Classical BSE dismissed as the cause of CWD in Norwegian red deer despite strain similarities between both prion agents.

The first case of CWD in a Norwegian red deer was detected by a routine ELISA test and confirmed by western blotting and immunohistochemistry in the brain stem of the animal. Two different western blotting tests were conducted independently in two different laboratories, showing that the red deer glycoprofile was different from the Norwegian CWD reindeer and CWD moose and from North American CWD. The isolate showed nevertheless features similar to the classical BSE (BSE-C) strain. Furthermore, BSE-C could not be excluded based on the PrPSc immunohistochemistry staining in the brainstem and the absence of detectable PrPSc in the lymphoid tissues. Because of the known ability of BSE-C to cross species barriers as well as its zoonotic potential, the CWD red deer isolate was submitted to the EURL Strain Typing Expert Group (STEG) as a BSE-C suspect for further investigation. In addition, different strain typing in vivo and in vitro strategies aiming at identifying the BSE-C strain in the red deer isolate were performed independently in three research groups and BSE-C was not found in it. These results suggest that the Norwegian CWD red deer case was infected with a previously unknown CWD type and further investigation is needed to determine the characteristics of this potential new CWD strain.

RNA editing alterations define manifestation of prion diseases.

Prion diseases are fatal neurodegenerative disorders caused by misfolding of the normal prion protein into an infectious cellular pathogen. Clinically characterized by rapidly progressive dementia and accounting for 85% of human prion disease cases, sporadic Creutzfeldt-Jakob disease (sCJD) is the prevalent human prion disease. Although sCJD neuropathological hallmarks are well-known, associated molecular alterations are elusive due to rapid progression and absence of preclinical stages. To investigate transcriptome alterations during disease progression, we utilized tg340-PRNP129MM mice infected with postmortem material from sCJD patients of the most susceptible genotype (MM1 subtype), a sCJD model that faithfully recapitulates the molecular and pathological alterations of the human disease. Here we report that transcriptomic analyses from brain cortex in the context of disease progression, reveal epitranscriptomic alterations (specifically altered RNA edited pathway profiles, eg., ER stress, lysosome) that are characteristic and possibly protective mainly for preclinical and clinical disease stages. Our results implicate regulatory epitranscriptomic mechanisms in prion disease neuropathogenesis, whereby RNA-editing targets in a humanized sCJD mouse model were confirmed in pathological human autopsy material.

The emergence of classical BSE from atypical/Nor98 scrapie.

Atypical/Nor98 scrapie (AS) is a prion disease of small ruminants. Currently there are no efficient measures to control this form of prion disease, and, importantly, the zoonotic potential and the risk that AS might represent for other farmed animal species remains largely unknown. In this study, we investigated the capacity of AS to propagate in bovine PrP transgenic mice. Unexpectedly, the transmission of AS isolates originating from 5 different European countries to bovine PrP mice resulted in the propagation of the classical BSE (c-BSE) agent. Detection of prion seeding activity in vitro by protein misfolding cyclic amplification (PMCA) demonstrated that low levels of the c-BSE agent were present in the original AS isolates. C-BSE prion seeding activity was also detected in brain tissue of ovine PrP mice inoculated with limiting dilutions (endpoint titration) of ovine AS isolates. These results are consistent with the emergence and replication of c-BSE prions during the in vivo propagation of AS isolates in the natural host. These data also indicate that c-BSE prions, a known zonotic agent in humans, can emerge as a dominant prion strain during passage of AS between different species. These findings provide an unprecedented insight into the evolution of mammalian prion strain properties triggered by intra- and interspecies passage. From a public health perspective, the presence of c-BSE in AS isolates suggest that cattle exposure to small ruminant tissues and products could lead to new occurrences of c-BSE.

Porcine Prion Protein as a Paradigm of Limited Susceptibility to Prion Strain Propagation.

