Stupendemys geographica (currently the largest known side-necked turtle) and a nearly complete skull of Caninemys tridentata found in Miocene rocks of the Tatacoa Desert in Colombia. These two specimens indicate that more than two large freshwater turtle species shared a single ecosystem during the middle Miocene in northern South America. We also show the changes in the shell and scutes that occurred along the ontogeny of S. geographica, including a flattening of the carapace, constriction of the vertebral scutes, and increase in the height and thickness of the nuchal upturn wall; some of these changes are also evident in extant representatives of Podocnemididae, and have implications for a better understanding of their phylogeny.There are many questions regarding the largest freshwater turtle that ever existed, including how its morphology changed during its ontogeny and how a single ecosystem was able to support more than one group of giant turtles. Here, we report the first individual preserving an associated skull and shell for   Giant turtles; Colombia; Miocene; Paleobiology; South America; Tatacoa So far,.5–7 Ma) . In the identata  from thescovered . Here, wormation . In addi22.1Peltocephalus dumerilianus, Erymnochelys madagascariensis, and Podocnemis spp., as well as those described in We found and prepared the fossil turtles described here  between 2018 to 2020. All specimens are housed at the Paleontological collection of VPPLT. We used specimens of the extant species 2.2Stupendemys geographica specimen VPPLT-1337 using Toshiba Aquilion CT at the Méderi Hospital X-ray facility, Bogotá, Colombia, under the following settings: zoom factor 1.2, 12 902 total images, 120 kV, 150 mA, and 2 mm inter-slice spacing. We analyzed the CT data using iQ-VIEW software (https://www.imagesystems.biz). We also scanned skulls from the extant podocnemidids Peltocephalus dumerilianus SMF-40169, Erymnochelys madagascariensis SMF-33056, and Podocnemis unifilis SMF-32915 using a Tomoscope HV 500 (Werth Messtechnik GmbH) with a 2k detector and a 225-kV μ-focus X-ray source in the industrial μCT facility at the Technical University in Deggendorf, Deggendorf, Germany. Scan parameters: CT mode 2, 150 μA, 190 kV, 1200 steps, voxel resolution 20.2 μm). CT data is available in (Supplementary Videos S1–S3). CT data for the extant taxa are available via direct contact to E-A. Cadena, as they are a part of a different research project currently in progress.We scanned the skull of https://doi.org/10.1016/j.heliyon.2021.e08591.Supplementary content related to this article has been published online at Supplementary_VideoS1.MOVSupplementary_VideoS2.movSupplementary_VideoS3.movSupplementary_VideoS4.movThe following are the supplementary data related to this article:2.32.3.1Stupendemys geographica, we examined 39 specimens including hatchlings (nine specimens), juveniles (11 specimens), and adults (19 specimens). We identified 24 landmarks for the vertebral scutes. Most of these landmarks have been defined and used in previous studies , including one specimens of S. geographica (VPLT 1337) and two specimens of C. tridentata (MCZ-4376 and CIAAP-2002-01). Landmarks were taken from pictures in lateral view using the PointPicker plugin in ImageJ 1.52q  and Caninemys tridentata was based on the new skull and shell fragments VPPTL-1720 described here, and the specimen described in S. geographica inside Pan-Pleurodira, using Proganochelys quenstedti as the outgroup taxon. The first analysis included all taxa, and the second was focused on the Podocnemidoidae clade, removing all other taxa except Progranochelys quenstedti, Notoemys laticentralis, and Platychelys oberndorferi. We used the following settings for the two analyses: traditional search, 20 trees to save per replication, and other parameters by default; memory tree increased to max. trees 10000; collapse of zero-length branches according to rule 1; light implied weighting k value of 12; and 1000 replicates of random addition sequences. All characters were equally weighted and some were treated as ordered .For the phylogenetic analysis, we used the matrix of 2.5Institutional abbreviations—CIAAP, Centro de Investigaciones Antropológicas, Arqueológicas y Palentológicas of the Universidad Nacional Experimental Francisco de Miranda, Coro, Falcón State, Venezuela; DNPM-MCT, Departmento Nacional de Produçaõ Mineral, Divisaõ de Geologia e Mineralogia, Ciências da Terra, Rio de Janeiro, Brazil; MCZ, Museum of Comparative Zoology, Harvard University, Cambridge, USA; SMF, Senckenberg Museum Frankfurt, herpetological collection, Frankfurt, Germany; VPPLT, Museo de Historia Natural La Tatacoa, La Victoria, Huila Department, Colombia.Anatomical abbreviations—Abd, abdominal scute; ac, anterior