THE PRODUCTION BY BACILLUS THURINGIENSIS BERLINER OF A HEAT-STABLE SUBSTANCE TOXIC FOR INSECTS

1959 ◽  
Vol 5 (2) ◽  
pp. 161-168 ◽  
Author(s):  
Ellicott McConnell ◽  
A. Glenn Richards
Keyword(s):  
Bacillus Thuringiensis ◽  
Inclusion Bodies ◽  
Galleria Mellonella ◽  
Culture Media ◽  
Water Soluble ◽  
Broth Culture ◽  
Toxic Principle ◽  
Heat Stable

Bacillus thuringiensis Berliner produces in vitro a heat-stable, dialyzable substance which is toxic for insects when injected. The same or a similar substance is produced in vivo. The toxic principle is of unknown composition. It is heat-stable, water-soluble, dialyzable, and resistant to low temperatures. It is probably neither a protein nor a lipid. Clearly it is distinct from the heat-labile inclusion bodies and from lecithinase. Growth-curve studies showed that the heat-stable toxin appeared in liver broth cultures during the active growth phase, prior to the formation of spores or inclusion bodies. An attempt to produce the toxic principle from culture media in the absence of bacteria was unsuccessful from sterile inocula both from in vivo and in vitro sources. The LD50 for larvae of Galleria mellonella injected with autoclaved supernatant from a 10-day-old liver broth culture of B. thuringiensis was determined to be 0.00036 ml per larva or 0.002 ml per gram of larvae. Approximately the same level of toxicity was found for another caterpillar, a fly larva, and cockroaches. After larvae of Galleria or Pyrausla have been dead for more than 2 days from infection with B. thuringiensis the bacillus could no longer be recovered. A sublethal amount of the heat-stable toxin injected into old larvae of Galleria delayed emergence of the adults by 30 to 40%. The non-pathogenic Bacillus cereus was found to produce a similar-acting, heat-stable toxin under the same conditions that one is produced by B. thuringiensis.

scholarly journals In vivo transcriptome analysis provides insights into host-dependent expression of virulence factors by Yersinia entomophaga MH96, during infection of Galleria mellonella

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Amber R Paulson ◽  
Maureen O’Callaghan ◽  
Xue-Xian Zhang ◽  
Paul B Rainey ◽  
Mark R H Hurst
Keyword(s):  
Galleria Mellonella ◽  
Functional Enrichment ◽  
Natural Environments ◽  
Insecticidal Toxin ◽  
Heat Stable ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Cell Densities

Abstract The function of microbes can be inferred from knowledge of genes specifically expressed in natural environments. Here, we report the in vivo transcriptome of the entomopathogenic bacterium Yersinia entomophaga MH96, captured during initial, septicemic, and pre-cadaveric stages of intrahemocoelic infection in Galleria mellonella. A total of 1285 genes were significantly upregulated by MH96 during infection; 829 genes responded to in vivo conditions during at least one stage of infection, 289 responded during two stages of infection, and 167 transcripts responded throughout all three stages of infection compared to in vitro conditions at equivalent cell densities. Genes upregulated during the earliest infection stage included components of the insecticidal toxin complex Yen-TC (chi1, chi2, and yenC1), genes for rearrangement hotspot element containing protein yenC3, cytolethal distending toxin cdtAB, and vegetative insecticidal toxin vip2. Genes more highly expressed throughout the infection cycle included the putative heat-stable enterotoxin yenT and three adhesins (usher-chaperone fimbria, filamentous hemagglutinin, and an AidA-like secreted adhesin). Clustering and functional enrichment of gene expression data also revealed expression of genes encoding type III and VI secretion system-associated effectors. Together these data provide insight into the pathobiology of MH96 and serve as an important resource supporting efforts to identify novel insecticidal agents.

scholarly journals A Bacillus thuringiensis Chitin-Binding Protein is Involved in Insect Peritrophic Matrix Adhesion and Takes Part in the Infection Process

Toxins ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 252
Author(s):  
Jiaxin Qin ◽  
Zongxing Tong ◽  
Yiling Zhan ◽  
Christophe Buisson ◽  
Fuping Song ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Galleria Mellonella ◽  
Binding Protein ◽  
Insect Pest ◽  
Infection Process ◽  
Peritrophic Matrix ◽  
Limited Information ◽  
Chitin Binding Protein

