scholarly journals Use of RNA Arbitrarily Primed-PCR Fingerprinting To Identify Vibrio cholerae Genes Differentially Expressed in the Host following Infection

2000 ◽  
Vol 68 (7) ◽  
pp. 3878-3887 ◽  
Author(s):  
Amit Chakrabortty ◽  
Soumita Das ◽  
Sabita Majumdar ◽  
Kanchan Mukhopadhyay ◽  
Susanta Roychoudhury ◽  
...  

ABSTRACT Evidence suggests that a repertoire of Vibrio cholerae genes are differentially expressed in vivo, and regulation of virulence factors in vivo may follow a different pathway. Our work was aimed at characterization of in vivo-grown bacteria and identification of genes that are differentially expressed following infection by RNA arbitrarily primed (RAP)-PCR fingerprinting. The ligated rabbit ileal loop model was used. The motility of in vivo-grown bacteria increased by 350% over that of in vitro-grown bacteria. Also, the in vivo-grown cells were more resistant to killing by human serum. By using the RAP-PCR strategy, five differentially expressed transcripts were identified. Two in vitro-induced transcripts encoded polypeptides for the leucine tRNA synthatase and the 50S ribosomal protein, and the three in vivo-induced transcripts encoded the SucA and MurE proteins and a polypeptide of unknown function. MurE is a protein involved in the peptidoglycan biosynthetic pathway. The lytic profiles of in vivo- and in vitro-grown cells suspended in distilled water were compared; the former was found to be slightly less sensitive to lysis. Ultrathin sections of both cells observed under the transmission electron microscope revealed that in contrast to the usual wavy discontinuous membrane structure of the in vitro-grown cells, in vivo-grown cells had a more rigid, clearly visible double-layered structure. The V. cholerae murE gene was cloned and sequenced. The sequence contained an open reading frame of 1,488 nucleotides with its own ribosome-binding site. A plasmid containing the murE gene of V. cholerae was transformed into V. cholerae 569B, and a transformed strain, 569BME, containing the plasmid was obtained. Ultrathin sections of 569BME viewed under a transmission electron microscope revealed a slightly more rigid cell wall than that of wild-type 569B. When V. cholerae 569B and 569BME cells were injected separately into ligated rabbit ileal loops, the transformed cells had a preference for growth in the ileal loops versus laboratory conditions.

Author(s):  
Gao Fengming

Transmission electron microscope(TEM) and scanning electron microscope(SEM) were widely used in experimental tumor studies. They are useful for evaluation of cellular transformation in vitro, classification of histological types of tumors and treating effect of tumors. We have obtained some results as follows:1. Studies on the malignant transformation of mammalian cells in vitro. Syrian golden hamster embryo cells(SGHEC) were transformed in vitro by ThO2 and/or ore dust. In a few days after dust added into medium, some dust crystals were phagocytized. Two weeks later, malignant transformation took place. These cells were of different size, nuclear pleomorphism, numerous ribosomes, increasing of microvilli on cell surface with various length and thickness, and blebs and ruffles(Figs. 1,2). Myelomonocytic leukemic transformation of mouse embryo cells(MEC) was induced in vitro by 3H-TdR. Transformed cells were become round from fusiform. The number of mitochondria and endoplasmic reticulum was reduced, ribosomes and nucleoli increased, shape of nuclei irregular, microvilli increased, and blebs and ruffles appeared(Fig. 3).


2007 ◽  
Vol 30 (6) ◽  
pp. 250 ◽  
Author(s):  
Jose Arellano Galindo ◽  
Maria Guadalupe Rodriquez Angeles ◽  
Norma Valazquez Guadarrama ◽  
Enrique Santos Esteban ◽  
Silvia Giono Cerezo

Purpose: To evaluate the hemolysin effect by ileal loop model produced by Vibrio cholerae O1 strains, compared with the cellular lysis or cytotoxic activity (CA) observed in cell culture. Method: We studied nine V. cholerae O1 strains, obtained during the Mexican outbreak of cholera (1990-1993), which had CA in Vero and CHO cells. Hemolysin was monitored with the hemolysis test. Titers of CA were calculated by CD50, and the association between CA and cholera toxin (CT) production was discarded by means of neutralization tests using an anti-CT polyclonal antibody. The CT production was measured with ELISA test. The LAL assay was performed in order to study relationships between the CA and bacterial lipopolysaccharide. Strains with CA were evaluated in rabbit and rat ileal loop models; hemorrhagic fluid was also measured. Tissues from ileal loop were included in paraffin to detect intestinal epithelial damage. Results: The hemolysin CA was not neutralized with the anti-CT polyclonal antibody. However, the associated factor of CA was heat labile. CA in cell cultures was not related to the bacterial lipopolysaccharide. The ileal loop test exhibited the presence of hemorrhagic tissue with inflammation. Conclusion: The V. cholerae O1 strains isolated were able to secrete hemolysin which, in turn, caused CA in cell cultures and produced the hemorrhagic and inflammatory effects observed in the ileal loop of rabbit and rat models.


