Cold Shock Genes cspA and cspB from Caulobacter crescentus Are Posttranscriptionally Regulated and Important for Cold Adaptation

Cold shock proteins (CSPs) are nucleic acid binding chaperones, first described as being induced to solve the problem of mRNA stabilization after temperature downshift. Caulobacter crescentus has four CSPs: CspA and CspB, which are cold induced, and CspC and CspD, which are induced only in stationary phase. In this work we have determined that the synthesis of both CspA and CspB reaches the maximum levels early in the acclimation phase. The deletion of cspA causes a decrease in growth at low temperature, whereas the strain with a deletion of cspB has a very subtle and transient cold-related growth phenotype. The cspA cspB double mutant has a slightly more severe phenotype than that of the cspA mutant, suggesting that although CspA may be more important to cold adaptation than CspB, both proteins have a role in this process. Gene expression analyses were carried out using cspA and cspB regulatory fusions to the lacZ reporter gene and showed that both genes are regulated at the transcriptional and posttranscriptional levels. Deletion mapping of the long 5'-untranslated region (5'-UTR) of each gene identified a common region important for cold induction, probably via translation enhancement. In contrast to what was reported for other bacteria, these cold shock genes have no regulatory regions downstream from ATG that are important for cold induction. This work shows that the importance of CspA and CspB to C. crescentus cold adaptation, mechanisms of regulation, and pattern of expression during the acclimation phase apparently differs in many aspects from what has been described so far for other bacteria.

The chemiluminescent peroxyoxalate system: state of the art almost 50 years from its discovery

Almost fifty years after the discovery of the peroxyoxalate reaction by E. A. Chandross in the early nineteen sixties, this review article intends to give a general overview on mechanistic aspects of this system and to describe the principles of its analytical application. After a short general introduction on the principles of chemiluminescence and the history of peroxyoxalate discovery, mechanistic aspects of high-energy intermediate formation, its structure and its reaction with an activator in the peroxyoxalate system are discussed. Finally, analytical applications of peroxyoxalate chemiluminescence are exemplified using representative recent examples, including oxalic acid detection in biological samples.

Human immunodeficiency virus prevalence, incidence, and residual risk of transmission by transfusions at Retrovirus Epidemiology Donor Study-II blood centers in Brazil

BACKGROUND: In Brazil nationally representative donor data are limited on human immunodeficiency virus (HIV) prevalence, incidence, and residual transfusion risk. The objective of this study was to analyze HIV data obtained over 24 months by the Retrovirus Epidemiology Donor Study-II program in Brazil. STUDY DESIGN AND METHODS: Donations reactive to third-and fourth-generation immunoassays (IAs) were further confirmed by a less-sensitive (LS) IA algorithm and Western blot (WB). Incidence was calculated for first-time (FT) donors using the LS-EIA results and for repeat donors with a model developed to include all donors with a previous negative donation. Residual risk was projected by multiplying composite FT and repeat donor incidence rates by HIV marker-negative infectious window periods. RESULTS: HIV prevalence among FT donors was 92.2/ 105 donations. FT and repeat donor and composite incidences were 38.5 (95% confidence interval [CI], 25.651.4), 22.5 (95% CI, 17.6-28.0), and 27.5 (95% CI, 22.0-33.0) per 100,000 person-years, respectively. Male and community donors had higher prevalence and incidence rates than female and replacement donors. The estimated residual risk of HIV transfusion transmission was 11.3 per 106 donations (95% CI, 8.4-14.2), which could be reduced to 4.2 per 106 donations (95% CI, 3.2-5.2) by use of individual-donation nucleic acid testing (NAT). CONCLUSION: The incidence and residual transfusion risk of HIV infection are relatively high in Brazil. Implementation of NAT will not be sufficient to decrease transmission rates to levels seen in the United States or Europe; therefore, other measures focused on decreasing donations by at-risk individuals are also necessary.