condyle; acp, acromial process; Ana, anal sute; ars, articular surface; ast, astragalus; axb, axillary buttress; axn, axis neural arch; bm, bite mark; bs, basisphenoid; bo, basioccipital; cal, calcaneum; cev, cervical; co, costal; cor, coracoid; dig, digit; dta, distal tarsus; ent, entoplastron; epi, epiplastron; Ext, extragular scute; Fem, femoral scute; fem, femur; fpp, foramen palatinum posterius; fpt, fossa pterygoidea; FR, frontal scute; fr, frontal; Hum, humeral scute; hyo, hyoplastron; hyp, hypoplastron; Int, intergular scute; ju, jugal; IP, interparietal scute; lp, lateral process; M, marginal scute; mes, mesoplastron; met, metatarso; mfo, musk foramen; mx, maxilla; posorbital scute; nc, nuchal upturn crest; ne, neural; nu, nuchal; op, opisthotic; P, pleural scute; PA, parietal scute; pa, parietal; pc, posterior condyle; pe, peripheral; Pec, pectoral scute; pha, phalange; pl, palatine; pm, premaxilla; PO, postorbital scute; po, postorbital; poz, postzygapophysis; pr, prefrontal; prz, postzygapophysis; pt, pterygoid; ptt, processus trochlearis pterygoidei; py, pygal; qj, quadratojugal; qu, quadrate; sap, superior articular process; so, supraoccipital; sp, suprapygal; sq, squamosal; tcp, thoracic process; tv, thoracic vertebra; V, vertebral scute; vfo, vertebral foramen; vo, vomer; vsm, visceral surface margin; xip, xiphiplastron.33.1TESTUDINES Batsch 1788 , lower segment of La Victoria Formation  sutural articulation of pelvis with shell; 2) loss of medial contact of mesoplastra; 3) well-developed anal notch; 4) fusion of gulars; 5) formed central articulations of cervical vertebrae; and 6) a well-developed processus trochlearis pterygoidei. It is a podocnemidid based on: 1) palatine forming moderate amount of the upper triturating surface and 2) a cervical centrum with saddle-shaped posterior condyle. Stupendemys geographica differs from all other podocnemidids in each of its anatomical elements as follows. Skull: 1) an upper triturating surface formed by the posteromedial portion of premaxillae, medial contact between maxillae, and palatines forming a secondary palate; 2) an extremely inflated snout formed by prefrontals; 3) anteroventrally descending wall of prefrontals onto the narium externa aperture; 4) striated surface of skull roof bones . Lower jaw: 1) a deep triturating surface, forming an oval concavity; 2) labial ridge curved anteriorly ending in an acute tip; 3) lingual ridge is a blunt margin forming an accessory ridge that increases in height and width anteriorly and runs as a narrow ridge at the medial symphysis; 4) high coronoid process; 5) large dorsal opening of fossa Meckelii, which fills the posterior end of the jaw to such an extent that the area articularis mandibularis forms part of the posterior margin, and the fossa opens posterolaterally next to the jaw articulation. Carapace: 1) low-arched carapace decreasing in height along ontogeny; 2) irregular nodular contours on external surface and deep median notch at front; 3) anterior border of nuchal-peripheral bones thickened and moderately (juvenile-adult) to strongly (adult) upturned; 4) extremely long peripherals 1 and 2 forming massive anterolateral horns slightly projected ventrally in forms attributed as male; 5) carapace dorsal bone surface smooth to striated or slightly pitted; 6) posterior peripheral bones moderately scalloped along margins; 7) vertebral scute 5 being the longest and widest of the series particularly in adult individuals. Plastron: 1) pectoral-abdominal sulcus very anterior to mesoplastra, reaching almost the hyoplastra lateral notch level in adults. Neck: 1) cervical vertebrae (probably 7 and 8) with neural arches relatively high in relation to anteroposterior lengths of centra; 2) articular facets of postzygapophyses forming acute angle of less than 90°; 3) cervical 8 neural arch with large horizontal plane, prezygapophyses directed perpendicularly, thin bladelike spine on anterior face of neural arch and no ventral keel on centrum. Humerus: 1) humerus squat, massive; 2) deep bicipital fossa between lateral and medial articular facets on ventral surface; 3) prominent ridge traversing ventral surface of shaft from medial process to distal end, terminating just above lateral condyle; 4) medial condyle broadest at anterior end; 5) medial and lateral condyles facing very ventrally; 6) straight to slightly slender shaft and more triangular in cross section than circular. Femur: 1) femur squat, massive, greatly flattened dorsoventrally particularly in adults; 2) breadth of tibial condyle approximately one-third total length of bone. Scapula: 1) dorsal strongly bowed scapular process with a flattened flange projecting laterally from the main axis.Peltocephalus dumerilianus, Erymnochelys madagascariensis and closer relatives: 