Bacillus thuringiensis (Bt) is used for insect pest control, and its larvicidal activity is primarily attributed to Cry toxins. Other factors participate in infection, and limited information is available regarding factors acting on the peritrophic matrix (PM). This study aimed to investigate the role of a Bt chitin-binding protein (CBPA) that had been previously shown to be expressed at pH 9 in vitro and could therefore be expressed in the alkaline gut of lepidopteron larvae. A ∆cbpA mutant was generated that was 10-fold less virulent than wild-type Bt HD73 towards Ostrinia furnacalis neonate larvae, indicating its important role in infection. Purified recombinant Escherichia coli CBPA was shown to have a chitin affinity, thus indicating a possible interaction with the chitin-rich PM. A translational GFP–CBPA fusion elucidated the localization of CBPA on the bacterial surface, and the transcriptional activity of the promoter PcbpA was immediately induced and confirmed at pH 9. Next, in order to connect surface expression and possible in vivo gut activity, last instar Galleria mellonella (Gm) larvae (not susceptible to Bt HD-73) were used as a model to follow CBPA in gut expression, bacterial transit, and PM adhesion. CBPA-GFP was quickly expressed in the Gm gut lumen, and more Bt HD73 strain bacteria adhered to the PM than those of the ∆cbpA mutant strain. Therefore, CBPA may help to retain the bacteria, via the PM binding, close to the gut surface and thus takes part in the early steps of Bt gut interactions.

scholarly journals Paroxysmal Nocturnal Hemoglobinuria

Blood ◽  
1953 ◽  
Vol 8 (5) ◽  
pp. 444-458 ◽  
Author(s):  
WILLIAM H. CROSBY
Keyword(s):  
Hemolytic Activity ◽  
Soluble Fraction ◽  
Insoluble Fraction ◽  
Water Soluble ◽  
Red Cells ◽  
Distilled Water ◽  
Heat Stable ◽  
Heat Labile

Abstract This report demonstrates the role and to some extent the interrelations of various factors that are active in the PNH hemolytic system. 1. Activity of four plasma factors, probably protein in nature, has been demonstrated. Two of these factors are hemolytic against PNH red cells, but not against normal red cells. The other two inhibit PNH hemolysis. (a). The heat labile hemolytic factor is water soluble and is therefore present in the soluble fraction of serum that has been dialyzed against distilled water. It is almost completely destroyed by heating at 53 C. for 10 minutes. It is slowly inactivated by incubation at 37 C. with 100 units per ml. of thrombin. It is rapidly destroyed by concentrations of thrombin in excess of 200 units per ml. It is inactive unless the heat stable hemolytic factor is also present. (b). The heat stable hemolytic factor is insoluble in water and is therefore precipitated from serum by dialysis against distilled water. It is quite resistant to 100 units per ml. of thrombin and to incubation at 53 C. It is inactive unless the heat labile hemolytic factor is also present. (c). The heat labile inhibitor is insoluble in water and is therefore found in the insoluble fraction of serum dialyzed against distilled water. It is inactivated by heating at 53 C. for 10 minutes but not by incubation with 100 units per ml. of thrombin. (d). The heat stable inhibitor is found in the water-soluble fraction of dialyzed serum. It withstands dialysis poorly, but it is not affected by 30 minutes incubation at 53 C. Incubation at 37 C. with 100 units per ml. of thrombin for 10 minutes destroys its inhibitory activity. Apparently the inhibitors are not interdependent. 2. Calcium in small amounts is probably essential to the PNH hemolytic system. The concentration of calcium that is optimum for hemolysis lies in the neighborhood of 2.5 mM. The optimum is a little less than the amount normally present in the plasma. Calcium in excess inhibits hemolysis in vitro, and no hemolysis occurs when the concentration exceeds 25 mM. per liter. 3. Magnesium is also essential to the PNH hemolytic system. As magnesium is added to the system in vitro hemolytic activity increases until the concentration exceeds 10 mM. per liter. Amounts greater than that have some dampening effect. Magnesium appears to antagonize the heat stable inhibitor of the PNH hemolytic system. 4. Thrombin is involved in this system insofar as the heat stable inhibitor and the heat labile hemolytic factor may be destroyed by thrombic activity. The inhibitor is rapidly destroyed, the hemolytic factor slowly. Therefore, the sum of the reaction to small amounts of thrombin in the PNH hemolytic system is to increase hemolytic activity. 5. Dilute heparin and protamine increase the activity of the PNH hemolytic system in vitro, probably by blocking the two inhibitors. Heparin appears to work against the heat stable inhibitor, protamine against the heat labile inhibitor. 6. The intensity of PNH hemolytic activity whether in vitro or in vivo is probably related to a balance that exists between the inhibitors and the hemolytic factors. Hemolytic crises may occur when the hemolytic factors are increased or when their antagonists are depressed.