Although experimental transmission of bovine spongiform encephalopathy (BSE) to pigs and transgenic mice expressing pig cellular prion protein (PrPC) (porcine PrP [PoPrP]-Tg001) has been described, no natural cases of prion diseases in pig were reported. This study analyzed pig-PrPC susceptibility to different prion strains using PoPrP-Tg001 mice either as animal bioassay or as substrate for protein misfolding cyclic amplification (PMCA). A panel of isolates representatives of different prion strains was selected, including classic and atypical/Nor98 scrapie, atypical-BSE, rodent scrapie, human Creutzfeldt-Jakob-disease and classic BSE from different species. Bioassay proved that PoPrP-Tg001-mice were susceptible only to the classic BSE agent, and PMCA results indicate that only classic BSE can convert pig-PrPC into scrapie-type PrP (PrPSc), independently of the species origin. Therefore, conformational flexibility constraints associated with pig-PrP would limit the number of permissible PrPSc conformations compatible with pig-PrPC, thus suggesting that pig-PrPC may constitute a paradigm of low conformational flexibility that could confer high resistance to the diversity of prion strains.

Met166 -Glu168 residues in human PrP ß2-α2 loop account for evolutionary resistance to prion infection.

AIMS: The amino acid sequence of prion protein (PrP) is a key determinant in the transmissibility of prion diseases. While PrP sequence is highly conserved among mammalian species, minor changes in the PrP amino acid sequence may confer alterations in the transmissibility of prion diseases. Classical bovine spongiform encephalopathy (C-BSE) is the only zoonotic prion strain reported to date causing variant Creutzfeldt-Jacob disease (vCJD) in humans, although experimental transmission points to atypical L-BSE and some classical scrapie isolates as also zoonotic. The precise molecular elements in the human PrP sequence that limit the transmissibility of prion strains such as sheep/goat scrapie or cervid chronic wasting disease (CWD) are not well known. METHODS: The transmissibility of a panel of diverse prions from different species was compared in transgenic mice expressing either wild-type human PrPC (MDE-HuTg340) or a mutated human PrPC harbouring Val166 -Gln168 amino acid changes (VDQ-HuTg372) in the ß2-α2 loop instead of Met166 -Glu168 wild-type variants. RESULTS: VDQ-HuTg372 mice were more susceptible to prions than MDE-HuTg340 mice in a strain-dependent manner. CONCLUSIONS: Met166 -Glu168 amino acid residues present in wild-type human PrPC are molecular determinants that limit the propagation of most prion strains assayed in the human PrP context.

TREM2 expression in the brain and biological fluids in prion diseases.

Triggering receptor expressed on myeloid cells 2 (TREM2) is an innate immune cell surface receptor that regulates microglial function and is involved in the pathophysiology of several neurodegenerative diseases. Its soluble form (sTREM2) results from shedding of the TREM2 ectodomain. The role of TREM2 in prion diseases, a group of rapidly progressive dementias remains to be elucidated. In the present study, we analysed the expression of TREM2 and its main sheddase ADAM10 in the brain of sporadic Creutzfeldt-Jakob disease (sCJD) patients and evaluated the role of CSF and plasma sTREM2 as a potential diagnostic marker of prion disease. Our data indicate that, compared to controls, TREM2 is increased in sCJD patient brains at the mRNA and protein levels in a regional and subtype dependent fashion, and expressed in a subpopulation of microglia. In contrast, ADAM10 is increased at the protein, but not the mRNA level, with a restricted neuronal expression. Elevated CSF sTREM2 is found in sCJD, genetic CJD with mutations E200K and V210I in the prion protein gene (PRNP), and iatrogenic CJD, as compared to healthy controls (HC) (AUC = 0.78-0.90) and neurological controls (AUC = 0.73-0.85), while CSF sTREM2 is unchanged in fatal familial insomnia. sTREM2 in the CSF of cases with Alzheimer's disease, and multiple sclerosis was not significantly altered in our series. CSF sTREM2 concentrations in sCJD are PRNP codon 129 and subtype-related, correlate with CSF 14-3-3 positivity, total-tau and YKL-40, and increase with disease progression. In plasma, sTREM2 is increased in sCJD compared with HC (AUC = 0.80), displaying positive correlations with plasma total-tau, neurofilament light, and YKL-40. We conclude that comparative study of TREM2 in brain and biological fluids of prion diseases reveals TREM2 to be altered in human prion diseases with a potential value in target engagement, patient stratification, and disease monitoring.