2009 ◽  
Vol 24 (8) ◽  
pp. 2499-2502 ◽  
Author(s):  
Junfeng Hui ◽  
Daidi Fan

Hydroxyapatite (HAp) and brushite (DCPD) are two important compounds of the calcium apatite family with excellent bioactivity and osteoconductive properties in vivo. This work aimed to investigate the stability of HAp nanorods synthesized by the hydrothermal method in acetic acid aqueous solution. The results illuminated that HAp nanorods were converted into hollow nanospheres, and it was found that the concentration and amount of the acetic acid and the reaction time significantly affected the degree of the morphological evolution. Transmission electron microscope, high-resolution transmission electron microscope, and x-ray diffraction were performed for characterizing the samples.


Coatings ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 388 ◽  
Author(s):  
Samy M. El-Megharbel ◽  
Mohammed Alsawat ◽  
Fawziah A. Al-Salmi ◽  
Reham Z. Hamza

A newly synthesized zinc (II) oxide nanoparticle (ZnO-NPs) has been used as a disinfectant Nano-spray for the emerging corona virus (SARS-CoV-2). The synthesized obtained nanomaterial of (ZnO) was fully chemically characterized by using different spectroscopic analysis (FT-IR, UV and XRD) and surface analysis techniques. ZnO-Nps surface morphology and chemical purity has been investigated by transmission electron microscope (TEM), high resolution transmission electron microscope (HR-TEM), scanning electron microscopy (SEM) as well as energy dispersive X-ray analysis (EDX), Additionally Zeta potential and Zeta size distribution were measured and evaluated to confirm its nano-range scale. The synthesized Zno-NPs have been tested using 10% DMSO and ddH2O for estimation of antiviral activity against (SARS-CoV-2) by using cytotoxicity assay (CC50) and inhibitory concentration (IC50). The results revealed that (Zno-NPs) has high anti-SARS-CoV-2 activity at cytotoxic concentrations in vitro with non-significant selectivity index (CC50/IC50 ≤ 1). The current study results demonstrated the (ZnO-NPs) has potent antiviral activity at low concentration (IC50 = 526 ng/mL) but with some cytotoxic effect to the cell host by (CC50 = 292.2 ng/mL). We recommend using of (ZnO-NPs) as potent disinfectant against (SARS-Cov-2), but there are slight side effects on the cellular host, so we recommend more prospective studies on complexation of other compounds with (ZnO-NPs) in different concentrations to reduce its cellular toxicity and elevate its antiviral activity against SARS-CoV-2 activities.


Author(s):  
C. A. M. Meszoely ◽  
R. L. Beaudoin ◽  
E. F. Erbe ◽  
R. L. Steere

Plasmodium lophurae grown in monolayers of cells derived from turkey brains was freeze-fractured after fixation in 1.25% gluteraldehyde. The fixed material was placed in an ascending series of glycerol buffer until a 40% concentration was reached. This preparation was then frozen and fractured at -190°C under vacuum. Both fracture surfaces of the specimen were simultaneously shadowed and carbon coated to obtain complementary replicas. The replicas were studied with the JEM 100-B transmission electron microscope.Previous studies on stained ultrathin sections of the merozoites of mature schizonts show the merozoite to be surrounded by two membranes and an innermost microtubular system. In addition to this pellicular system of the merozoites, the entire schizont is surrounded by a loose limiting membrane. Our studies revealed splitting of the two membranes enclosing the exoerythrocytic merozoite. This resulted in the presence of four fracture faces, each with its own distinct topography.


2016 ◽  
Vol 22 (4) ◽  
pp. 922-932 ◽  
Author(s):  
Saeed Akhtar ◽  
Mousa Alkhalaf ◽  
Adnan A. Khan ◽  
Turki M. Almubrad

AbstractWe report ultrastructural features and transmission electron tomography of the dhub lizard (Uromastyx aegyptia) cornea and its adaptation to hot and dry environments. Six corneas of dhub lizards were fixed in 2.5% glutaraldehyde and processed for electron microscopy and tomography. The ultrathin sections were observed with a JEOL 1400 transmission electron microscope. The cornea of the dhub lizard is very thin (~28–30 µm). The epithelium constitutes ~14% of the cornea, whereas the stroma constitutes 80% of the cornea. The middle stromal lamellae are significantly thicker than anterior and posterior stromal lamellae. Collagen fibril (CF) diameters in the anterior stroma are variable in size (25–75 nm). Proteoglycans (PGs) are very large in the middle and posterior stroma, whereas they are small in the anterior stroma. Three-dimensional electron tomography was carried out to understand the structure and arrangement of the PG and CFs. The presence of large PGs in the posterior and middle stroma might help the animal retain a large amount of water to protect it from dryness. The dhub corneal structure is equipped to adapt to the dry and hot desert environment.


