Induced expression of the Fragaria × ananassa Rapid alkalinization factor-33-like gene decreases anthracnose ontogenic resistance of unripe strawberry fruit stages

Rapid alkalinization factor (RALF) genes encode for ubiquitous small peptides that stimulate apoplastic alkalinization through interaction with malectin-like receptor kinase. RALF peptides may act as negative regulators of plant immune response, inhibiting the formation of the signal receptor complex for immune activation. Recently RALF homologues were identified in different fungal pathogen genomes contributing to host infection ability. Here, FaRALF-33-like gene expression was evaluated in strawberry fruits inoculated with Colletotrichum acutatum, Botrytis cinerea, or Penicillium expansum after 24 and 48 h post-infection. To investigate the role of FaRALF-33-like in strawberry susceptibility, transient transformation was used to overexpress it in white unripe fruits and silence it in red ripe fruits. Agroinfiltrated fruits were inoculated with C. acutatum and expression, and histological analysis of infection were performed. Silencing of FaRALF-33-like expression in C. acutatum-inoculated red fruits led to a delay in fruit colonization by the fungal pathogen, and infected tissues showed less penetrated infective hyphae than in wild-type fruits. In contrast, C. acutatum-inoculated white unripe fruits overexpressing the FaRALF-33-like gene decreased the ontogenic resistance of these fruits, leading to the appearance of disease symptoms and penetrated subcuticular hyphae, normally absent in white unripe fruits. The different response of transfected strawberry fruits to C. acutatum supports the hypothesis that the FaRALF-33-like gene plays an important role in the susceptibility of fruits to the fungal pathogen C. acutatum.Fil: Merino, Maria Cecilia. Universidad de Bologna; Italia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Guidarelli, Michela. Universidad de Bologna; ItaliaFil: Negrini, Francesca. Universidad de Bologna; ItaliaFil: De Biase, Dario. Universidad de Bologna; ItaliaFil: Pession, Annalisa. Universidad de Bologna; ItaliaFil: Baraldi, Elena. Universidad de Bologna; Itali

Allele Specific Locked Nucleic Acid Quantitative PCR (ASLNAqPCR): An Accurate and Cost-Effective Assay to Diagnose and Quantify KRAS and BRAF Mutation

The use of tyrosine kinase inhibitors (TKIs) requires the testing for hot spot mutations of the molecular effectors downstream the membrane-bound tyrosine kinases since their wild type status is expected for response to TKI therapy. We report a novel assay that we have called Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR). The assay uses LNA-modified allele specific primers and LNA-modified beacon probes to increase sensitivity, specificity and to accurately quantify mutations. We designed primers specific for codon 12/13 KRAS mutations and BRAF V600E, and validated the assay with 300 routine samples from a variety of sources, including cytology specimens. All were analyzed by ASLNAqPCR and Sanger sequencing. Discordant cases were pyrosequenced. ASLNAqPCR correctly identified BRAF and KRAS mutations in all discordant cases and all had a mutated/wild type DNA ratio below the analytical sensitivity of the Sanger method. ASLNAqPCR was 100% specific with greater accuracy, positive and negative predictive values compared with Sanger sequencing. The analytical sensitivity of ASLNAqPCR is 0.1%, allowing quantification of mutated DNA in small neoplastic cell clones. ASLNAqPCR can be performed in any laboratory with real-time PCR equipment, is very cost-effective and can easily be adapted to detect hot spot mutations in other oncogenes

Immediate or delayed retrieval of the displaced third molar : A review

The displacement of a third molar is a rare occurrence, but it could lead to serious and/or life threatening complication. Aim of this review is to understand the most correlated causes of displacement and the possible solutions proposed in literature to avoid and solve this complication for maxillary and mandibular third molars at the appropriate time. A search for ?third molar displacement? was performed by using Pubmed database. Articles referred to soft tissues displacement, from 1957 to 2018, were included in the review. The references lists of all eligible articles were examined and additional studies were added to the review only if indexed on Pubmed. All the articles on maxillary sinus displacement and the dislocation of dental fragments or surgical equipment were excluded. From a total of 134 results, 68 articles were examined for satisfying inclusion criteria. 18 articles were excluded because not inherent with the topic; 19 articles on infratemporal space, 11 on sublingual space, 9 on submandibular space, 11 on lateral pharyngeal space displacement were considered congruent for the review and included. The displacement of the third molar in deeper tissues could be avoided by the use of proper surgical procedures and instrumentarium. If displacement occurs, and the operator could not reach the tooth in safe conditions, the patient should be immediately referred to a maxillo-facial surgeon, because of the possibility of further displacement or the onset of hazardous or potentially fatal infections in vital regions