Classical scrapie in small ruminants is caused by at least four different prion strains.

The diversity of goat scrapie strains in Europe has recently been studied using bioassays in a wide collection of rodent models, resulting in the classification of classical scrapie into four different categories. However, the sole use of the first passage does not lead to isolate adaptation and identification of the strains involved and might therefore lead to misclassification of some scrapie isolates. Therefore, this work reports the complete transmission study of a wide collection of goat transmissible spongiform encephalopathy (TSE) isolates by intracranial inoculation in two transgenic mouse lines overexpressing either small ruminant (TgGoat-ARQ) or bovine (TgBov) PrPC. To compare scrapie strains in sheep and goats, sheep scrapie isolates from different European countries were also included in the study. Once the species barrier phenomenon was overcome, an accurate classification of the isolates was attained. Thus, the use of just two rodent models allowed us to fully differentiate at least four different classical scrapie strains in small ruminants and to identify isolates containing mixtures of strains. This work reinforces the idea that classical scrapie in small ruminants is a prion disease caused by multiple different prion strains and not by a single strain, as is the case for epidemic classical bovine spongiform encephalopathy (BSE-C). In addition, the clear dissimilarity between the different scrapie strains and BSE-C does not support the idea that classical scrapie is the origin of epidemic BSE-C.

Radical Change in Zoonotic Abilities of Atypical BSE Prion Strains as Evidenced by Crossing of Sheep Species Barrier in Transgenic Mice.

Classical bovine spongiform encephalopathy (BSE) is the only zoonotic prion disease described to date. Although the zoonotic potential of atypical BSE prions have been partially studied, an extensive analysis is still needed. We conducted a systematic study by inoculating atypical BSE isolates from different countries in Europe into transgenic mice overexpressing human prion protein (PrP): TgMet129, TgMet/Val129, and TgVal129. L-type BSE showed a higher zoonotic potential in TgMet129 mice than classical BSE, whereas Val129-PrP variant was a strong molecular protector against L-type BSE prions, even in heterozygosis. H-type BSE could not be transmitted to any of the mice. We also adapted 1 H- and 1 L-type BSE isolate to sheep-PrP transgenic mice and inoculated them into human-PrP transgenic mice. Atypical BSE prions showed a modification in their zoonotic ability after adaptation to sheep-PrP producing agents able to infect TgMet129 and TgVal129, bearing features that make them indistinguishable of sporadic Creutzfeldt-Jakob disease prions.

Nonpathogenic Heterologous Prions Can Interfere with Prion Infection in a Strain-Dependent Manner.

Co-occurrence of different prion strains into the same host has been recognized as a natural phenomenon for several sporadic Creutzfeldt-Jakob disease (sCJD) patients and natural scrapie cases. The final outcome of prion coinfection is not easily predictable. In addition to the usual factors that influence prion conversion, the replication of one strain may entail positive or negative consequences to the other. The main aim of this study was to gain insights into the prion coinfection and interference concepts and their potential therapeutic implications. Here, different mouse models were challenged with several combinations of prion strains coupled on the basis of the lengths of their incubation periods and the existence/absence of a species barrier in the tested animal model. We found that nontransmissible strains can interfere the replication of fully transmissible strains when there is a species transmission barrier involved, as happened with the combination of a mouse (22L) and a human (sCJD) strain. However, this phenomenon seems to be strain dependent, since no interference was observed when the human strain coinoculated was vCJD. For the other combinations tested in this study, the results suggest that both strains replicate independently without effect on the replication of one over the other. It is common that the strain with more favorable conditions (e.g., a higher speed of disease development or the absence of a species barrier) ends being the only one detectable at the terminal stage of the disease. However, this does not exclude the replication of the least favored strain, leading to situations of the coexistence of prion strains.IMPORTANCE As a general conclusion, the outcome of prion coinfection is strongly dependent on the strain combination and the model utilized and is therefore difficult to predict. The coexistence of several prion strains may remain undetected if one of the strains has more favorable conditions to replicate in the host. The use of several models (such as a transgenic mouse expressing PrP from different species) to analyze field prion isolates is recommended to avoid this situation. The inference effect exerted by nonreplicative prion strains should be considered an interesting tool to advance in new therapeutic strategies for treating prion diseases; it may even be a proper therapeutic strategy.