2018 ◽  
Vol 14 (1) ◽  
pp. 16
Author(s):  
Qonita Awliya Hanif ◽  
Reva Edra Nugraha ◽  
Witri Wahyu Lestari

<p><em>Metal–Organic Frameworks</em> (MOFs) merupakan material berpori baru yang berpotensi sebagai pengantar dan pelepas lambat obat. Strukturnya yang fleksibel, ukuran pori kristalin yang teratur, dan sisi koordinasi yang beragam merupakan beberapa kelebihan dari MOFs yang mendukung dalam enkapsulasi berbagai obat. Metode yang dapat digunakan untuk sintesis MOFs diantaranya nanopresipitasi, <em>solvothermal</em>, <em>reverse microemulsion</em>, dan reaksi <em>solvothermal</em> dengan template surfaktan. Karakterisasi material hasil sintesis maupun profil setelah enkapsulasi (<em>loading</em>) dapat dilakukan menggunakan <em>Scanning Electron Micrscope</em> (SEM), <em>Transmission Electron Microscope</em> (TEM), <em>Differential Scanning Calorymetry</em> (DSC), <em>Fourier Transform Infra Red Spectroscopy</em> (FTIR), dan <em>Powder X-Ray Diffraction </em>(PXRD). Metode <em>loading</em> obat terdiri dari dua kategori, yakni penggabungan agen biomedis secara langsung dan <em>loading</em> secara <em>post synthesis</em>. Sebelum MOFs diaplikasikan, perlu dilakukan aktivasi dan penempelan material obat. Pengujian lepas lambat dapat dijalankan pada beberapa kondisi seperti dalam <em>Simulated Body Fluid</em> (SBF), <em>Phosphate Buffer Saline</em> (PBS), <em>Bovine Serum Albumin</em> (BSA) maupun simulasi menggunakan <em>Grand Canonical Monte Carlo</em> (GCMC). Pengujian secara <em>in vivo</em> dan <em>in vitro</em> juga dapat dilakukan untuk mengetahui dampaknya pada tubuh makhluk hidup dan aktivitasnya terhadap sel patogen. Kombinasi organik <em>linker</em> dan ion logam pusat yang berbeda akan menghasilkan ukuran pori, fleksibilitas, kapasitas <em>loading</em>, profil pelepasan obat, toksisitas, dan kemampuan menginhibisi yang berbeda pula. Pada review kali ini akan dibahas tentang kajian singkat terkait struktur dan desain MOFs, bio-MOFs, nano bio MOFs, strategi sintesis, dan strategi <em>loading</em> dan pelepasan obat untuk aplikasi dalam biomedis. Selanjutnya akan diberikan beberapa contoh aplikasi yang sudah dilakukan sejauh ini misalnya beberapa jenis MOFs yang sudah dienkapsulasi dengan beberapa material obat, seperti 5-fluoracil, ibuprofen, doxorubicin, dan dikaji waktu pelepasannya dan interaksinya dengan permodelan komputasi.</p><p><strong>Study of Metal–Organic Frameworks (M</strong><strong>OF</strong><strong>s) as </strong><strong>a</strong><strong> Novel Material for Drug Delivery</strong>. Metal–Organic Frameworks (MOFs) are a novel class of porous material that has wide potential applications including in drug delivery and slow release. Its flexible structure, regular crystalline pore size, and various coordination sites are some of the advantages of supporting MOFs properties in the encapsulation of various drugs. Various methods can be used for the MOFs synthesis include nanoprecipitation, solvothermal, reverse micro emulsion, and surfactant-templated solvothermal. Both characterization for synthesized materials and profile after encapsulation can be done using Scanning Electron Microscope (SEM), Transmission Electron Microscope (TEM), Differential Scanning Calorimetry (DSC), Fourier Transform Infra-Red Spectroscopy (FTIR), and Powder X-Ray Diffraction (PXRD). The drug loading method consists of two categories, namely the direct incorporation of biomedical agents and post-synthesis method. Before MOFs are applied in biomedical application, activation and attachment of medicinal materials should be performed. Meanwhile, for slow release testing can be run on several conditions such as in Simulated Body Fluid (SBF), Phosphate Buffer Saline (PBS), Bovine Serum Albumin (BSA) and simulation using Grand Canonical Monte Carlo (GCMC). In vivo and in vitro testing can also be done to determine the impact on the body of living creatures and their activity on pathogen cells. Different organic linker and metal center combinations will result in pore size, flexibility, loading capacity, drug release profiles, toxicity, and different inhibiting ability. Herein, we will discuss a brief review of the structure and design of MOFs, bio-MOFs, nano-bio MOFs, synthesis, drug loading and release strategies for applications in biomedicine. Furthermore, there will be some examples of applications that have been done so far, e.g. some types of MOFs that have been encapsulated with some medicinal materials, such as 5-fluorouracil, ibuprofen, doxorubicin, and reviewed its release time and interaction with computational modeling.</p>


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