scholarly journals Selection of Single-Chain Antibodies against the VP8* Subunit of Rotavirus VP4 Outer Capsid Protein and Their Expression in Lactobacillus casei

2004 ◽  
Vol 70 (11) ◽  
pp. 6936-6939 ◽  
Author(s):  
Vicente Monedero ◽  
Jes�s Rodr�guez-D�az ◽  
Rosa Viana ◽  
Javier Buesa ◽  
Gaspar P�rez-Mart�nez
Keyword(s):  
Lactobacillus Casei ◽  
Probiotic Bacteria ◽  
Extracellular Expression ◽  
Outer Capsid Protein ◽  
Single Chain ◽  
Single Chain Antibodies ◽  
Outer Capsid ◽  
Selection Of

ABSTRACT Single-chain antibodies (scFv) recognizing the VP8* fraction of rotavirus outer capsid and blocking rotavirus infection in vitro were isolated by phage display. Vectors for the extracellular expression in Lactobacillus casei of one of the scFv were constructed. L. casei was able to secrete active scFv to the growth medium, showing the potential of probiotic bacteria to be engineered to express molecules suitable for in vivo antirotavirus therapies.

scholarly journals Putative Autocleavage of Outer Capsid Protein μ1, Allowing Release of Myristoylated Peptide μ1N during Particle Uncoating, Is Critical for Cell Entry by Reovirus

2004 ◽  
Vol 78 (16) ◽  
pp. 8732-8745 ◽  
Author(s):  
Amy L. Odegard ◽  
Kartik Chandran ◽  
Xing Zhang ◽  
John S. L. Parker ◽  
Timothy S. Baker ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Structural Changes ◽  
General Mechanism ◽  
Membrane Penetration ◽  
Outer Capsid Protein ◽  
Core Proteins ◽  
Peptide Release ◽  
Outer Capsid

ABSTRACT Several nonenveloped animal viruses possess an autolytic capsid protein that is cleaved as a maturation step during assembly to yield infectious virions. The 76-kDa major outer capsid protein μ1 of mammalian orthoreoviruses (reoviruses) is also thought to be autocatalytically cleaved, yielding the virion-associated fragments μ1N (4 kDa; myristoylated) and μ1C (72 kDa). In this study, we found that μ1 cleavage to yield μ1N and μ1C was not required for outer capsid assembly but contributed greatly to the infectivity of the assembled particles. Recoated particles containing mutant, cleavage-defective μ1 (asparagine → alanine substitution at amino acid 42) were competent for attachment; processing by exogenous proteases; structural changes in the outer capsid, including μ1 conformational change and σ1 release; and transcriptase activation but failed to mediate membrane permeabilization either in vitro (no hemolysis) or in vivo (no coentry of the ribonucleotoxin α-sarcin). In addition, after these particles were allowed to enter cells, the δ region of μ1 continued to colocalize with viral core proteins in punctate structures, indicating that both elements remained bound together in particles and/or trapped within the same subcellular compartments, consistent with a defect in membrane penetration. If membrane penetration activity was supplied in trans by a coinfecting genome-deficient particle, the recoated particles with cleavage-defective μ1 displayed much higher levels of infectivity. These findings led us to propose a new uncoating intermediate, at which particles are trapped in the absence of μ1N/μ1C cleavage. We additionally showed that this cleavage allowed the myristoylated, N-terminal μ1N fragment to be released from reovirus particles during entry-related uncoating, analogous to the myristoylated, N-terminal VP4 fragment of picornavirus capsid proteins. The results thus suggest that hydrophobic peptide release following capsid protein autocleavage is part of a general mechanism of membrane penetration shared by several diverse nonenveloped animal viruses.

scholarly journals Ribosome display and selection of single-chain variable fragments effectively inhibit growth and progression of microspheres in vitro and in vivo

2018 ◽  
Vol 109 (5) ◽  
pp. 1503-1512
Author(s):  
Shangke Huang ◽  
Lu Feng ◽  
Gaili An ◽  
Xiaojin Zhang ◽  
Zixuan Zhao ◽  
...  
Keyword(s):  
Single Chain ◽  
Ribosome Display ◽  
Single Chain Variable Fragments ◽  
Selection Of

In vivo selection of single-chain antibodies using a yeast two-hybrid system

2000 ◽  
Vol 238 (1-2) ◽  
pp. 161-172 ◽  
Author(s):  
Antje Pörtner-Taliana ◽  
Marijane Russell ◽  
Karen J Froning ◽  
Paul R Budworth ◽  
John D Comiskey ◽  
...  
Keyword(s):  
Hybrid System ◽  
Single Chain ◽  
Yeast Two Hybrid ◽  
Single Chain Antibodies ◽  
In Vivo Selection ◽  
Yeast Two Hybrid System ◽  
Two Hybrid ◽  
Selection Of

scholarly journals Metatranscriptome profiling of the dynamic transcription of mRNA and sRNA of a probioticLactobacillusstrain in human gut