First description of group A rotavirus from fecal samples of ostriches (Struthio camelus)

This study investigated the occurrence of rotavirus infections in ostriches (Struthio camelus) reared in Northern Parana, Brazil. Fecal (n = 66) and serum (n = 182) samples from nine farms located in four different cities were analyzed by silver stained-polyacrylamide gel electrophoresis (ss-PAGE), RT-PCR assay, virus isolation, and counterimmunoelectroosmophoresis (CIE). Rotavirus group A seropositivity occurred in 5.49% (10/182) of serum samples of ostriches originated from two farms. Only 9.09% (6/66) of fecal samples from ostriches with diarrhea maintained in one farm were positive by ss-PAGE, RT-PCR, and virus isolation. The G (VP7) and P (VP4) genotypes of rotavirus wild strains isolated in cell culture were determined by multiplex-nested PCR. The genotyping identified two rotavirus strains: G6P[1] and G10P[1]. In three rotavirus strains it was only possible to identify the P type; one strain being P[1] and two strains that presented the combination of P[1] + P[7]. These findings might represent the first characterization of rotavirus in ostriches, and the finding of porcine and bovine-like rotavirus genotypes in ostriches might suggest virus reassortment and possible interspecies transmission. (C) 2011 Elsevier Ltd. All rights reserved.

DNA-Interactive Properties of Crotamine, a Cell-Penetrating Polypeptide and a Potential Drug Carrier

Crotamine, a 42-residue polypeptide derived from the venom of the South American rattlesnake Crotalus durissus terrificus, has been shown to be a cell-penetrating protein that targets chromosomes, carries plasmid DNA into cells, and shows specificity for actively proliferating cells. Given this potential role as a nucleic acid-delivery vector, we have studied in detail the binding of crotamine to single- and double-stranded DNAs of different lengths and base compositions over a range of ionic conditions. Agarose gel electrophoresis and ultraviolet spectrophotometry analysis indicate that complexes of crotamine with long-chain DNAs readily aggregate and precipitate at low ionic strength. This aggregation, which may be important for cellular uptake of DNA, becomes less likely with shorter chain length. 25-mer oligonucleotides do not show any evidence of such aggregation, permitting the determination of affinities and size via fluorescence quenching experiments. The polypeptide binds non-cooperatively to DNA, covering about 5 nucleotide residues when it binds to single (ss) or (ds) double stranded molecules. The affinities of the protein for ss-vs. ds-DNA are comparable, and inversely proportional to salt levels. Analysis of the dependence of affinity on [NaCl] indicates that there are a maximum of,3 ionic interactions between the protein and DNA, with some of the binding affinity attributable to non-ionic interactions. Inspection of the three-dimensional structure of the protein suggests that residues 31 to 35, Arg-Trp-Arg-Trp-Lys, could serve as a potential DNA-binding site. A hexapeptide containing this sequence displayed a lower DNA binding affinity and salt dependence as compared to the full-length protein, likely indicative of a more suitable 3D structure and the presence of accessory binding sites in the native crotamine. Taken together, the data presented here describing crotamine-DNA interactions may lend support to the design of more effective nucleic acid drug delivery vehicles which take advantage of crotamine as a carrier with specificity for actively proliferating cells. Citation: Chen P-C, Hayashi MAF, Oliveira EB, Karpel RL (2012) DNA-Interactive Properties of Crotamine, a Cell-Penetrating Polypeptide and a Potential Drug Carrier. PLoS ONE 7(11): e48913. doi:10.1371/journal.pone.0048913

Molecular characterization of group A bovine rotavirus in southeastern and central-western Brazil, 2009-2010

Rotavirus is an important cause of neonatal diarrhea in humans and several animal species, including calves. A study was conducted to examine 792 fecal samples collected from calves among 65 dairy and beef herds distributed in two of Brazil's major livestock producing regions, aiming to detect the occurrence of rotavirus and perform a molecular characterization of the rotavirus according to G and P genotypes in these regions. A total of 40 (5.05%) samples tested positive for rotavirus by the polyacrylamide gel electrophoresis (PAGE) technique. The molecular characterization was performed by multiplex semi-nested RT-PCR reactions, which indicated that the associations of genotypes circulating in herds in Brazil's southeastern region were G6P[11], G10P[11], G[-]P[5] + [11], G[-]P[6] in the state of Sao Paulo and G6P[11], G8P[5], G11P[11], G10P[11] in the state of Minas Gerais. In the central-western region, the genotypes G6P[5] + [11], G6P[5], G8P[-], G6P[11], G [-] P[1], G[-] P[11], and G[-] P[5] were detected in the state of Goias, while the genotypes G6P[5], G8[P11], G6[P11], G8[P1], G8[P5], G6[P1] were circulating in herds in the state of Mato Grosso do Sul. The genotypic diversity of bovine rotavirus found in each region under study underlines the importance of characterizing the circulating samples in order to devise the most effective prophylactic measures.

