role ◽  
Membrane Damage ◽  
Bacterial Strains ◽  
Late Promoter ◽  
Transmission Electron ◽  
Dipeptidyl Aminopeptidase

ABSTRACT A novel system that leaks β-galactosidase (β-gal) without a requirement for secretion or export signals was developed inLactococcus lactis by controlled expression of integrated phage holin and lysin cassettes. The late promoter of the lytic lactococcal bacteriophage φ31 is an 888-bp fragment (P15A10) encoding the transcriptional activator. When a high-copy-number P15A10::lacZ.stfusion was introduced into L. lactis strains C10, ML8, NCK203, and R1/r1t, high levels of the resultant β-gal activity were detected in the supernatant (approximately 85% of the total β-gal activity for C10, ML8, and NCK203 and 45% for R1/r1t). Studies showed that the phenotype resulted from expression of Tac31A from the P15A10 fragment, which activated a homologous late promoter in prophages harbored by the lactococcal strains. Despite the high levels of β-gal obtained in the supernatant, the growth of the strains was not significantly affected, nor was there any evidence of severe membrane damage as determined by using propidium iodide or transmission electron microscopy. Integration of the holin-lysin cassette of phage r1t, under the control of the phage φ31 late promoter, into the host genome of MG1363 yielded a similar “leaky” phenotype, indicating that holin and lysin might play a critical role in the release of β-gal into the medium. In addition to β-gal, tetanus toxin fragment C was successfully delivered into the growth medium by this system. Interestingly, the X-prolyl dipeptidyl aminopeptidase PepXP (a dimer with a molecular mass of 176 kDa) was not delivered at significant levels outside the cell. These findings point toward the development of bacterial strains able to efficiently release relevant proteins and enzymes outside the cell in the absence of known secretion and export signals.

Interaction mechanisms of bacterial strains isolated from extreme habitats with uranium

2006 ◽  
Vol 94 (9-11) ◽  
Author(s):  
Mohamed Merroun ◽  
M. Nedelkova ◽  
Andre Rossberg ◽  
C. Hennig ◽  
Sonja Selenska-Pobell
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Cellular Localization ◽  
Bacterial Strains ◽  
X Ray ◽  
Deep Well ◽  
Transmission Electron ◽  
Microbiological Methods ◽  
Extreme Habitats ◽  
X Ray Absorption

This paper summarizes the effect of pH on the speciation and cellular localization of uranium bound by bacterial strains isolated from the S15 deep-well montoring site, located at the Siberian radioactive subsurface depository Tomsk-7, Russia. Microbiological methods in combination with extended X-ray absorption fine structure (EXAFS) spectroscopy, transmission electron microscopy (TEM), and energy dispersive X-ray spectroscopy (EDX) were applied. EXAFS analysis showed that the cells of the two isolates,

scholarly journals Isolation and Characterization of Outer Membrane Vesicles of Pectobacterium brasiliense 1692

2021 ◽  
Vol 9 (9) ◽  
pp. 1918
Author(s):  
Silindile Maphosa ◽  
Lucy Novungayo Moleleki
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Outer Membrane ◽  
Critical Role ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Broad Host Range ◽  
Isolation And Characterization ◽  
Transmission Electron ◽  
Animal Pathogens

Pectobacterium brasiliense (Pbr) 1692 is an aggressive phytopathogen affecting a broad host range of crops and ornamental plants, including potatoes. Previous research on animal pathogens, and a few plant pathogens, revealed that Outer Membrane Vesicles (OMVs) are part of Gram-negative bacteria’s (GNB) adaptive toolkit. For this reason, OMV production and subsequent release from bacteria is a conserved process. Therefore, we hypothesized that OMVs might transport proteins that play a critical role in causing soft rot disease and in the survival and fitness of Pbr1692. Here, we show that the potato pathogen, Pbr1692, releases OMVs of various morphologies in Luria Bertani media at 31 °C. Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) confirmed the production of OMVs by Pbr1692 cells. Transmission Electron Microscopy showed that these exist as chain-, single-, and double-membrane morphologies. Mass spectrometry followed by Gene Ontology, Clusters of Orthologous Groups, Virulence Factor, CAZymes, Antibiotic Resistance Ontology, and Bastion6 T6SE annotations identified 129 OMV-associated proteins with diverse annotated roles, including antibiotic stress response, virulence, and competition. Pbr1692 OMVs contributed to virulence in potato tubers and elicited a hypersensitive response in Nicotiana benthamiana leaves. Furthermore, Pbr1692 OMVs demonstrated antibacterial activity against Dickeya dadantii.