1) large postorbital bones avoiding a contact between jugals and parietals; 2) cheek emargination extremely reduced to almost absent; 3) anterior overhang of prefrontals completely covering the narium externa aperture ending in a straight to convex edge; 4) laterally facing orbits in dorsal view of the skull, with a very narrow to almost complete absent dorsal exposure of the maxilla and jugal; and 5) an articular with a processus retroarticularis posteroventromedially projected, differing from the ventrally projected Podocnemis spp. It shares with Peltocephalus dumerilianus: 1) an interparietal scute with lateral margins tapering posteriorly; 2) ventral premaxillae forming a very deep medial concavity ; 3) pinched snout; 4) long contact between prefrontal and maxilla in lateral view ; 5) vertebral 1 scute nearly square in shape with parallel margins ontogenetically conservative; and 6) axillary buttress reaching peripheral 3.It shares with Remarks—The specific epithet ‘geographicus’ is incorrect because Stupendemys constitutes a feminine generic name. Its associated specific epithets therefore must also be feminine following the International Commission on Zoological Nomenclature , lowermost segment of La Victoria Formation .MCT-1496  the follMCT-1496 . An addi44.1Skull. The skull of VPPLT-1337 is broken at the level of the processus trochlearis pterygoidei missing completely its midposterior region. The preserved portion of the skull has not suffered of crushing, only missing some small portions of the lateral margin of the right frontal and the most ventral margins of both maxillae. In dorsal view . However, it differs from this taxon and all other podocnemidids in having an upper triturating surface formed by a medial contact between maxillae and palatines that creates a secondary palate, completely hiding the aperture narum interna  placed at the positive end. PCA 1 is driven largely by the position of the orbit, the anterior projection of the snout, the overall height of the skull, and the position of landmark 7 . PC 2 contains 17.12% of the variation in the sample and largely separates one specimen of P. expansa at the positive end, and all P. lewyana at the negative end of the axis. PC 2 is driven by the shape of the orbit, the slope of the dorsal most aspect of the skull, and particularly the position of landmark 1 (anteriormost tip of premaxillae) and landmark 7 . S. geographica occupies a unique portion of the morphospace, grouping with the Peltocephalus and Erymnochelys on PC 1 and P. lewyana on the negative end of PC 2.There are three major distinct snout architectures among the podocnemidid turtles studied, as supported by the geometric morphometric analysis that we conducted A. FigureC. tridentata and Podocnemis spp. fit on the negative end of the axis having relatively small orbits and very narrow prefrontal-frontal lateral exposure  component of shape related to phylogeny. In the phylomorphospace plot C, 40.6% exposure D; whereaexposure E. PC 2 sgination F, whereaon level G. Shape 5.2Stupendemys geographica represents the first fossil shell measuring less than 1 m long. All previously described shells have measured greater than 1 m and reach a maximum of 2.86 m  skulls vary considerably  produced 27 most parsimonious trees (MPT), from which a strict consensus was obtained (Supplementary File S6). However, the support values for this analysis were poor: Tree Length (TL) = 1315; Consistency Index (CI) = 0.272; and Retention Index (RI) = 0.738. This resembled the topology presented in Podocnemis spp. and different relationships inside the clade formed by Erymnochelys madagascariensis, Neochelys spp. Turkanemys pattersoni, Kenyemys williamsi and Papoulemys laurenti. All of these differences are expected due to an update in coding we performed, which involved fixing some errors and coding for polymorphisms related to ontogeny as discussed above. In this strict consensus, Caninemys tridentata and Stupendemys geographica were found in different positions inside a larger clade that included the extant Peltocephalus dumerilianus, Bairdemys spp., and other taxa of the Stereogenyini clade following The first known skull and shell from the same individual of Brasilemys josai and above). This analysis produced a single most parsimonious tree  and is supported by more advanced cover of the adductor fossa  (character 19); premaxillae reaching aperture narum interna (character 47); a large anterior opening of the cavum pterygoidei (character 96); and the lack of a horizontal occipital shelf (character 117). S. geographica is found inside a clade that also includes the extant Pe. dumerilianus at its base  coexisted in the same habitat (Pebas wetland system) during part of the middle to late Miocene; despite being