2018 ◽  
Author(s):  
Jicheng Wang ◽  
Zhihong Sun ◽  
Jianmin Qiao ◽  
Dong Chen ◽  
Chao Cheng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lactobacillus Casei ◽  
Lactic Acid Production ◽  
Expression Patterns ◽  
Probiotic Bacteria ◽  
Gi Tract ◽  
Human Gut ◽  
Bacterial Gene

AbstractMetatranscriptomic sequencing has recently been applied to study how pathogens and probiotics affect human gastrointestinal (GI) tract microbiota, which provides new insights into their mechanisms of action. In this study, metatranscriptomic sequencing was applied to deduce thein vivoexpression patterns of an ingestedLactobacillus caseistrain, which was compared with itsin vitrogrowth transcriptomes. Extraction of the strain-specific reads revealed that transcripts from the ingestedL. caseiwere increased, while those from the residentL. paracaseistrains remained unchanged. Mapping of all metatranscriptomic reads and transcriptomic reads toL. caseigenome showed that gene expressionin vitroandin vivodiffered dramatically. About 39% (1163) mRNAs and 45% (93) sRNAs ofL. caseiwell-expressed were repressed after ingested into human gut. Expression of ABC transporter genes and amino acid metabolism genes was induced at day-14 of ingestion; and genes for sugar and SCFA metabolisms were activated at day-28 of ingestion. Moreover, expression of sRNAs specific to thein vitrolog phase was more likely to be activated in human gut. Expression of rli28c sRNA with peaked expression during thein vitrostationary phase was also activated in human gut; this sRNA repressedL. caseigrowth and lactic acid productionin vitro. These findings implicate that the ingestedL. caseimight have to successfully change its transcription patterns to survive in human gut, and the time-dependent activation patterns indicate a highly dynamic cross-talk between the probiotic and human gut including its microbe community.ImportanceProbiotic bacteria are important in food industry and as model microorganisms in understanding bacterial gene regulation. Although probiotic functions and mechanisms in human gastrointestinal tract are linked to the unique probiotic gene expression, it remains elusive how transcription of probiotic bacteria is dynamically regulated after being ingested. Previous study of probiotic gene expression in human fecal samples has been restricted due to its low abundance and the presence of of closely related species. In this study, we took the advantage of the good depth of metatranscriptomic sequencing reads and developed a strain-specific read analysis method to discriminate the transcription of the probioticLactobacillus caseiand those of its resident relatives. This approach and additional bioinformatics analysis allowed the first study of the dynamic transcriptome profiles of probioticL casei in vivo. The novel findings indicate a highly regulated repression and dynamic activation of probiotic genome in human GI tract.

Flow cytometry as an auxiliary tool for the selection of probiotic bacteria

2015 ◽  
Vol 6 (5) ◽  
pp. 727-734 ◽  
Author(s):  
D. Mudroňová
Keyword(s):  
Flow Cytometry ◽  
Antimicrobial Agents ◽  
Probiotic Bacteria ◽  
In Vitro Tests ◽  
Good Growth ◽  
Bacterial Strains ◽  
Functional Characterisation ◽  
Selection Of

Selection of appropriate bacterial strains is crucial for development of new probiotic preparations. The fundamental prerequisite for potential efficacy of a probiotic preparation for oral application is the selection of appropriate bacterial strains with good gastrointestinal colonisation abilities, antimicrobial activity, and tolerance of conditions in the gastrointestinal tract, resistance to different antimicrobial agents, survival during processing and storage. The strain should be genetically stable, it should have good growth properties, to maintain its high viability at processing and when in storage. Mostly, the properties of promising strains are tested in the first phase in vitro, and only the best ones undergo subsequent in vivo testing. in vitro tests are often performed by classical microbiological cultivation methods which are material and time consuming, and they are not able to distinguish between ‘viable but nonculturable’ and dead bacteria. Flow cytometry is usually used for counting, phenotyping or functional characterisation of immune cells. Nowadays, flow cytometry is increasingly used in microbiology for counting bacteria, determining their viability and metabolic activity, detecting specific strains or testing their adherence abilities. The utilisation of flow cytometry in combination with an appropriate fluorescent labelling represents an effective and rapid method for the selection of probiotic bacteria.

scholarly journals The Effectiveness of Candidate Probiotic Bacteria to Control Vibriosis Disease

2017 ◽  
Vol 5 (2) ◽  
pp. 1
Author(s):  
Mulyati Mulyati ◽  
Suryati Suryati ◽  
Irfani Baga
Keyword(s):  
Survival Rate ◽  
Sea Water ◽  
Challenge Test ◽  
Probiotic Bacteria ◽  
Pathogenicity Test ◽  
Inhibitory Ability ◽  
Significant Difference ◽  
Portunid Crab