Postextractive alveolar ridge preservation using L-PRF: clinical and histological evaluation

Leukocyte- and platelet-rich fibrin (L-PRF) is an autologous platelet concentrate rich in growth factors and plasma proteins, obtained by centrifugation of patient whole blood, and widely used in oral surgery. This report describes a case of alveolar ridge preservation with L-PRF membranes. Postextractive alveolar healing was then assessed through a histologic and histomorphometric analysis. A patient requiring tooth extraction and subsequent implant rehabilitation was treated with simple extraction and socket filling with L-PRF membranes. Implant placement was performed at 3 months, and a bone biopsy was obtained for histomorphometric analysis. Histological examination of the grafted sites showed that the use of L-PRF could achieve good results in terms of bone dimension and quality and soft tissue healing. The results of this study support the use of L-PRF membranes to preserve hard and soft tissues after tooth extraction

RET mutation and increased angiogenesis in medullary thyroid carcinomas

Advanced medullary thyroid cancers (MTCs) are now being treated with drugs that inhibit receptor tyrosine kinases, many of which involved in angiogenesis. Response rates vary widely, and toxic effects are common, so treatment should be reserved for MTCs likely to be responsive to these drugs. RET mutations are common in MTCs, but it is unclear how they influence the microvascularization of these tumors. We examined 45 MTCs with germ-line or somatic RET mutations (RETmut group) and 34 with wild-type RET (RETwt). Taqman Low-Density Arrays were used to assess proangiogenic gene expression. Immunohistochemistry was used to assess intratumoral, peritumoral and nontumoral expression levels of VEGFR1, R2, R3, PDGFRa, PDGFB and NOTCH3. We also assessed microvessel density (MVD) and lymphatic vessel density (LVD) based on CD31-positive and podoplanin-positive vessel counts, respectively, and vascular pericyte density based on staining for a-smooth muscle actin (a-SMA), a pericyte marker. Compared with RETwt tumors, RETmut tumors exhibited upregulated expression of proangiogenic genes (mRNA and protein), especially VEGFR1, PDGFB and NOTCH3. MVDs and LVDs were similar in the two groups. However, microvessels in RETmut tumors were more likely to be a-SMA positive, indicating enhanced coverage by pericytes, which play key roles in vessel sprouting, maturation and stabilization. These data suggest that angiogenesis in RETmut MTCs may be more intense and complete than that found in RETwt tumors, a feature that might increase their susceptibility to antiangiogenic therapy. Given their increased vascular pericyte density, RETmut MTCs might also benefit from combined or preliminary treatment with PDGF inhibitors

Papillary thyroid carcinoma tall cell variant shares accumulation of mitochondria, mitochondrial DNA mutations, and loss of oxidative phosphorylation complex I integrity with oncocytic tumors

open12siMDL was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC) ‘Bruna Martelli’ Fellowship.Papillary thyroid carcinoma tall cell variant (PTC-TCV), a form of PTC regarded as an aggressive subtype, shares several morphologic features with oncocytic tumors. Pathogenic homoplasmic/highly heteroplasmic somatic mitochondrial DNA (mtDNA) mutations, usually affecting oxidative phosphorylation (OXPHOS) complex I subunits, are hallmarks of oncocytic cells. To clarify the relationship between PTC-TCV and oncocytic thyroid tumors, 17 PTC-TCV and 16 PTC non-TCV controls (cPTC) were subjected to: (1) transmission electron microscopy (TEM) to assess mitochondria accumulation, (2) next-generation sequencing to analyze mtDNA and nuclear genes frequently mutated in thyroid carcinoma, and (3) immunohistochemistry (IHC) for prohibitin and complex I subunit NDUFS4 to evaluate OXPHOS integrity. TEM showed replacement of cytoplasm by mitochondria in PTC-TCV but not in cPTC cells. All 17 PTC-TCV had at least one mtDNA mutation, totaling 21 mutations; 3/16 cPTC (19%) had mtDNA mutations (p < 0.001). PTC-TCV mtDNA mutations were homoplasmic/highly heteroplasmic, 16/21 (76%) mapping within mtDNA-encoded complex I subunits. MtDNA mutations in cPTC were homoplasmic in 2 cases and at low heteroplasmy in the third case, 2/3 mapping to mtDNA-encoded complex I subunits; 2/3 cPTC with mtDNA mutations had small tall cell subpopulations. PTC-TCV showed strong prohibitin expression and cPTC low-level expression, consistent with massive and limited mitochondrial content, respectively. All 17 PTC-TCV showed NDUFS4 loss (partial or complete) and 3 of 16 cPTC (19%) had (partial) NDUFS4 loss, consistent with lack of complex I integrity in PTC-TCV (p < 0.001). IHC loss of NDUFS4 was associated with mtDNA mutations (p < 0.001). Four BRAF V600E mutated PTCs had loss of NDUSF4 expression limited to neoplastic cell subpopulations with tall cell features, indicating that PTCs first acquire BRAF V600E and then mtDNA mutations. Similar to oncocytic thyroid tumors, PTC-TCV is characterized by mtDNA mutations, massive accumulation of mitochondria, and loss of OXPHOS integrity. IHC loss of NDUFS-4 can be used as a surrogate marker for OXPHOS disruption and to reliably diagnose PTC-TCV.openTsybrovskyy, Oleksiy; De Luise, Monica; Biase, Dario; Caporali, Leonardo; Fiorini, Claudio; Gasparre, Giuseppe; Carelli, Valerio; Hackl, Dominik; Imamovic, Larisa; Haim, Silke; Sobrinho‐Simões, Manuel; Tallini, GiovanniTsybrovskyy, Oleksiy; De Luise, Monica; Biase, Dario; Caporali, Leonardo; Fiorini, Claudio; Gasparre, Giuseppe; Carelli, Valerio; Hackl, Dominik; Imamovic, Larisa; Haim, Silke; Sobrinho‐Simões, Manuel; Tallini, Giovann

