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(A) Schematic of the 15,409-base viral genome of VR-2332 (open boxes) with sequenced cDNA clones (shaded boxes). (B) Sequence of the VR-2332 region between ORF 1a and 1b and its resulting predicted RNA pseudoknot tertiary structure involved in ribosome frameshifting, as modeled on the predicted pseudoknot of LV (1, 38). The proposed heptanucleotide slippery sequence (boxed), UAG stop codon of ORF 1a (bold), and differences between VR-2332 and LV (italics) are indicated.
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(A) Primer extension analysis of strain VR-2332. RNA from VR-2332-infected MA-104 cells was hybridized to γ-32P-radiolabeled VR-2332 leader reverse primer /658P4 and reverse transcribed. The primer extension products were electrophoresed alongside the known sequencing products obtained from clone 712 and forward primer P71/. The primer extension product migrated with the thymidine residue located at nucleotide 2965 of clone 712, resulting in an extension product of 98 nucleotides. (B) Comparison of PRRSV leader sequences. VR-2332 leader (190 bases in length) and LV leader (221 bases in length) sequences exhibit 61.0% identity as analyzed by the GCG GAP program, with a gap weight of 5 and a length weight of 5 (lines between the sequences indicate identity). The leader-body junction sequence utilized for transcription of each mRNA is boxed.
(A) Northern blot analysis of PRRSV mRNA 7. Total RNA from cells infected with VR-2332 (lane 1) or LV (lane 2) were electrophoresed through an agarose gel and blotted onto a nylon membrane. The membrane was probed sequentially with a VR-2332 ORF 7 oligomer (lane 1) and then an oligomer to LV ORF 7 (lane 2). No nonspecific hybridization was detected in several analyses. (B) Northern blot analysis of total RNA from VR-2332-infected MA-104 (CL2621) cells and alveolar macrophages (AM) shows that junction site 1 is used to transcribe the majority of mRNA 7 (7.1) and that junction site 2 is used to transcribe a minority of mRNA 7 (7.2). Mock-, VR-2332-, and LV-infected-cell total RNA populations were electrophoresed through a 2% agarose gel and transferred to membranes. The membranes were probed with reverse complement oligomers to VR-2332 ORF 7 (a), VR-2332 ORF 7 junction site 1 (b), VR-2332 ORF 7 junction site 2 (c), and LV ORF 7 (d). An RNA ladder (Gibco BRL) was used to assess sgmRNA size.
In this report, we have examined the subgenomic messages produced in cells, and not only did we find that the leader-body junction sequences used for each mRNA of strain VR-2332 are different from those for LV mRNAs, but we also found evidence in infected swine macrophages that the VR-2332 sgmRNA junction sites utilized are variable, particularly in the case of mRNA 7. Although both VR-2332 and LV genomic sequences naturally contain potential leader junction sequences for mRNA 7 at three sites, only one particular site is used preferentially for transcription in each isolate. It appears that the leader sequence, ending in UUAACC, is joined to downstream leader-body junction motifs by more than simple base pair homology, since only a specific subset of potential leader-body motifs are utilized. Thus, we suggest that another viral nucleotide or protein sequence(s), secondary structure of the viral RNA, or other host factors may play a role in site selection. The differences in PRRSV polymerase proteins described in this report may play a key role in determining the choice of junction site utilized.
TK drafted the manuscript. TK, MA, HT, YA, and HK performed pathological diagnosis. MT treated the patient. HT and HK supervised the writing of the report. All authors read and approved the final manuscript.
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.
This clinimetric study was approved by the ethics committee of the Brindisi ASL (Italy). All procedures were conducted according to the declaration of Helsinki, and all patients provided informed consent prior to study inclusion. Authorization to adapt the CNFDS into Italian was obtained from the original developer.
A booklet asking information about demographic (e.g. age, sex) and clinical characteristics (e.g. pain duration) was administered to each patient. The same booklet contained the Italian versions of the Neck Disability Index (NDI-I) [14], of the Neck Bournemouth Questionnaire (NBQ-I) [15], and of the CNFDS-I; a Visual Analogue Scale (VAS) to assess pain intensity was also included [19].
Structural validity is the degree to which scores of an instrument adequately reflect the dimensionality of the construct to be measured [23]. Since this measurement property was not previously assessed for the CNFDS, an exploratory factor analysis was performed on the 15 items of the questionnaire, using a principal component estimator. The eigenvalue of each extracted factor was calculated, and a scree plot was drawn. A ratio between the first and the second eigenvalue larger than 4 was considered as an indication of unidimensionality [24]. Factor loadings for each item were also calculated, and loadings smaller than 0.4 were considered for item reduction of the questionnaire [25].