MicroRNA miR-146b-5p regulates signal transduction of TGF-beta by repressing SMAD4 in thyroid cancer

MicroRNAs (miRNA) are small non-coding RNAs involved in post-transcriptional gene regulation that have crucial roles in several types of tumors, including papillary thyroid carcinoma (PTC). miR-146b-5p is overexpressed in PTCs and is regarded as a relevant diagnostic marker for this type of cancer. A computational search revealed that miR-146b-5p putatively binds to the 3' untranslated region (UTR) of SMAD4, an important member of the transforming growth factor beta (TGF-beta) signaling pathway. The TGF-beta pathway is a negative regulator of thyroid follicular cell growth, and the mechanism by which thyroid cancer cells evade its inhibitory signal remains unclear. We questioned whether the modulation of the TGF-beta pathway by miR-146b-5p can contribute to thyroid tumorigenesis. Luciferase reporter assay confirmed the direct binding of miR-146b-5p on the SMAD4 3'UTR. Specific inhibition of miR-146b-5p with a locked nucleic acid-modified anti-miR-146b oligonucleotide significantly increased SMAD4 levels in the human papillary carcinoma cell lines, TPC-1 and BCPAP. Moreover, suppression of miR-146b-5p increased the cellular response to the TGF-beta anti-proliferative signal, significantly decreasing the proliferation rate. The overexpression of miR-146b-5p in normal rat follicular PCCL3 cells decreased SMAD4 levels and disrupted TGF-beta signal transduction. MiR-146b-5p overexpression in PCCL3 cells also significantly increased cell proliferation in the absence of thyroid-stimulating hormone and conferred resistance to TGF-beta-mediated cell-cycle arrest. Additionally, the activation of thyroid most common oncogenes RET/PTC3 and BRAF in PCCL3 cells upregulated miR-146b-5p expression. Our results confirm the oncogenic role of miR-146b-5p in thyroid follicular cells and contribute to knowledge regarding the modulation of TGF-beta signal transduction by miRNAs in PTCs. Oncogene (2012) 31, 1910-1922; doi:10.1038/onc.2011.381; published online 29 August 2011

Interphase Chromosome Flow-FISH

A 2-day method using flow cytometry and FISH for interphase cells was developed to detect monosomy 7 cells in myelodysplastic syndrome patients. The method, Interphase Chromosome Flow-FISH (IC Flow-FISH), involves fixation of leukocytes from blood, membrane permeabilization, hybridization of cellular DNA with peptide nucleic acid probes with cells intact, and analysis by flow cytometry. Hundreds to thousands of monosomy 7 cells were consistently detected from 10-20 mL of blood in patients with monosomy 7. Proportions of monosomy 7 cells detected in IC Flow-FISH were compared with results from conventional cytogenetics; identification of monosomy 7 populations was verified with FACS; and patient and donor cells were mixed to test for sensitivity. IC Flow-FISH allows for detecting monosomy 7 without requiring bone marrow procurement or the necessity of metaphase spreads, and wider applications to other chromosomal abnormalities are in development. (Blood. 2012; 120(15): e54-e59)

Mesoscale eddies: Hotspots of prokaryotic activity and differential community structure in the ocean

[EN] To investigate the effects of mesoscale eddies on prokaryotic assemblage structure and activity, we sampled two cyclonic eddies (CEs) and two anticyclonic eddies (AEs) in the permanent eddy-field downstream the Canary Islands. The eddy stations were compared with two far-field (FF) stations located also in the Canary Current, but outside the influence of the eddy field. The distribution of prokaryotic abundance (PA), bulk prokaryotic heterotrophic activity (PHA), various indicators of single-cell activity (such as nucleic acid content, proportion of live cells, and fraction of cells actively incorporating leucine), as well as bacterial and archaeal community structure were determined from the surface to 2000m depth. In the upper epipelagic layer (0?200 m), the effect of eddies on the prokaryotic community was more apparent, as indicated by the higher PA, PHA, fraction of living cells, and percentage of active cells incorporating leucine within eddies than at FF stations. Prokaryotic community composition differed also between eddy and FF stations in the epipelagic layer. In the mesopelagic layer (200?1000 m), there were also significant differences in PA and PHA between eddy and FF stations, although in general, there were no clear differences in community composition or single-cell activity. The effects on prokaryotic activity and community structure were stronger in AE than CE, decreasing with depth in both types of eddies. Overall, both types of eddies show distinct community compositions (as compared with FF in the epipelagic), and represent oceanic ?hotspots? of prokaryotic activity (in the epi- and mesopelagic realms).