similar in size, with larger individuals reaching more than 2 m long. The completely different skull morphologies of C. tridentata and S. geographica indicate also potentially different paleodiets for each taxon. A vacuum feeding strategy combined with a powerfully biting supported by the three toothlike structures of the maxillae in C. tridentata .Dr Edwin-Alberto Cadena and Dr. Andrés Link were supported by 10.13039/501100008793Universidad del Rosario .Dr. Edwin-Alberto Cadena was supported by 10.13039/100005966Leakey Foundation.Dr. Siobhán B. Cooke, Dr. Melisa Tallman, and Dr. Laura K. Stroik were supported by the National Geographic Foundation.Dr. Siobhán B. Cooke was supported by Data included in article/supplementary material/referenced in article.The authors declare no conflict of interest.No additional information is available for this paper.


Salmonella has resulted in treatment failure and high mortality rates. In this study, we estimated the presence of plasmid-mediated quinolone resistance (PMQR) genes in Salmonella enterica isolated from human salmonellosis patients in South Korea from 2016 to 2019. We evaluated the association of these genes with fluoroquinolone susceptibility. Antimicrobial susceptibility tests for Salmonella isolates were performed using the Vitek II system, and the minimum inhibitory concentrations (MIC) of ciprofloxacin and levofloxacin were determined using the E-test method. Plasmid-mediated quinolone resistance (PMQR) genes were detected by PCR amplification and quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes were analyzed following Sanger sequencing of the PCR products. Thirty-four Salmonella strains with reduced susceptibility to fluoroquinolones (ciprofloxacin MIC ≥ 0.125 µg/mL and levofloxacin MIC ≥ 0.25 µg/mL) were selected from 208 human clinical Salmonella isolates. Among them, 22 Salmonella strains harbored one PMQR gene , and three Salmonella strains carried two PMQR genes (qnrS and aac(6′)-Ib-cr or qnrA and qnrB). qnrS was the most common PMQR gene. Serotyping revealed that Salmonella 4,[5]12:i:-  and Salmonella Typhimurium  were the two most predominant serovars, and Multi-locus sequence typing (MLST) showed that ST19 and ST34 were the most frequent sequence types. In conclusion, qnr gene-positive Salmonella 4,[5],12:i:- and Salmonella Typhimurium were the main serovars responsible for reduced susceptibility to fluoroquinolones. Therefore, our findings suggest that PMQR-positive Salmonella strains, which can be isolated from various samples including human, food, and the environment, should be carefully monitored.Non-typhoidal salmonellosis remains a pressing public health problem worldwide. Quinolones, particularly fluoroquinolones, are widely used to treat various infections, including non-typhoidal salmonellosis, which can be a serious illness. The emergence of fluoroquinolone-resistant The online version contains supplementary material available at 10.1186/s13099-021-00431-7. Salmonella that usually produces acute onset of fever, abdominal pain, diarrhea, and vomiting. Antimicrobial therapy is not recommended in healthy individuals with mild or moderate infection ,12:i:- were recently isolated from pigs, chickens, humans, geese, and cats in China ,12:i:- has rapidly emerged worldwide ,12:i:- was reported in 2008 ,12:i:- isolates from chickens, geese, and cats in China were resistant to nalidixic acid (52.5%) ,12:i:- from swine in the United States have also been reported ,12:i:-), 9 isolates of ST34 type , 2 isolates of ST36 type , ST27 type , ST13 type  and ST469 type  were identified. ST19 and ST34 were the predominant STs in Salmonella Typhimurium and Salmonella 4,[5],12:i:-, which had high genetic diversity but were over 80% similar to each other  was conducted. Nine isolates of ST19 type (seven orldwide , 29. ST1sistance . It has sistance  and thatsistance . Salmoneri Lanka , and a SPortugal , 36. MorPortugal . HoweverSalmonella strains with reduced susceptibility to fluoroquinolones from human salmonellosis. Among them, Salmonella 4,[5],12:i:- and Salmonella Typhimurium were the most common serovars, and MLST revealed that ST19 and ST34 were the predominant lineages, with a high genetic similarity of over 80%. The spread of plasmid-mediated antibiotic resistance in ST19 and ST34 strains requires careful attention in South Korea. Furthermore, all isolates carried one or two of the PMQR genes, suggesting that various genes associated with quinolone resistance can be transferred horizontally among Enterobacteriaceae, causing human infections in South Korea.We isolated 34 Additional file 1: Table S1.Samonella serovars isolated from clinical samples in Gyeonggi-do, South Korea.Additional file 2: Table S2. Primer sequences used for this experiment.