Qualitative evaluation of ferritin in serum samples by Raman spectroscopy and principal component analysis.

Iron molecule is of great importance in the synthesis of hemoglobin which is essential for oxygen transport. Iron levels are quantified by accurately high sensitivity tests, such as serum ferritin (SF). However, common studies to quantify SF are long and strenuous (~ 5 h), for example enzyme-linked immunosorbent assay (ELISA). In this paper, blood serum samples were analyzed by Raman spectroscopy (RS), and a computational analysis of spectra is proposed to detect differences in SF as an alternative procedure. Serum samples were obtained from 22 patients, 9 who were clinically diagnosed with anemia and 13 controls. Patients with anemia had low levels of SF (< 30 ng/ml), and a control group had levels between 30 and 500 ng/ml. The spectra obtained were conditioned with a baseline correction and smoothing, then evaluated by principal component analysis (PCA), and a predictive model was estimated by lineal discrimination analysis (LDA). The results showed a clear differentiation of the study groups by PCA, also 99.69% sensitivity and 100% specificity by LDA. This study suggest that Raman spectroscopy is a fast (~ 5 min) and a powerful tool capable to qualitative differentiate ferritin concentrations.

Protective Effect of Val129-PrP against Bovine Spongiform Encephalopathy but not Variant Creutzfeldt-Jakob Disease.

Bovine spongiform encephalopathy (BSE) is the only known zoonotic prion that causes variant Creutzfeldt-Jakob disease (vCJD) in humans. The major risk determinant for this disease is the polymorphic codon 129 of the human prion protein (Hu-PrP), where either methionine (Met129) or valine (Val129) can be encoded. To date, all clinical and neuropathologically confirmed vCJD cases have been Met129 homozygous, with the exception of 1 recently reported Met/Val heterozygous case. Here, we found that transgenic mice homozygous for Val129 Hu-PrP show severely restricted propagation of the BSE prion strain, but this constraint can be partially overcome by adaptation of the BSE agent to the Met129 Hu-PrP. In addition, the transmission of vCJD to transgenic mice homozygous for Val129 Hu-PrP resulted in a prion with distinct strain features. These observations may indicate increased risk for vCJD secondary transmission in Val129 Hu-PrP-positive humans with the emergence of new strain features.

Goat K222-PrPC polymorphic variant does not provide resistance to atypical scrapie in transgenic mice.

Host prion (PrPC) genotype is a major determinant for the susceptibility to prion diseases. The Q/K222-PrPC polymorphic variant provides goats and mice with high resistance against classical scrapie and bovine spongiform encephalopathy (BSE); yet its effect against atypical scrapie is unknown. Here, transgenic mice expressing the goat wild-type (wt) or the K222-PrPC variant were intracerebrally inoculated with several natural cases of atypical scrapie from sheep and goat and their susceptibility to the prion disease was determined. Goat wt and K222-PrPC transgenic mice were 100% susceptible to all the atypical scrapie isolates, showing similar survival times and almost identical disease phenotypes. The capacity of the K222-PrPC variant to replicate specifically the atypical scrapie strain as efficiently as the goat wt PrPC, but not the classical scrapie or cattle-BSE as previously reported, further suggests the involvement of concrete areas of the host PrPC in the strain-dependent replication of prions.

An assessment of the long-term persistence of prion infectivity in aquatic environments.