scholarly journals Assessment of in vitro and in vivo Antimicrobial Activity of an Isonitrosomalononitrile Silver(I) Salt

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Colmont M ◽  
Brunel JM
Keyword(s):  
Antimicrobial Activity ◽  
Animal Model ◽  
Galleria Mellonella ◽  
Silver Salt ◽  
Antimicrobial Activities ◽  
Water Soluble ◽  
Gram Positive ◽  
Minimum Inhibitory Concentrations

The design and evaluation of antimicrobial activities of an isonitrosomalononitrile silver(I) salt was reported. This highly stable water-soluble silver salt shows Minimum Inhibitory Concentrations (MIC) values ranging from 0.15 to 5 µg/mL towards both sensitive and resistant Gram-positive and negative bacteria. Furthermore, this silver salt has been investigated for its ability to treat a S. aureus infected Galleria mellonella larvae animal model with promising results. Thus, our results demonstrated that 80% of the treated larvae survived after 24h with respect to 10% of the untreated ones, respectively.

Fenofibrate Solid Dispersion for Improving Oral Bioavailability: Preparation, and Characterization and in vivo Evaluation

2020 ◽  
Vol 13 (5) ◽  
pp. 5102-5109
Author(s):  
Venu Madhav K ◽  
Somnath De ◽  
Chandra Shekar Bonagiri ◽  
Sridhar Babu Gummadi
Keyword(s):  
Oral Bioavailability ◽  
Oral Delivery ◽  
Solid Dispersions ◽  
In Vitro Release ◽  
Aqueous Solubility ◽  
Water Soluble ◽  
Scanning Calorimetry ◽  
In Vivo Evaluation

Fenofibrate (FN) is used in the treatment of hypercholesterolemia. It shows poor dissolution and poor oral bioavailability after oral administration due to high liphophilicity and low aqueous solubility. Hence, solid dispersions (SDs) of FN (FN-SDs) were develop that might enhance the dissolution and subsequently oral bioavailability. FN-SDs were prepared by solvent casting method using different carriers (PEG 4000, PEG 6000, β cyclodextrin and HP β cyclodextrin) in different proportions (0.25%, 0.5%, 0.75% and 1% w/v). FN-SDs were evaluated solubility, assay and in vitro release studies for the optimization of SD formulation. Differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD) and scanning electron microscopy (SEM) analysis was performed for crystalline and morphology analysis, respectively. Further, optimized FN-SD formulation evaluated for pharmacokinetic performance in Wistar rats, in vivo in comparison with FN suspension.  From the results, FN-SD3 and FN-SD6 have showed 102.9 ±1.3% and 105.5±3.1% drug release, respectively in 2 h. DSC and PXRD studies revealed that conversion of crystalline to amorphous nature of FN from FT-SD formulation. SEM studies revealed the change in the orientation of FN when incorporated in SDs. The oral bioavailability FN-SD3 and FN-SD6 formulations exhibited 2.5-folds and 3.1-folds improvement when compared to FN suspension as control. Overall, SD of FN could be considered as an alternative dosage form for the enhancement of oral delivery of poorly water-soluble FN.

Chemosensitizing Activity of Histone Deacetylases Inhibitory Cyclic Hydroxamic Acids for Combination Chemotherapy of Lymphatic Leukemia

2018 ◽  
Vol 18 (4) ◽  
pp. 365-371 ◽  
Author(s):  
Denis V. Mishchenko ◽  
Margarita E. Neganova ◽  
Elena N. Klimanova ◽  
Tatyana E. Sashenkova ◽  
Sergey G. Klochkov ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Acute Toxicity ◽  
Histone Deacetylases ◽  
Hydroxamic Acids ◽  
Water Soluble ◽  
Male Rat ◽  
Hybrid Male ◽  
Cyclic Hydroxamic Acids