Amyotrophic lateral sclerosis (ALS) is a neurodegenerative neuromuscular disease that affects motor neurons controlling voluntary muscles. Survival is usually 2–5 years after onset, and death occurs due to respiratory failure. The identification of biomarkers would be very useful to help in disease diagnosis and for patient stratification based on, e.g., progression rate, with implications in therapeutic trials. Neurofilaments constitute already-promising markers for ALS and, recently, chitinases have emerged as novel marker targets for the disease. Here, we investigated cerebrospinal fluid (CSF) chitinases as potential markers for ALS. Chitotriosidase (CHIT1), chitinase-3-like protein 1 (CHI3L1), chitinase-3-like protein 2 (CHI3L2) and the benchmark marker phosphoneurofilament heavy chain (pNFH) were quantified by an enzyme-linked immunosorbent assay (ELISA) from the CSF of 34 ALS patients and 24 control patients with other neurological diseases. CSF was also analyzed by UHPLC-mass spectrometry. All three chitinases, as well as pNFH, were found to correlate with disease progression rate. Furthermore, CHIT1 was elevated in ALS patients with high diagnostic performance, as was pNFH. On the other hand, CHIT1 correlated with forced vital capacity (FVC). The three chitinases correlated with pNFH, indicating a relation between degeneration and neuroinflammation. In conclusion, our results supported the value of CHIT1 as a diagnostic and progression rate biomarker, and its potential as respiratory function marker. The results opened novel perspectives to explore chitinases as biomarkers and their functional relevance in ALS. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects upper and lower motor neurons in the motor cortex, brainstem and spinal cord. Typical age of onset is between 50 and 70 years of age. It is an infrequent disease, with an incidence of 2–3/100,000; however, the lifetime risk is estimated at 1/350 in Europe  compatible with HexNAc-HexNAc was detected  and graphite Hypercarb (that binds oligosaccharides) cartridges to obtain an oligosaccharide enriched fraction. Pools of CSF available from patients tested for CHIT1 were used. Hypercarb-bound fractions were analyzed by UHPLC-MS using a Hypercarb column. We screened for putative CHIT1 products, which included chito-oligosaccharides as hydrolysis products of chitin (linear polymer of β1-4 linked GlcNAc) , the disdetected . This coIn this study, we presented evidence supporting the relevance of chitinases as ALS biomarkers. This comparative study highlighted the importance of different chitinases as potentially useful markers for distinct disease characteristics.CHIT1 levels were elevated in ALS compared to disease controls, in agreement with reports by other groups ,29,30,38For CHI3L2, increased levels have been reported in the literature ,32 but, Although concentration values for all proteins were within the range described elsewhere ,30, therThe three tested chitinases correlated with disease progression rate in ALS patients, and CHIT1 and CHI3L2 showed the strongest correlations. These results were in agreement with other authors ,29,30,38We found that all chitinases correlated with pNFH, with the correlation being highest for CHIT1 and CHI3L2. Since pNFH is a marker of neuronal damage , most imhttps://www.proteinatlas.org/ENSG00000064886-CHI3L2/tissue/cerebral+cortex#img), but its expression in ALS tissues has not been described so far. As such, the origin of CSF CHI3L2 is currently unknown. Possible interplay between pro-inflammatory cytokines and chitinases has been discussed [Chitinases are secreted by activated glial cells, as well as by other cells of the immune system ,13,16. Ciscussed , with imiscussed . In lineiscussed . By contiscussed . Additioiscussed . More stm/z signal compatible with the disaccharide HexNAc-HexNAc, which could consist of di-N-acetylchitobiose  or LacdiNAc , which are hydrolyzed by CHIT1 [CHIT1 has chitinolytic activity towards chitin and artificial substrates , as wellby CHIT1 ,36. Humaby CHIT1 , which cby CHIT1 . Howeverby CHIT1 , we alsoby CHIT1 . LimitatIn conclusion, our results supported the importance of chitinases as biomarker targets in ALS. Particularly, CHIT1 was a promising biomarker for diagnosis, progression rate and respiratory function. These results provided novel perspectives to further explore the potential of chitinases as ALS biomarkers and their functional relevance, they and may have implications in other neurological diseases.