Inibizione selettiva del gene MYCN mediante PNA (acidi peptido nucleici) anti-gene nel rabdomiosarcoma umano

MYCN oncogene amplification/expression is a feature of many childhood tumors, and some adult tumors, and it is associated with poor prognosis. While MYC expression is ubiquitary, MYCN has a restricted expression after birth and it is an ideal target for an effective therapy. PNAs belong to the latest class of nucleic acid-based therapeutics, and they can bind chromosomal DNA and block gene transcription (anti-gene activity). We have developed an anti-gene PNA that targets specifically the MYCN gene to block its transcription. We report for the first time MYCN targeted inhibition in Rhabdomyosarcoma (RMS) by the anti-MYCN-PNA in RMS cell lines (four ARMS and four ERMS) and in a xenograft RMS mouse model. Rhabdomyosarcoma is the most common pediatric soft-tissue sarcoma, comprising two main subgroups [Alveolar (ARMS) and Embryonal (ERMS)]. ARMS is associated with a poorer prognosis. MYCN amplification is a feature of both the ERMS and ARMS, but the MYCN amplification and expression levels shows a significant correlation and are greater in ARMS, in which they are associated with adverse outcome. We found that MYCN mRNA and protein levels were higher in the four ARMS (RH30, RH4, RH28 and RMZ-RC2) than in the four ERMS (RH36, SMS-CTR, CCA and RD) cell lines. The potent inhibition of MYCN transcription was highly specific, it did not affect the MYC expression, it was followed by cell-growth inhibition in the RMS cell lines which correlated with the MYCN expression rate, and it led to complete cell-growth inhibition in ARMS cells. We used a mutated- PNA as control. MYCN silencing induced apoptosis. Global gene expression analysis (Affymetrix microarrays) in ARMS cells treated with the anti-MYCN-PNA revealed genes specifically induced or repressed, with both genes previously described as targets of N-myc or Myc, and new genes undescribed as targets of N-myc or Myc (mainly involved in cell cycle, apoptosis, cell motility, metastasis, angiogenesis and muscle development). The changes in the expression of the most relevant genes were confirmed by Real-Time PCR and western blot, and their expression after the MYCN silencing was evaluated in the other RMS cell lines. The in vivo study, using an ARMS xenograft murine model evaluated by micro-PET, showed a complete elimination of the metabolic tumor signal in most of the cases (70%) after anti-MYCN-PNA treatment (without toxicity), whereas treatment with the mutated-PNA had no effect. Our results strongly support the development of MYCN anti-gene therapy for the treatment of RMS, particularly for poor prognosis ARMS, and of other MYCN-expressing tumors.

Immunoconiugati contenenti tossine vegetali o siRNA per la deplezione selettiva di cellule leucemiche. Preparazione, citotossicità e studi di mutagenesi sito-specifica.

CD33 is a myeloid cell surface marker absent on normal hematopoietic stem cells and normal tissues but present on leukemic blasts in 90% of adult and paediatric acute myeloid leukaemia (AML) cases. By virtue of its expression pattern and its ability to be rapidly internalized after antibody binding, CD33 has become an attractive target for new immunotherapeutic approaches to treat AML. In this study two immunoconjugates were constructed to contain a humanised single-chain fragment variable antibody (scFv) against CD33 in order to create new antibody-derived therapeutics for AML. The first immunoconjugate was developed to provide targeted delivery of siRNAs as death effectors into leukemic cells. To this purpose, a CD33-specific scFv, modified to include a Cys residue at its C-terminal end (scFvCD33-Cys), was coupled through a disulphide bridge to a nona-d-arginine (9R) peptide carrying a free Cys to the N-terminal. The scFvCD33-9R was able to completely bind siRNAs at a protein to nucleic acid ratio of about 10:1, as confirmed by electrophoretic gel mobility-shift assay. The conjugate was unable to efficiently transduce cytotoxic siRNA (siTox) into the human myeloid cell line U937. We observed slight reductions in cell viability, with a reduction of 25% in comparison to the control group only at high concentration of siTox (300 nM). The second immunoconjugate was constructed by coupling the scFvCD33-Cys to the type 1 ribosome inactivating protein Dianthin 30 (DIA30) through a chemical linking The resulting immunotoxin scFvCD33-DIA30 caused the rapid arrest of protein synthesis, inducing apoptosis and leading ultimately to cell death. In vitro dose-dependent cytotoxicity assays demonstrated that scFvCD33-DIA30 was specifically toxic to CD33-positive cell U937. The concentration needed to reach 50 % of maximum killing efficiency (EC50) was approximately 0.3 nM. The pronounced antigen-restricted cytotoxicity of this novel agent makes it a candidate for further evaluation of its therapeutic potential.