The study aims to isolate, characterize, and examine probiotic bacteria's inhibitory ability against Vibrio harveyi bacteria, both in-vitro and in vivo. Methods used in the study consist of 1) An Isolation of Candidate Probiotic Bacteria, 2) An Antagonistic Test of Candidate Probiotic Bacteria in vitro, 3) An Identification of Bacteria, 4) A Pathogenicity Test of Candidate Probiotic Bacteria, 5) An Antagonistic Test of Candidate Probiotic Bacteria against V. harveyi in vivo. According to the isolation of candidate probiotic bacteria, there are 18 isolated candidate probiotic. After being tested for its inhibitory ability in vitro, there are 8 isolates with zone of inhibition as follows: isolate MM 7 from intestine (22 mm), isolate MM 6 from intestine (12 mm), isolate MM 10 from sea water (10 mm), isolate MM 5 from intestine (9 mm), isolate MM 4 from intestine (8 mm), isolate MM 3 from intestine (7 mm), isolate MM 2.2 from intestine (7 mm), isolate MM 2.1 from intestine (7 mm). Eight genera of the candidate probiotic bacteria is derived from Portunid crab, they are Staphylococcus, Streptococcus, bacillus, vibrio, Alcaligenes, Lactobacillus, micrococcus. Before proceeding the V. harveyi bacterial challenge test in vivo, three potential isolates consisting of MM6, MM7 and MM10 as the probiotic bacteria are pathogenicity-tested against V. harveyi. The survival rate of Portunid crab on pathogenicity test using MM6, MM7 and MM10 generates 91.11-100%, while the control generates 100% survival rate. Variance analysis result through post-hoc Tukey's Honest Significant Difference (HSD) test at 95% confidence interval indicates that isolate MM7 and MM10 are significantly able to increase hatchling Portunid crab's survival rate.

scholarly journals In vitro and in vivo inhibition of malaria parasite infection by monoclonal antibodies against Plasmodium falciparum circumsporozoite protein (CSP)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Merricka C. Livingstone ◽  
Alexis A. Bitzer ◽  
Alish Giri ◽  
Kun Luo ◽  
Rajeshwer S. Sankhala ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Monoclonal Antibodies ◽  
Disease Burden ◽  
Vaccine Candidate ◽  
Specific Binding ◽  
Circumsporozoite Protein ◽  
Target Epitope ◽  
Selection Of

AbstractPlasmodium falciparum malaria contributes to a significant global disease burden. Circumsporozoite protein (CSP), the most abundant sporozoite stage antigen, is a prime vaccine candidate. Inhibitory monoclonal antibodies (mAbs) against CSP map to either a short junctional sequence or the central (NPNA)n repeat region. We compared in vitro and in vivo activities of six CSP-specific mAbs derived from human recipients of a recombinant CSP vaccine RTS,S/AS01 (mAbs 317 and 311); an irradiated whole sporozoite vaccine PfSPZ (mAbs CIS43 and MGG4); or individuals exposed to malaria (mAbs 580 and 663). RTS,S mAb 317 that specifically binds the (NPNA)n epitope, had the highest affinity and it elicited the best sterile protection in mice. The most potent inhibitor of sporozoite invasion in vitro was mAb CIS43 which shows dual-specific binding to the junctional sequence and (NPNA)n. In vivo mouse protection was associated with the mAb reactivity to the NANPx6 peptide, the in vitro inhibition of sporozoite invasion activity, and kinetic parameters measured using intact mAbs or their Fab fragments. Buried surface area between mAb and its target epitope was also associated with in vivo protection. Association and disconnects between in vitro and in vivo readouts has important implications for the design and down-selection of the next generation of CSP based interventions.

scholarly journals Preclinical Testing of Radiopharmaceuticals for the Detection and Characterization of Osteomyelitis: Experiences from a Porcine Model

Molecules ◽  
2021 ◽  
Vol 26 (14) ◽  
pp. 4221
Author(s):  
Aage Kristian Olsen Alstrup ◽  
Svend Borup Jensen ◽  
Ole Lerberg Nielsen ◽  
Lars Jødal ◽  
Pia Afzelius
Keyword(s):  
Animal Model ◽  
Animal Models ◽  
Porcine Model ◽  
Animal Experiments ◽  
Radioactive Tracers ◽  
The Individual ◽  
Selection Of

The development of new and better radioactive tracers capable of detecting and characterizing osteomyelitis is an ongoing process, mainly because available tracers lack selectivity towards osteomyelitis. An integrated part of developing new tracers is the performance of in vivo tests using appropriate animal models. The available animal models for osteomyelitis are also far from ideal. Therefore, developing improved animal osteomyelitis models is as important as developing new radioactive tracers. We recently published a review on radioactive tracers. In this review, we only present and discuss osteomyelitis models. Three ethical aspects (3R) are essential when exposing experimental animals to infections. Thus, we should perform experiments in vitro rather than in vivo (Replacement), use as few animals as possible (Reduction), and impose as little pain on the animal as possible (Refinement). The gain for humans should by far exceed the disadvantages for the individual experimental animal. To this end, the translational value of animal experiments is crucial. We therefore need a robust and well-characterized animal model to evaluate new osteomyelitis tracers to be sure that unpredicted variation in the animal model does not lead to a misinterpretation of the tracer behavior. In this review, we focus on how the development of radioactive tracers relies heavily on the selection of a reliable animal model, and we base the discussions on our own experience with a porcine model.

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