Picornaviridae presently comprises 68 approved genera with 158 species plus many unassigned viruses. In order to better match picornavirus taxonomy to the functional and genomic groupings between genera, the establishment of five subfamilies  is proposed. The subfamilies are defined by phylogenetic analyses of 3CD (precursor of virus-encoded proteinase and polymerase) and P1 (capsid protein precursor) coding sequences and comprise between 7 and 22 currently approved virus genera. Due to the high within-subfamily and between-subfamily divergences of the picornavirus genera, p-distance estimates are unsuited for the demarcation of subfamilies. Members of the proposed subfamilies typically show some commonalities in their genome organisations, including VP1/2A cleavage mechanisms and possession of leader proteins. Other features, such as internal ribosomal entry site types, are more variable within and between members of genera. Some subfamilies are characterised by homology of proteins 1A, 2A, 2B and 3A encoded by members, which do not belong to the canon of orthologous picornavirus proteins. The proposed addition of a subfamily layer to the taxonomy of picornaviruses provides a valuable additional organisational level to the family that acknowledges the existence of higher-level evolutionary groupings of its component genera.The highly diverse virus family The online version contains supplementary material available at 10.1007/s00705-021-05178-9. Picornaviridae have small (~30 nm), non-enveloped capsids with T=1/pseudo T=3 symmetry [hel, a helicase with a typical fold of P-loop ATPases, 3BVPg, a small genome-linked oligopeptide, 3Cpro, a chymotrypsin-like cysteine proteinase, and 3Dpol, an RNA-dependent RNA polymerase (RdRP) . Dicipie small ~0 nm, nonavirales .Picornaviridae are currently classified into 68 assigned genera with 158 species. However, many yet unassigned viruses are awaiting classification. All picornaviruses share orthologous proteins, which exhibit the characteristic picornavirus hallmarks GDD and FLK(R) of the 3Dpol). More-variable and often genetically non-homologous picornavirus proteins include the leader protein (L), where present, 1A, 2A, 2B, 3A and 3B, although some may play similar functional roles in replication.Picornaviruses are genetically highly diverse and infect members of six vertebrate classes on all continents. Members of the family pol) and cluster together in phylogenetic analysis. In general, they also share a common – although not invariant – genome layout. An idealised genome layout, the L434 schema, was proposed by Rueckert and Wimmer [Picornavirus genera are distinguished on the basis of genetic relationships. Members of a given genus share significant sequence similarity  , but thCaphthovirinae, Kodimesavirinae, Ensavirinae, Paavivirinae, and Heptrevirinae. The results indicate that the picornavirus 'supergroups' based on analysis of the proteinase/polymerase precursor 3CD are useful to define the taxonomic rank of picornavirus subfamilies. These proposed assignments to subfamilies have the additional value of defining virus groups with distinct features of their genome layouts and replication strategies.Here, we investigate the potential taxonomic utility of the 'supergroup' concept and accordingly propose the establishment of five picornavirus subfamilies to be named http://tree.bio.ed.ac.uk/software/figtree). Divergence was estimated using MEGA X. Results obtained using GRAViTy type of 158 currently accepted picornavirus species revealed eight clades (comprising between one and 354 sequences). Five clades correspond to the previously defined picornavirus “supergroups”, while further lineages clades 6-8) incorporate the recently described (i) harkavirus A1 and three yet unassigned picornaviruses , (ii) three currently unclassified reptile- and primate-derived picornaviruses , and (iii) ampivirus A1 [-8 incorpA phylogenetic analysis of the CP-encoding gene region (P1) yielded clades with significant support corresponding to SG1, SG3, SG4, and SG5. Only SG2 lacked monophyly, but all of the nodes showed low posterior probability, indicating poor robustness of the tree. Two additional clades identified in 3CD analysis