The environment plays a key role in horizontal transmission of prion diseases, since prions are extremely resistant to classical inactivation procedures. In prior work, we observed the high stability of bovine spongiform encephalopathy (BSE) infectivity when these prions were incubated in aqueous media such as phosphate-buffered saline (PBS) or wastewater for nearly nine months. As a continuation of this experiment, the same samples were maintained in PBS or wastewater for five additional years and residual BSE infectivity was assessed in bovine PrPC transgenic mice. Over this long time period (more than six years), BSE infectivity was reduced by three and one orders of magnitude in wastewater and PBS respectively. To rule out a possible agent specific effect, sheep scrapie prions were subjected to the same experimental protocol, using eight years as the experimental end-point. No significant reduction in scrapie infectivity was observed over the first nine months of wastewater incubation while PBS incubation for eight years only produced a two logarithmic unit reduction in infectivity. By contrast, the dynamics of PrPRes persistence was different, disappearing progressively over the first year. The long persistence of prion infectivity observed in this study for two different agents provides supporting evidence of the assumed high stability of these agents in aquatic environments and that environmental processes or conventional wastewater treatments with low retention times would have little impact on prion infectivity. These results could have great repercussions in terms of risk assessment and safety for animals and human populations.

Breast cancer detection based on serum sample surface enhanced Raman spectroscopy.

Raman spectroscopy is a vibrational technique which provides information about the chemical structure. Nevertheless, since many chemicals are present in a sample at very low concentration, the Raman signal observed is extremely weak. In surface enhanced Raman scattering (SERS), Raman signals can be enhanced by many orders of magnitude when nanoparticles are used. To the best of our knowledge, this is the first report in the breast cancer detection based on serum SERS. The serum samples were obtained from 12 patients who were clinically diagnosed with advanced breast cancer and 15 controls. In the same proportion, the serum samples were mixed with colloidal gold nanoparticles of 40 nm using sonication. At least 10 spectra were collected of each serum sample using a Jobin-Yvon LabRAM Raman Spectrometer with a laser of 830 nm. Raw spectra were processed by carrying baseline correction, smoothing, and normalization and then analyzed using principle component analysis (PCA) and linear discriminant analysis (LDA). Raman spectra showed strongly enhanced bands in the 600-1800 cm (-1) range due to the nanoparticle colloidal clusters observed. These Raman bands allowed identifying biomolecules present at low concentration as amide I and III, ß carotene, glutathione, tryptophan, tyrosine, and phenylalanine. Preliminary results demonstrated that SERS and PCA-LDA can be used to discriminate between control and cancer samples with high sensitivity and specificity. SERS allowed short exposures and required a minimal sample preparation. The preliminary results suggest that SERS and PCA-LDA could be an excellent support technique for the breast cancer detection using serum samples.

Effect of Q211 and K222 PRNP Polymorphic Variants in the Susceptibility of Goats to Oral Infection With Goat Bovine Spongiform Encephalopathy.

BACKGROUND: The prion protein-encoding gene (PRNP) is one of the major determinants for scrapie occurrence in sheep and goats. However, its effect on bovine spongiform encephalopathy (BSE) transmission to goats is not clear. METHODS: Goats harboring wild-type, R/Q211 or Q/K222 PRNP genotypes were orally inoculated with a goat-BSE isolate to assess their relative susceptibility to BSE infection. Goats were killed at different time points during the incubation period and after the onset of clinical signs, and their brains as well as several peripheral tissues were analyzed for the accumulation of pathological prion protein (PrP(Sc)) and prion infectivity by mouse bioassay. RESULTS: R/Q211 goats displayed delayed clinical signs compared with wild-type goats. Deposits of PrP(Sc) were detected only in brain, whereas infectivity was present in peripheral tissues too. In contrast, none of the Q/K222 goats showed any evidence of clinical prion disease. No PrP(Sc) accumulation was observed in their brains or peripheral tissues, but very low infectivity was detected in some tissues very long after inoculation (44-45 months). CONCLUSIONS: These results demonstrate that transmission of goat BSE is genotype dependent, and they highlight the pivotal protective effect of the K222 PRNP variant in the oral susceptibility of goats to BSE.