DNA block copolymers - synthesis, morphologies and applications

The last decades have witnessed significant and rapid progress in polymer chemistry and molecular biology. The invention of PCR and advances in automated solid phase synthesis of DNA have made this biological entity broadly available to all researchers across biological and chemical sciences. Thanks to the development of a variety of polymerization techniques, macromolecules can be synthesized with predetermined molecular weights and excellent structural control. In recent years these two exciting areas of research converged to generate a new type of nucleic acid hybrid material, consisting of oligodeoxynucleotides and organic polymers. By conjugating these two classes of materials, DNA block copolymers are generated exhibiting engineered material properties that cannot be realized with polymers or nucleic acids alone. Different synthetic strategies based on grafting onto routes in solution or on solid support were developed which afforded DNA block copolymers with hydrophilic, hydrophobic and thermoresponsive organic polymers in good yields. Beside the preparation of DNA block copolymers with a relative short DNA-segment, it was also demonstrated how these bioorganic polymers can be synthesized exhibiting large DNA blocks (>1000 bases) applying the polymerase chain reaction. Amphiphilic DNA block copolymers, which were synthesized fully automated in a DNA synthesizer, self-assemble into well-defined nanoparticles. Hybridization of spherical micelles with long DNA templates that encode several times the sequence of the micelle corona induced a transformation into rod-like micelles. The Watson-Crick motif aligned the hydrophobic polymer segments along the DNA double helix, which resulted in selective dimer formation. Even the length of the resulting nanostructures could be precisely adjusted by the number of nucleotides of the templates. In addition to changing the structural properties of DNA-b-PPO micelles, these materials were applied as 3D nanoscopic scaffolds for organic reactions. The DNA strands of the corona were organized by hydrophobic interactions of the organic polymer segments in such a fashion that several DNA-templated organic reactions proceeded in a sequence specific manner; either at the surface of the micelles or at the interface between the biological and the organic polymer blocks. The yields of reactions employing the micellar template were equivalent or better than existing template architectures. Aside from its physical properties and the morphologies achieved, an important requirement for a new biomaterial is its biocompatibility and interaction with living systems, i.e. human cells. The toxicity of the nanoparticles was analyzed by a cell proliferation assay. Motivated by the non-toxic nature of the amphiphilic DNA block copolymers, these nanoobjects were employed as drug delivery vehicles to target the anticancer drug to a tumor tissue. The micelles obtained from DNA block copolymers were easily functionalized with targeting units by hybridization. This facile route allowed studying the effect of the amount of targeting units on the targeting efficacy. By varying the site of functionalization, i.e. 5’ or 3’, the outcome of having the targeting unit at the periphery of the micelle or in the core of the micelle was studied. Additionally, these micelles were loaded with an anticancer drug, doxorubicin, and then applied to tumor cells. The viability of the cells was calculated in the presence and absence of targeting unit. It was demonstrated that the tumor cells bearing folate receptors showed a high mortality when the targeting unit was attached to the nanocarrier.

Modulation of hypoxia-inducible factors as a therapeutic target for multiple myeloma

Hypoxia-inducible factor-1 alpha (HIF-1α) plays a critical role in survival and is associated with poor prognosis in solid tumors. The role of HIF-1α in multiple myeloma is not completely known. In the present study, we explored the effect of EZN2968, an locked nucleic acid antisense oligonucleotide against HIF-1α, as a molecular target in MM. A panel of MM cell lines and primary samples from MM patients were cultured in vitro in the presence of EZN2968 . Under normoxia culture condition, HIF-1α mRNA and protein expression was detectable in all MM cell lines and in CD138+ cells from newly diagnosed MM patients samples. Significant up-regulation of HIF-1α protein expression was observed after incubation with IL6 or IGF-I, confirming that HIF-1α can be further induced by biological stimuli. EZN2968 efficiently induces a selective and stable down-modulation of HIF-1α and decreased the secretion of VEGF released by MM cell. Treatment with EZN2968 gave rise to a progressive accumulation of cells in the S and subG0 phase. The analysis of p21, a cyclin-dependent kinase inhibitors controlling cell cycle check point, shows upregulation of protein levels. These results suggest that HIF-1α inhibition is sufficient for cell cycle arrest in normoxia, and for inducing an apoptotic pathways.. In the presence of bone marrow microenvironment, HIF-1α inhibition blocks MAPK kinase pathway and secretion of pro-surviaval cytokines ( IL6,VEGF,IL8) In this study we provide evidence that HIF-1α, even in the absence of hypoxia signal, is expressed in MM plasma cells and further inducible by bone marrow milieu stimuli; moreover its inhibition is sufficient to induce a permanent cell cycle arrest. Our data support the hypothesis that HIF-1α inhibition may suppress tumor growth by preventing proliferation of plasma cells through p21 activation and blocking pro-survival stimuli from bone marrow microenvironment.