containing the sequences of harkavirus A1 and the six unassigned picornaviruses showed a similarly distinct clustering in P1 . The name was derived from the two earliest described genera Cardiovirus and Aphthovirus. This subfamily includes the genera Ailurivirus, Aphthovirus, Bopivirus, Cardiovirus, Cosavirus, Erbovirus, Hunnivirus, Malagasivirus, Mischivirus, Mosavirus, Mupivirus, Senecavirus, Teschovirus, Torchivirus, Tottorivirus, and Marsupivirus (35 species). A characteristic feature of virus genome expression in this subfamily is the cleavage of the CP precursor 1AB (VP0) into 1A (VP4) and 1B (VP2)  and a 2A protein with an NPG↓P motif . Aphthoviruses and erboviruses possess a leader proteinase (Lpro). Mosaviruses have two copies of 3BVPg, while foot-and-mouth disease virus (genus Aphthovirus) has three copies. Viruses in the majority of genera possess a type II IRES.upivirus 5 speciesKodimesavirinae (SG2). The name was derived from the four earliest described genera of this subfamily, Kobuvirus, Dicipivirus, Megrivirus, and Salivirus. This subfamily includes the genera Danipivirus, Dicipivirus, Gallivirus, Hemipivirus, Kobuvirus, Livupivirus, Ludopivirus, Megrivirus, Myrropivirus, Oscivirus, Passerivirus, Pemapivirus, Poecivirus, Pygoscepivirus, Rafivirus, Rajidapivirus, Rosavirus, Sakobuvirus, Salivirus, Sicinivirus, Symapivirus, and Tropivirus (38 species). A characteristic feature is that the 2B protein is conserved (orthologous) among the members of the subfamily , an uncleaved 1AB , the presence of a 2A protein with an H-box/NC motif . Viruses of the Kodimesavirinae have a type II or type IV IRES.opivirus 8 speciesEnsavirinae (SG3). The name was derived from the two earliest described genera, Enterovirus and Sapelovirus. This subfamily includes the genera Anativirus, Boosepivirus, Diresapivirus, Enterovirus, Felipivirus, Parabovirus, Rabovirus, and Sapelovirus (30 species). Characteristic features are the cleavage of 1AB and the presence of a 2A protein with proven or assumed chymotrypsin-like cysteine proteinase activity . Besides the 1A and 2A proteins, the 2B and 3A proteins are also conserved (orthologous) among the members of this subfamily . IRES types I, II and IV are found.irinae SG. The namPaavivirinae (SG4). The name was derived from the two earliest described genera, Parechovirus and Avihepatovirus. This subfamily includes the genera Aalivirus, Aquamavirus, Avihepatovirus, Avisivirus, Crohivirus, Grusopivirus, Kunsagivirus, Limnipivirus, Orivirus, Parechovirus, Pasivirus, Potamipivirus, and Shanbavirus (29 species). A characteristic feature of all members is that the polyprotein lacks an L protein and that 1AB remains uncleaved  pasiviruses have a protein with an NPG↓P motif, and (iii) oriviruses and grusopi B viruses have a 2A protein with unknown function. All known aquamaviruses have two copies of 3BVPg. The IRES belongs to type II or IV.ved Fig. . The 2B Heptrevirinae (SG5). The name was derived from the two earliest described genera, Hepatovirus and Tremovirus. This subfamily includes the genera Caecilivirus, Crahelivirus, Fipivirus, Gruhelivirus, Hepatovirus, Rohelivirus, and Tremovirus (21 species). Characteristic features include a very short 1A peptide (14-35 amino acids) and the occurrence of 1AB cleavage  to 0.639 (S.E. 0.01), whereas the between-group divergence is greater 0.7 (S.E. 0.01-0.013) .The mean  S.E. 0.02 to 0.63within-group and between-group divergence yield results that allow the distinction of five 'supergroups' corresponding to the respective clades shown in Figure Comparisons of averaged https://talk.ictvonline.org/taxonomy/vmr/) along with a compilation of 75 unassigned full-length candidate picornavirus sequences representing members of eight clades shown in Fig. http://gravity.cvr.gla.ac.uk). The program (“Genome Relationships Applied to Virus Taxonomy") compared the picornavirus sequences with all full genome sequences of currently classified +stranded viruses through computation of composite generalized Jaccard (CGJ) distances derived from hidden Markov model profile similarities of translated sequences, a metric derived from similarities in genome organizational features (gene orders and