Role of the goat K222-PrP(C) polymorphic variant in prion infection resistance.

UNLABELLED: The prion protein-encoding gene (prnp) strongly influences the susceptibility of small ruminants to transmissible spongiform encephalopathies (TSEs). Hence, selective breeding programs have been implemented to increase sheep resistance to scrapie. For goats, epidemiological and experimental studies have provided some association between certain polymorphisms of the cellular prion protein (PrP(C)) and resistance to TSEs. Among them, the Q/K polymorphism at PrP(C) codon 222 (Q/K222) yielded the most promising results. In this work, we investigated the individual effects of the K222-PrP(C) variant on the resistance/susceptibility of goats to TSEs. For that purpose, we generated two transgenic mouse lines, expressing either the Q222 (wild type) or K222 variant of goat PrP(C). Both mouse lines were challenged intracerebrally with a panel of TSE isolates. Transgenic mice expressing the wild-type (Q222) allele were fully susceptible to infection with all tested isolates, whereas transgenic mice expressing similar levels of the K222 allele were resistant to all goat scrapie and cattle BSE isolates but not to goat BSE isolates. Finally, heterozygous K/Q222 mice displayed a reduced susceptibility to the tested panel of scrapie isolates. These results demonstrate a highly protective effect of the K222 variant against a broad panel of different prion isolates and further reinforce the argument supporting the use of this variant in breeding programs to control TSEs in goat herds. IMPORTANCE: The objective of this study was to determine the role of the K222 variant of the prion protein (PrP) in the susceptibility/resistance of goats to transmissible spongiform encephalopathies (TSEs). Results showed that transgenic mice expressing the goat K222-PrP polymorphic variant are resistant to scrapie and bovine spongiform encephalopathy (BSE) agents. This protective effect was also observed in heterozygous Q/K222 animals. Therefore, the single amino acid exchange from Q to K at codon 222 of the cellular prion protein provides resistance against TSEs. All the results presented here support the view that the K222 polymorphic variant is a good candidate for selective breeding programs to control and eradicate scrapie in goat herds.

Transmission characteristics of variably protease-sensitive prionopathy.

Variably protease-sensitive prionopathy (VPSPr), a recently identified and seemingly sporadic human prion disease, is distinct from Creutzfeldt-Jakob disease (CJD) but shares features of Gerstmann-Sträussler-Scheinker disease (GSS). However, contrary to exclusively inherited GSS, no prion protein (PrP) gene variations have been detected in VPSPr, suggesting that VPSPr might be the long-sought sporadic form of GSS. The VPSPr atypical features raised the issue of transmissibility, a prototypical property of prion diseases. We inoculated VPSPr brain homogenate into transgenic mice expressing various levels of human PrP (PrPC). On first passage, 54% of challenged mice showed histopathologic lesions, and 34% harbored abnormal PrP similar to that of VPSPr. Surprisingly, no prion disease was detected on second passage. We concluded that VPSPr is transmissible; thus, it is an authentic prion disease. However, we speculate that normal human PrPC is not an efficient conversion substrate (or mouse brain not a favorable environment) and therefore cannot sustain replication beyond the first passage.

Monitoring of chemotherapy leukemia treatment using Raman spectroscopy and principal component analysis.