Molecular genetics of inherited cystic kidney diseases: new diagnostic approaches

Background. Hhereditary cystic kidney diseases are a heterogeneous spectrum of disorders leading to renal failure. Clinical features and family history can help to distinguish the recessive from dominant diseases but the differential diagnosis is difficult due the phenotypic overlap. The molecular diagnosis is often the only way to characterize the different forms. A conventional molecular screening is suitable for small genes but is expensive and time-consuming for large size genes. Next Generation Sequencing (NGS) technologies enables massively parallel sequencing of nucleic acid fragments. Purpose. The first purpose was to validate a diagnostic algorithm useful to drive the genetic screening. The second aim was to validate a NGS protocol of PKHD1 gene. Methods. DNAs from 50 patients were submitted to conventional screening of NPHP1, NPHP5, UMOD, REN and HNF1B genes. 5 patients with known mutations in PKHD1 were submitted to NGS to validate the new method and a not genotyped proband with his parents were analyzed for a diagnostic application. Results. The conventional molecular screening detected 8 mutations: 1) the novel p.E48K of REN in a patient with cystic nephropathy, hyperuricemia, hyperkalemia and anemia; 2) p.R489X of NPHP5 in a patient with Senior Loken Syndrome; 3) pR295C of HNF1B in a patient with renal failure and diabetes.; 4) the NPHP1 deletion in 3 patients with medullar cysts; 5) the HNF1B deletion in a patient with medullar cysts and renal hypoplasia and in a diabetic patient with liver disease. The NGS of PKHD1 detected all known mutations and two additional variants during the validation. The diagnostic NGS analysis identified the patient’s compound heterozygosity with a maternal frameshift mutation and a paternal missense mutation besides a not transmitted paternal missense mutation. Conclusions. The results confirm the validity of our diagnostic algorithm and suggest the possibility to introduce this NGS protocol to clinical practice.

Variabilita' ed evoluzione dei prioni: Mutabilita' in vitro

I prioni, privi di acidi nucleici, esistono come ceppi e possono mutare, in particolare quando attraversano una barriera di specie. Numerosi studi convergono sulla conclusione che le caratteristiche ceppo-specifiche siano inscritte nella conformazione della PrPSc, con la variabilità di ceppo associata a varianti conformazionali della PrPSc. In questo studio ci siamo avvalsi del PMCA, tecnica che riproduce in vitro molti aspetti della biologia dei prioni, per mettere a punto condizioni sperimentali di replicazione che permettessero di osservare fenomeni di mutazione e selezione, onde investigare i meccanismi molecolari e di popolazione alla base della mutabilità dei prioni. In condizioni di replicazione eterologa, che mima la trasmissione tra diverse specie, è stato inizialmente possibile identificare un mutante difettivo della scrapie, caratterizzato da una diversa conformazione della PrPSc e capace di replicare in vitro ma non più in vivo. Le condizioni in cui tale mutante è emerso hanno permesso di sviluppare ulteriori ipotesi di lavoro, basate sul concetto della quasi-specie. Impartendo diversi regimi di replicazione e seguendo l’evoluzione di due ceppi, è stato possibile evidenziare fenomeni di mutazione anche in condizioni di replicazione omologa, in assenza di forti pressioni selettive. In entrambi i ceppi sono emerse varianti conformazionali di PrPSc durante passaggi replicativi ad ampia popolazione, mentre le popolazioni sottoposte a ripetuti colli di bottiglia hanno mostrato un rapido declino del tasso di replicazione. Sono stati infine investigati l’efficacia e il potenziale mutageno di molecole anti-prioniche, ottenendo importanti risultati preliminari sull’efficacia di molecole che legano la PrPC. Questi risultati evidenziano come la mutabilità sia una caratteristica intrinseca dei prioni e supportano l’idea che i prioni siano molto variabili, similmente alle quasi-specie virali, e perciò adattabili e proni a fenomeni di mutazione e selezione. Tali conclusioni hanno impatto su problematiche sanitarie quali lo studio del potenziale zoonotico e i fenomeni di farmaco-resistenza dei prioni.