scholarly journals Scanning Transmission Electron Microscopy for Polymer Blends

2017 ◽  
Vol 4 (1) ◽  
pp. 31-36
Author(s):  
Pradeep Singh ◽  
B. R. Venugopal ◽  
Radha Kamalakaran
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Polymer Blends ◽  
Scanning Transmission Electron Microscopy ◽  
Critical Role ◽  
Polymer Materials ◽  
Scanning Transmission Electron ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Butadiene Styrene

Physical properties of the polymer can be altered by mixing one or more polymers together also known as polymer blending. The miscibility of polymers is a key parameter in determining the properties of polymer blend. Conventional transmission electron microscopy (CTEM) plays a critical role in determining the miscibility and morphology of the polymers in blend system. One of the most difficult part in polymer microscopy is the staining by heavy metals to generate contrast in CTEM. RuO4 and OsO4 are commonly used to stain the polymer materials for CTEM imaging. CTEM imaging is difficult to interpret for blends due to lack of clear distinction in contrast. Apart from having difficulty in contrast generation, staining procedures are extremely dangerous as improper handling could severely damage skin, eyes, lungs etc. We have used scanning transmission electron microscopy (STEM) to image polymer blends without any staining processes. In current work, Acrylonitrile Butadiene Styrene (ABS)/Methacrylate Butadiene Styrene (MBS) and Styrene Acrylonitrile (SAN) along with filler additive were dispersed on Polycarbonate (PC) matrix and studied by STEM/HAADF (high angle annular dark field). By using HAADF, contrast was generated through molecular density difference to differentiate components in the blend.

scholarly journals Mice with LMAN1 Deficiency Exhibit Thrombocytopenia and Reduced Serum Thrombopoietin Level

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 412-412
Author(s):  
Rami Khoriaty ◽  
Lesley Everett ◽  
Jennifer Chase ◽  
Guojing Zhu ◽  
Bin Zhang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Coagulation Factors ◽  
Hematopoietic Stem ◽  
Platelet Counts ◽  
Normal Platelet ◽  
Transmission Electron ◽  
Wide Range