This is the first study to perform a factor analysis of the CNFDS as the original study [8], while the previous ones [33,34,35,36] did not do that. The CNFDS-I resulted to be unidimensional and this represents a key finding, as it indicates that it is appropriate to use its total sum score [24]. Meanwhile, this finding does not support the use of the subscales proposed by the French developers [35]. In contrast with the CNFDS-I, other neck-related disability tools available in Italian (i.e. NDI-I and NPQ-I) were not shown to be unidimensional [14, 15, 37], questioning the suitability of using their total sum scores.
The unidimensionality of the NDI has been questioned also by other studies in other language versions [38, 39]. A future study can aim at comparing directly the unidimensionality of all these tools, to check if the CNFDS is indeed the best performing method from a psychometric point of view. Item 9 was the only CNFDS-I item with a low factor loading; future studies should assess if this result is repeated in other languages.
DA designed the study, acted as project manager, led the development of the CNFDS-I, contributed substantially to the data collection and drafted the first version of the manuscript; MM performed the statistical analysis, discussed the interpretation of results and revised the manuscript for important intellectual content; AC contributed to the statistical analysis, discussed the interpretation of results and revised the manuscript for important intellectual content; AD contributed in designing the study, in the data collection and reviewed the manuscript for important intellectual content; GG contributed in designing the study, in the data collection and reviewed the manuscript for important intellectual content; FB contributed in designing the study, in organizing and conducting the data collection, in interpreting the results, and in providing important intellectual revision of the manuscript. All authors read and approved the submission of the manuscript.
KRAS-mutant cancer cells rely on high levels of ROS and on a labile iron pool (LIP) to sustain their growth10,11,12. Increasing evidence supports the link between ROS production and free iron10,13,14. In particular, ROS could release iron from iron-containing proteins, thus expanding the LIP, which in turn could enhance ROS formation through redox chemistry10,13,14. Moreover, RAS oncogenic activation further increases cellular iron content by upregulating iron-uptake proteins, such as transferrin receptor (Trf1), and by downregulating iron export and storage proteins, in particular ferritin10,11,12. In this context, the stress inducible protein heme-oxygenase-1 (HO-1), regulates ferritin expression to avoid an excessive free iron content, which can damage macromolecules15.
To assess whether the alteration in cellular iron content contributes to STS-mediated sensitization to vitamin C, KRAS-mutant CRC cells grown in STS conditions were treated with the iron chelator desferrioxamine (DFO) before vitamin C exposure. Consistent with our hypothesis, DFO treatment preceding vitamin C exposure rescued vitamin C-induced cell cytotoxicity (Fig. 3e), thus confirming that the increase in intracellular free iron mediated by FMD/STS and vitamin C is, at least in part, responsible for their cytotoxic effect.
Several studies suggested a potential role of ferritin in protecting cells from oxidative damage through the sequestration of intracellular free iron10,11,12. Among enzymes promoting ferritin expression, the stress-inducible HO-1 has been implicated in promoting cell survival during cell exposure to oxidative insults29,30. Since the FMD/STS downregulates the FTH protein expression level, we investigated whether HO-1 is implicated in FTH regulation in response to STS and vitamin C treatment. In one recent study from our group, the FMD sensitized breast cancer cells to chemotherapy in part by downregulating HO-1, further supporting a possible role of this stress-inducible protein in mediating FMD beneficial effects31.
An analysis of TCGA database indicated that patients with KRAS mutated CRC but not wild-type KRAS CRC expressing low levels of ferritin displayed longer 3- and 5-year survival when compared with patients bearing tumors with high ferritin levels, supporting a role of intracellular iron and free radical scavenging mechanisms in protecting CRC cells also in patients. The current results pointing to the iron binding and transporting proteins as mediators of cancer cell resistance to therapy, together with our previous works, support that FMD cycles, by altering a wide range of nutrients, growth factors, plasma and cellular proteins and metals, are able to affect a wide range of escape routes required for the survival or resistance acquisition of different tumors. The high toxicity of the combination of the FMD together with vitamin C against KRAS mutated tumors, provides a clear example of how a non-toxic therapy combining both wide-acting (FMD) and targeted (vitamin C) antitumor mechanisms can be as or more effective in delaying cancer progression in mice as/than standard antitumor therapies that are much more toxic to normal cells and organs. 2ff7e9595c
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