Background: Anti-tumor effect of hydroxamic acid derivatives is largely connected with its properties as efficient inhibitors of histone deacetylases, and other metalloenzymes involved in carcinogenesis. Objective: The work was aimed to (i) determine the anti-tumor and chemosensitizing activity of the novel racemic spirocyclic hydroxamic acids using experimental drug sensitive leukemia P388 of mice, and (ii) determine the structure-activity relationships as metal chelating and HDAC inhibitory agents. Method: Outbreed male rat of 200-220 g weights were used in biochemical experiments. In vivo experiments were performed using the BDF1 hybrid male mice of 22-24 g weight. Lipid peroxidation, Fe (II) -chelating activity, HDAC fluorescent activity, anti-tumor and anti-metastatic activity, acute toxicity techniques were used in this study. Results: Chemosensitizing properties of water soluble cyclic hydroxamic acids (CHA) are evaluated using in vitro activities and in vivo methods and found significant results. These compounds possess iron (II) chelating properties, and slightly inhibit lipid peroxidation. CHA prepared from triacetonamine (1a-e) are more effective Fe (II) ions cheaters, as compared to CHA prepared from 1- methylpiperidone (2a-e). The histone deacetylase (HDAC) inhibitory activity, lipophilicity and acute toxicity were influenced by the length amino acids (size) (Glycine < Alanine < Valine < Leucine < Phenylalanine). All compounds bearing spiro-N-methylpiperidine ring (2a-e) are non-toxic up to 1250 mg/kg dose, while compounds bearing spiro-tetramethylpiperidine ring (1a-e) exhibit moderate toxicity which increases with increasing lipophility, but not excite at 400 mg/kg. Conclusion: It was shown that the use of combination of non-toxic doses of cisplatin (cPt) or cyclophosphamide with CHA in most cases result in the appearance of a considerable anti-tumor effect of cytostatics. The highest chemosensitizing activity with respect to leukemia Р388 is demonstrated by the CHA derivatives of Valine 1c or 2c.

scholarly journals A Peptide Found in Human Serum, Derived from the C-Terminus of Albumin, Shows Antifungal Activity In Vitro and In Vivo

2020 ◽  
Vol 8 (10) ◽  
pp. 1627
Author(s):  
Tecla Ciociola ◽  
Pier Paolo Zanello ◽  
Tiziana D’Adda ◽  
Serena Galati ◽  
Stefania Conti ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Human Serum ◽  
Fungicidal Activity ◽  
Galleria Mellonella ◽  
Serum Proteins ◽  
Morphological Alterations ◽  
C Terminus ◽  
Different Sources

The growing problem of antimicrobial resistance highlights the need for alternative strategies to combat infections. From this perspective, there is a considerable interest in natural molecules obtained from different sources, which are shown to be active against microorganisms, either alone or in association with conventional drugs. In this paper, peptides with the same sequence of fragments, found in human serum, derived from physiological proteins, were evaluated for their antifungal activity. A 13-residue peptide, representing the 597–609 fragment within the albumin C-terminus, was proved to exert a fungicidal activity in vitro against pathogenic yeasts and a therapeutic effect in vivo in the experimental model of candidal infection in Galleria mellonella. Studies by confocal microscopy and transmission and scanning electron microscopy demonstrated that the peptide penetrates and accumulates in Candida albicans cells, causing gross morphological alterations in cellular structure. These findings add albumin to the group of proteins, which already includes hemoglobin and antibodies, that could give rise to cryptic antimicrobial fragments, and could suggest their role in anti-infective homeostasis. The study of bioactive fragments from serum proteins could open interesting perspectives for the development of new antimicrobial molecules derived by natural sources.

scholarly journals Hexapod Assassins’ Potion: Venom Composition and Bioactivity from the Eurasian Assassin Bug Rhynocoris iracundus

Biomedicines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 819
Author(s):  
Nicolai Rügen ◽  
Timothy P. Jenkins ◽  
Natalie Wielsch ◽  
Heiko Vogel ◽  
Benjamin-Florian Hempel ◽  
...  
Keyword(s):  
Biomedical Applications ◽  
Galleria Mellonella ◽  
Systemic Reactions ◽  
Venom Composition ◽  
Assassin Bug ◽  
Molecular Components ◽  
First Time ◽  
Tissue Digestion