Abstract LMAN1 and MCFD2 encode the components of a mammalian cargo-receptor that facilitates the ER-to-Golgi transport of coagulation factors V (FV) and VIII (FVIII) for secretion to the plasma. Mutations in LMAN1 or MCFD2 result in a rare bleeding disorder known as combined deficiency of coagulation factors V and VIII (F5F8D), characterized by FV and FVIII levels that are ~10% of normal. No other clinical phenotypes are known in human patients. Lman1 null mice have ~50% of normal FV and FVIII levels and exhibit a partially penetrant, perinatal lethality, suggesting a critical role for yet unknown LMAN1 secretory cargo(s). To further characterize the function of the LMAN1/MCFD2 complex and identify new cargos, we generated several murine models of F5F8D, including ubiquitous null Lman1 (Lman1-/-) and Mcfd2 (Mcfd2-/-) mice maintained on a C57BL/6J genetic background. Adult Lman1-/- mice were mildly thrombocytopenic, exhibiting a 25% decrease in platelet count relative to wild-type (WT) mice (9.3 x 105 vs. 12.3 x 105 cells/uL, p < 0.004), but no other CBC abnormalities. In contrast, Lman1 heterozygous and Mcfd2-/- mice exhibited normal platelet counts. To further explore the role of LMAN1 in megakaryocyte/platelet development or survival, bone marrow (BM) histology and platelet transmission electron microscopy were performed. Lman1-/-mice had no platelet morphologic abnormalities by light or transmission electron microscopy, as well as normal number and morphology of BM megakaryocytes. Hematopoietic stem cells and megakaryocyte progenitors were indistinguishable between WT and Lman1-/- mice by flow cytometry. In order to determine whether the thrombocytopenia phenotype results from LMAN1 deficiency specifically in the hematopoietic compartment, mice with tissue-specific knockout of Lman1 in hematopoietic and endothelial cells (Tie2-Cre) were generated. Platelet counts of mice with LMAN1 deficiency restricted to the hematopoietic compartment were indistinguishable from those in WT controls. In contrast, hepatocyte-specific (Alb-Cre) Lman1 deficiency, resulted in significant thrombocytopenia relative to WT controls (p < 0.017), with platelet counts comparable to those observed in ubiquitous Lman1 null mice. Since thrombopoietin (TPO) is a major hepatocyte-derived regulator of platelet synthesis, plasma TPO levels were measured by ELISA in ubiquitous Lman1 and Mcfd2 null mice. Plasma TPO levels in Lman1-/- mice were ~56% lower than those in WT levels (128.7 x 103 vs. 229.5 x 103 pg/mL, p < 0.0025). However, TPO levels were not reduced in Mcfd2-/- mice (p > 0.17). TPO mRNA expression in the liver of Lman1-/-mice was indistinguishable from livers of WT littermate controls. In conclusion, global LMAN1-deficient and hepatocyte-specific LMAN1 deficient mice exhibit thrombocytopenia, a phenotype not previously reported in F5F8D patients. Lman1-/- mice, but not Mcfd2-/- mice, exhibit low plasma TPO levels, suggesting that TPO may be a novel LMAN1-dependent secretory cargo. These results raise the possibility that F5F8D patients with LMAN1 mutations may have mild thrombocytopenia, previously unappreciated as a result of the small number of F5F8D patients and the wide range of clinically normal platelet counts. Disclosures No relevant conflicts of interest to declare.

High-resolution Transmission Electron Microscopy of dislocations in intermetallic compounds

1995 ◽  
Vol 53 ◽  
pp. 76-77
Author(s):  
M.J. Mills
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
High Resolution ◽  
Intermetallic Compounds ◽  
Structural Information ◽  
Critical Role ◽  
Atomic Level ◽  
Low Energy ◽  
Transmission Electron ◽  
Line Defects

The fine structure of dislocations plays a critical role in determining the macroscopic mechanical behavior Intermetallic compounds. Many of the technologically important characteristics of these compounds, an example their strength at high temperatures, appear to be determined by intricate details of dislocation stucture at the atomic level. High resolution transmission electron microscopy (HREM) offers the etential to obtain structural information at this level by observing these line defects in an "end-on" configuration.Samples of HREM images of several important dislocation types in Ni3Al and TiAl are shown in Figures through 3. Each of these particular dislocation types (i.e. Burgers vectors and line directions) tend to be longly favored in these compounds, indicating that along these line directions the dislocations are likely have either low mobility or low energy.

Direct Observation by Transmission Electron Microscopy of the Early Stages of Growth of Superconducting Thin Films

1989 ◽  
Vol 169 ◽  
Author(s):  
M. Grant Norton ◽  
Lisa A. Tietz ◽  
Scott R. Summerfelt ◽  
C. Barry Carter
Keyword(s):  
Electron Microscopy ◽  
Thin Films ◽  
Transmission Electron Microscopy ◽  
Substrate Surface ◽  
Critical Role ◽  
Specimen Preparation ◽  
Superconducting Thin Films ◽  
Ion Milling ◽  
Early Stages ◽  
Transmission Electron

AbstractThe fabrication of high quality thin films often depends on the early stages of the growth process during which epitaxy is established. The substrate surface structure generally plays a critical role at this stage. Many observations of the high‐Tc superconductor film‐substrate interface structure and chemistry have been made by transmission electron microscopy (TEM) of cross‐section samples. Ion‐milling induced damage, however, can be severe in these specimens. In the present study, the early stages of the growth of high Tc superconducting thin films of YBa2Cu3O7&#X03B4; have been studied by TEM using a technique which requires no post‐deposition specimen preparation.