SARS-CoV-2 vaccines: What we know, what we can do to improve them and what we could learn from other well-known viruses

: In recent weeks, the rate of SARS-CoV-2 infections has been progressively increasing all over the globe, even in countries where vaccination programs have been strongly implemented. In these regions in 2021, a reduction in the number of hospitalizations and deaths compared to 2020 was observed. This decrease is certainly associated with the introduction of vaccination measures. The process of the development of effective vaccines represents an important challenge. Overall, the breakthrough infections occurring in vaccinated subjects are in most cases less severe than those observed in unvaccinated individuals. This review examines the factors affecting the immunogenicity of vaccines against SARS-CoV-2 and the possible role of nutrients in modulating the response of distinct immune cells to the vaccination

Different Methods in HPV Genotyping of Anogenital and Oropharyngeal Lesions: Comparison between VisionArray® Technology, Next Generation Sequencing, and Hybrid Capture Assay

(1) Background: Human papillomaviruses (HPVs) are known to be related to the development of about 5% of all human cancers. The clinical relevance of HPV infection has been deeply investigated in carcinomas of the oropharyngeal area, uterine cervix, and anogenital area. To date, several different methods have been used for detecting HPV infection. The aim of the present study was to compare three different methods for the diagnosis of the presence of the HPV genome. (2) Methods: A total of 50 samples were analyzed. Twenty-five of them were tested using both next generation sequencing (NGS) and VisionArray® technology, the other 25 were tested using Hybrid Capture (HC) II assay and VisionArray® technology. (3) Results: A substantial agreement was obtained using NGS and VisionArray® (κ = 0.802), as well as between HC II and VisionArray® (κ = 0.606). In both analyses, the concordance increased if only high risk HPVs I(HR-HPVs) were considered as “positive”. (4) Conclusions: Our data highlighted the importance of technical choice in HPV characterization, which should be guided by the clinical aims, costs, starting material, and turnaround time for results

High MYC Levels Favour Multifocal Carcinogenesis

The term “field cancerisation” describes the formation of tissue sub-areas highly susceptible to multifocal tumourigenesis. In the earlier stages of cancer, cells may indeed display a series of molecular alterations that allow them to proliferate faster, eventually occupying discrete tissue regions with irrelevant morphological anomalies. This behaviour recalls cell competition, a process based on a reciprocal fitness comparison: when cells with a growth advantage arise in a tissue, they are able to commit wild-type neighbours to death and to proliferate at their expense. It is known that cells expressing high MYC levels behave as super-competitors, able to kill and replace less performant adjacent cells; given MYC upregulation in most human cancers, MYC-mediated cell competition is likely to pioneer field cancerisation. Here we show that MYC overexpression in a sub-territory of the larval wing epithelium of Drosophila is sufficient to trigger a number of cellular responses specific to mammalian pre-malignant tissues. Moreover, following induction of different second mutations, high MYC-expressing epithelia were found to be susceptible to multifocal growth, a hallmark of mammalian pre-cancerous fields. In summary, our study identified an early molecular alteration implicated in field cancerisation and established a genetically amenable model which may help study the molecular basis of early carcinogenesis