Assassin bug venoms are potent and exert diverse biological functions, making them potential biomedical goldmines. Besides feeding functions on arthropods, assassin bugs also use their venom for defense purposes causing localized and systemic reactions in vertebrates. However, assassin bug venoms remain poorly characterized. We collected the venom from the assassin bug Rhynocoris iracundus and investigated its composition and bioactivity in vitro and in vivo. It caused lysis of murine neuroblastoma, hepatoma cells, and healthy murine myoblasts. We demonstrated, for the first time, that assassin bug venom induces neurolysis and suggest that it counteracts paralysis locally via the destruction of neural networks, contributing to tissue digestion. Furthermore, the venom caused paralysis and melanization of Galleria mellonella larvae and pupae, whilst also possessing specific antibacterial activity against Escherichia coli, but not Listeria grayi and Pseudomonas aeruginosa. A combinatorial proteo-transcriptomic approach was performed to identify potential toxins responsible for the observed effects. We identified neurotoxic Ptu1, an inhibitory cystin knot (ICK) toxin homologous to ω-conotoxins from cone snails, cytolytic redulysins homologous to trialysins from hematophagous kissing bugs, and pore-forming hemolysins. Additionally, chitinases and kininogens were found and may be responsible for insecticidal and cytolytic activities. We demonstrate the multifunctionality and complexity of assassin bug venom, which renders its molecular components interesting for potential biomedical applications.

scholarly journals In Silico Predicted Antifungal Peptides: In Vitro and In Vivo Anti-Candida Activity

2021 ◽  
Vol 7 (6) ◽  
pp. 439
Author(s):  
Tecla Ciociola ◽  
Walter Magliani ◽  
Tiziano De Simone ◽  
Thelma A. Pertinhez ◽  
Stefania Conti ◽  
...  
Keyword(s):  
In Silico ◽  
Mammalian Cells ◽  
Galleria Mellonella ◽  
Ex Vivo ◽  
Consensus Sequence ◽  
In Silico Analysis ◽  
Significant Activity ◽  
Synthetic Antibody

It has been previously demonstrated that synthetic antibody-derived peptides could exert a significant activity in vitro, ex vivo, and/or in vivo against microorganisms and viruses, as well as immunomodulatory effects through the activation of immune cells. Based on the sequence of previously described antibody-derived peptides with recognized antifungal activity, an in silico analysis was conducted to identify novel antifungal candidates. The present study analyzed the candidacidal and structural properties of in silico designed peptides (ISDPs) derived by amino acid substitutions of the parent peptide KKVTMTCSAS. ISDPs proved to be more active in vitro than the parent peptide and all proved to be therapeutic in Galleria mellonella candidal infection, without showing toxic effects on mammalian cells. ISDPs were studied by circular dichroism spectroscopy, demonstrating different structural organization. These results allowed to validate a consensus sequence for the parent peptide KKVTMTCSAS that may be useful in the development of novel antimicrobial molecules.

scholarly journals Development, Characterization and In Vivo Pharmacokinetic Assessment of Rectal Suppositories Containing Combination Antiretroviral Drugs for HIV Prevention

Pharmaceutics ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1110
Author(s):  
Kunal Jhunjhunwala ◽  
Charles W. Dobard ◽  
Sunita Sharma ◽  
Natalia Makarova ◽  
Angela Holder ◽  
...  
Keyword(s):  
Hiv Prevention ◽  
Antiretroviral Drugs ◽  
Water Soluble ◽  
Fixed Dose ◽  
Receptive Anal Intercourse ◽  
On Demand ◽  
Dose Combination ◽  
Rectal Suppositories

Receptive anal intercourse (RAI) contributes significantly to HIV acquisition underscoring the need to develop HIV prevention options for populations engaging in RAI practices. We explored the feasibility of formulating rectal suppositories with potent antiviral drugs for on-demand use. A fixed-dose combination of tenofovir (TFV) and elvitegravir (EVG) (40 mg each) was co-formulated in six different suppository bases (three fat- and three water-soluble). Fat-soluble witepsol H15 and water-soluble polyethylene glycol (PEG) based suppositories demonstrated favorable in vitro release and were advanced to assess in vivo pharmacokinetics following rectal administration in macaques. In vivo drug release profiles were similar for both suppository bases. Median concentrations of TFV and EVG detected in rectal fluids at 2 h were 1- and 2-logs higher than the in vitro IC50, respectively; TFV-diphosphate levels in rectal tissues met or exceeded those associated with high efficacy against rectal simian HIV (SHIV) exposure in macaques. Leveraging on these findings, a PEG-based suppository with a lower dose combination of tenofovir alafenamide (TAF) and EVG (8 mg each) was developed and found to achieve similar rectal drug exposures in macaques. This study establishes the utility of rectal suppositories as a promising on-demand strategy for HIV PrEP and supports their clinical development.

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