scholarly journals Structure of Coal-Derived Metal-Supported Few-Layer Graphene Composite Materials Synthesized Using a Microwave-Assisted Catalytic Graphitization Process

Nanomaterials ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1672
Author(s):  
Faridul Islam ◽  
Arash Tahmasebi ◽  
Rou Wang ◽  
Jianglong Yu
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Bituminous Coal ◽  
Critical Role ◽  
Fabricated Metal ◽  
Catalytic Graphitization ◽  
Microwave Assisted ◽  
Layer Graphene ◽  
Transmission Electron ◽  
Few Layer Graphene

Metal-supported few-layer graphene (FLG) was synthesized via microwave-assisted catalytic graphitization owing to the increasing demand for it and its wide applications. In this study, we quickly converted earth-abundant and low-cost bituminous coal to FLG over Fe catalysts at a temperature of 1300 °C. X-ray diffraction, Raman spectroscopy, transmission electron microscopy, and N2 adsorption–desorption experiments were performed to analyze the fabricated metal-supported FLG. The results indicated that the microwave-irradiation temperature at a set holding-time played a critical role in the synthesis of metal-supported FLG. The highest degree of graphitization and a well-developed pore structure were fabricated at 1300 °C using a S10% Fe catalyst for 20 min. High-resolution transmission electron microscopy analysis confirmed that the metal-supported FLG fabricated via microwave-assisted catalytic graphitization consisted of 3–6 layers of graphene nanosheets. In addition, the 2D band at 2700 cm−1 in the Raman spectrum of the fabricated metal-supported FLG samples were observed, which indicated the presence of few-layer graphene structure. Furthermore, a mechanism was proposed for the microwave-assisted catalytic graphitization of bituminous coal. Here, we developed a cost-effective and environmental friendly metal-supported FLG method using a coal-based carbonaceous material.

scholarly journals 258ULTRASTRUCTURAL STUDY OF STALLION SPERM FOLLOWING THAWING, CAPACITATING AND IVF PROCEDURES: EFFECT OF THE PRESENCE OF CUMULUS-ENCLOSED OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 249
Author(s):  
F. Abbate ◽  
P.G. Germanà ◽  
S. Colleoni ◽  
F. Gandolfi
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Membrane Damage ◽  
Membrane Integrity ◽  
Developmental Competence ◽  
Lower Percentage ◽  
Sperm Cells ◽  
Transmission Electron ◽  
Intact Membrane

The use of IVF in horses has a limited efficiency, reflecting low oocyte developmental competence and inadequate sperm capacitation procedures. In a preliminary study, using carboxyfluorescein diacetate/propidium iodide staining, we determined that the freezing-thawing procedure left only 56.6±3.4 % of the sperm cells with an intact membrane. The following incubation in TALP-IVF induced membrane damage at high rates with only 9.58±1.8 % of them intact after 18h. However, the presence of at least four cumulus-enclosed oocytes (CEO) in the medium significantly increased the number of membrane-intact spermatozoa at the end of incubation (53.87±1.99%). This indicated that the sperm thawing and capacitating procedures can damage the cell membrane but the presence of four or more CEO in TALP-IVF could prevent further damages. The aim of the study was to investigate in detail the membrane damages and to analyze the differences induced by the presence of CEO. Spermatozoa were thawed in water at 37°C, and centrifuged for 30 minutes at 600g in a 45–90% Percoll gradient made with modified Tyrode’s medium. The sperm pellet was washed once in the same medium and diluted to a final concentration of 1×106 spermatozoa/ml TALP supplemented with 0.6% (w/v) BSA fatty acid free and 12μgmL−1 heparin (TALP-IVF). Sperm cells were incubated with 0 or 4 in vitro-matured CEO. Sperm cells were examined after thawing, 0, 2 and 18h from the beginning of incubation in TALP-IVF. Each experiment was replicated at least 3 times. Both scanning and transmission electron microscopy were performed on sperm samples fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer, pH 7.2, using standard procedures. Specimens for scanning electron microscopy were examined under a field emission gun JEOL JSM 6301 microscope. For transmission electron microscopy the samples were examined with a JEOL JEM 100 SX. A minimum of 25 cells were analyzed for each group. Immediately after thawing, damaged spermatozoa showed, on the surface of their heads, small vesicles correlated to a progressive process of vacuolisation and degeneration of membrane integrity. The same lesions were visible at all the successive time points taken into account. Moreover, a loss of the acrosome integrity with acrosomal swelling and a decrease of content homogeneity were observed particularly in the spermatozoa cultured for 18 h without CEO. When CEO were present in the IVF medium lesions were visible in a lower percentage of spermatozoa but the type of lesions did not differ from those observed in their absence. These observations confirmed our previous data and gave more details on the lesions that occur during the IVF procedures in the horse. Supported by MURST COFIN grant n. 2001078849.