In this research, we used the Raman spectroscopy to distinguish between normal and leukemia blood serum and identify the different types of leukemia based on serum biochemistry. In addition, monitoring of patients under chemotherapy leukemia treatment (CHLT) was studied. Blood samples were obtained from seven patients who were clinically diagnosed with three leukemia types and 21 healthy volunteers. In addition, other five leukemia patients were monitored during the CHLT, two patients were declared healthy, one patient suspended it; the health of the other two patients worsened, and no improvement was observed along CHLT. The serum samples were put under an Olympus microscope integrated to the Raman system, and several points were chosen for the Raman measurement. The Horiba Jobin Yvon LabRAM HR800 Raman system is equipped with a liquid nitrogen-cooled detector and a laser of 830 nm with a power irradiation of 17 mW. It is shown that the serum samples from patient with leukemia and from the control group can be discriminated when multivariate statistical methods of principal component analysis (PCA) and linear discriminant analysis (LDA) are applied to their Raman spectra obtaining two large clusters corresponding to the control and leukemia serum samples and three clusters inside the leukemia group associated with the three leukemia types. The major differences between leukemia and control spectra were at 1,338 (Trp, α-helix, phospholipids), 1,447 (lipids), 1,523 (ß-carotene), 1,556 (Trp), 1,587 (protein, Tyr), 1,603 (Tyr, Phe), and 1,654 (proteins, amide I, α-helix, phospholipids) cm(-1), where these peaks were less intense in the leukemia spectrum. Minor differences occurred at 661 (glutathione), 890 (glutathione), 973 (glucosamine), 1,126 (protein, phospholipid C-C str), 1,160 (ß-carotene), 1,174 (Trp, Phe), 1,208 (Trp), 1,246 (amide III), 1,380 (glucosamine), and 1,404 (glutathione) cm(-1). Leukemia spectrum showed a peak at 917 cm(-1) associated with glutathione, but it was absent in the control spectrum. The results suggest that the Raman spectroscopy and PCA could be a technique with a strong potential of support for current techniques to detect and identify the different leukemia types by using a serum sample. Nevertheless, with the construction of a data library integrated with a large number of leukemia and control Raman spectra obtained from a wide range of healthy and leukemic population, the Raman-PCA technique could be converted into a new technique for minimally invasive real-time diagnosis of leukemia from serum samples. In addition, complementary results suggest that using these techniques is possible to monitor CHLT.

Cervical cancer detection based on serum sample Raman spectroscopy.

The use of Raman spectroscopy to analyze the biochemical composition of serum samples and hence distinguish between normal and cervical cancer serum samples was investigated. The serum samples were obtained from 19 patients who were clinically diagnosed with cervical cancer, 3 precancer, and 20 healthy volunteer controls. The imprint was put under an Olympus microscope, and around points were chosen for Raman measurement.All spectra were collected at a Horiba Jobin-Yvon LabRAM HR800 Raman Spectrometer with a laser of 830-nm wavelength and 17-mW power irradiation. Raw spectra were processed by carrying out baseline correction, smoothing, and normalization to remove noise, florescence, and shot noise and then analyzed using principal component analysis (PCA). The control serum spectrum showed the presence of higher amounts of carotenoids indicated by peaks at 1,002, 1,160, and 1,523 cm(-1)and intense peaks associated with protein components at 754, 853, 938, 1,002, 1,300-1,345, 1,447, 1,523, 1,550, 1,620, and 1,654 cm(-1). The Raman bands assigned to glutathione (446, 828, and 1,404 cm(-1)) and tryptophan (509, 1,208, 1,556, 1,603, and 1,620 cm(-1)) in cervical cancer were higher than those of control samples, suggesting that their presence may also play a role in cervical cancer. Furthermore, weak bands in the control samples attributed to tryptophan (545, 760, and 1,174 cm(-1)) and amide III (1,234-1,290 cm(-1)) seem to disappear and decrease in the cervical cancer samples, respectively. It is shown that the serum samples from patients with cervical cancer and from the control group can be discriminated with high sensitivity and specificity when the multivariate statistical methods of PCA is applied to Raman spectra. PCA allowed us to define the wavelength differences between the spectral bands of the control and cervical cancer groups by confirming that the main molecular differences among the control and cervical cancer samples were glutathione, tryptophan, ß carotene, and amide III. The preliminary results suggest that Raman spectroscopy could be a highly effective technique with a strong potential of support for current techniques as Papanicolaou smear by reducing the number of these tests; nevertheless, with the construction of a data library integrated with a large number of cervical cancer and control Raman spectra obtained from a wide range of healthy and cervical cancer population, Raman-PCA technique could be converted into a new technique for noninvasive real-time diagnosis of cervical cancer from serum samples.