scholarly journals Bone–titanium oxide interface in humans revealed by transmission electron microscopy and electron tomography

2011 ◽  
Vol 9 (67) ◽  
pp. 396-400 ◽  
Author(s):  
Anders Palmquist ◽  
Kathryn Grandfield ◽  
Birgitta Norlindh ◽  
Torsten Mattsson ◽  
Rickard Brånemark ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Bone Tissue ◽  
Titanium Oxide ◽  
Bone Growth ◽  
Electron Tomography ◽  
Critical Role ◽  
Light And Electron Microscopy ◽  
Implant Surface ◽  
Transmission Electron

Osseointegration, the direct contact between an implant surface and bone tissue, plays a critical role in interfacial stability and implant success. Analysis of interfacial zones at the micro- and nano-levels is essential to determine the extent of osseointegration. In this paper, a series of state-of-the-art microscopy techniques are used on laser-modified implants retrieved from humans. Partially laser-modified implants were retrieved after two and a half months' healing and processed for light and electron microscopy. Light microscopy showed osseointegration, with bone tissue growing both towards and away from the implant surface. Transmission electron microscopy revealed an intimate contact between mineralized bone and the laser-modified surface, including bone growth into the nano-structured oxide. This novel observation was verified by three-dimensional Z-contrast electron tomography, enabling visualization of an apatite layer, with different crystal direction compared with the apatite in the bone tissue, encompassing the nano-structured oxide. In conclusion, the present study demonstrates the nano-scale osseointegration and bonding between apatite and surface-textured titanium oxide. These observations provide novel data in human specimens on the ultrastructure of the titanium–bone interface.

Techniques for Transmission Electron Microscopy of Fiber-Matrix Interfaces in Aluminum Alloy-Boron Composites by Ion Erosio

1971 ◽  
Vol 29 ◽  
pp. 204-205
Author(s):  
G. G. Shaw
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Aluminum Alloy ◽  
Electron Beam ◽  
Pure Aluminum ◽  
Ion Milling ◽  
Transmission Electron ◽  
Fiber Matrix ◽  
Ion Erosion ◽  
Row Of Fibers

The morphology and composition of the fiber-matrix interface can best be studied by transmission electron microscopy and electron diffraction. For some composites satisfactory samples can be prepared by electropolishing. For others such as aluminum alloy-boron composites ion erosion is necessary.When one wishes to examine a specimen with the electron beam perpendicular to the fiber, preparation is as follows: A 1/8 in. disk is cut from the sample with a cylindrical tool by spark machining. Thin slices, 5 mils thick, containing one row of fibers, are then, spark-machined from the disk. After spark machining, the slice is carefully polished with diamond paste until the row of fibers is exposed on each side, as shown in Figure 1.In the case where examination is desired with the electron beam parallel to the fiber, preparation is as follows: Experimental composites are usually 50 mils or less in thickness so an auxiliary holder is necessary during ion milling and for easy transfer to the electron microscope. This holder is pure aluminum sheet, 3 mils thick.

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