orientations) [Sequences representative of each classified picornavirus from the ICTV Virus Metadata Resource (tations) .Caphthovirinae, Kodimesavirinae, Ensavirinae, Paavivirinae and Heptrevirinae  and did not display the same differentiation from each other and other Heptrevirinae members as observed in other subfamilies. Furthermore, Fujian spotted paddle-tail newt picornavirus (MG600085), a proposed member of clade 6, failed to cluster with the two other picornaviruses assigned to this clade. Apart from these two exceptions, there was a close correlation in branching order and branching lengths between genome relationships inferred by GRAViTy with those obtained by phylogenetic analysis of P1 and 3CD regions for all five proposed subfamilies, and almost all genera and species.Picornaviruses formed a monophyletic group in a dendrogram constructed from CGJ distances Fig. , with thnae Fig. . Each suKodimesavirinae, the P1 sequences of the members of all remaining subfamilies also cluster in clades comprising at least three sequences . Hence, other criteria must be applied. The presence or absence of one or more non-structural proteins that are unique to individual genera could be an expedient alternative, such as the extremely short 1A protein of the members of the Heptrevirinae, the 2Apro protein of the Ensavirinae, the 2B proteins of the members of the Ensavirinae, Kodimesavirinae and Paavivirinae, and the 3A of the Ensavirinae. Shared possession of such proteins within a subfamily provides strong support for their evolutionary relatedness to each other. Indeed, phylogenetic analysis of the 2Apro of the Ensavirinae, the 2B protein of the Kodimesavirinae, Ensavirinae, and Paavivirinae, and the 3A protein of the Ensavirinae  yields tree topologies that resemble those of the respective 3CD trees (data not shown). The recognition that several picornavirus proteins in different subfamilies may have separate origins and share no sequence homology is relevant for the selection of genome regions for virus classification – degrees of similarity computed between sequences in alignments that are non-homologous are actually meaningless and should be excluded from phylogenetic comparisons.The estimation of p-distances, which proved to be helpful for the assignment of picornaviruses at the levels of types, species and genera, is of limited use for classification in the proposed subfamilies. Pairwise amino acid frequency distributions of P1, 3CD, and 3D proteins showed considerable overlaps in both Kodimesavirinae even though the P1 tree for these viruses was polyphyletic  in the P1 phylogeny and in GRAViTy analysis  accounts for its highly divergent placement in the P1 phylogenetic tree types. Future investigations may contribute to elaborating a fully consistent description of the subfamilies. At present, picornavirus subfamilies are defined by phylogenetic analysis of the 3CD protein and supported by additional phylogenetic analysis of the P1 polyprotein as well as whole genome analysis by GRAViTy and coherent features of CP, 2A, 2B, and 3A proteins.The formal establishment of picornavirus subfamilies that may replace the previously unofficially introduced 'supergroups' will increase the informativeness of picornavirus taxonomy. The family Picornaviridae to include five new subfamilies described in this paper has been submitted on behalf of the Study Group to the ICTV for discussion and possible formal approval.An official taxonomic proposal for reorganization of the family Supplementary file1Supplementary Fig. S1 Phylogenetic analysis based on picornavirus 3CD proteins. A total of 578 sequences representing the 3CD region (3162 nt) of members of all known picornavirus species and types were analysed with MrBayes v3.2 (nucleotide substitution model HKY+G+I). Convergence was reached after 13 million generations. Sequences cluster in clades 1 to 8 (indicated in different colors). Presented are GenBank accession number, species name , virus name/type, common name , and strain designation (in square brackets). Posterior probabilities of major clades are presented. The scale indicates substitutions per nucleotide (PDF 816 KB)Supplementary file2Supplementary Fig. S2 Phylogenetic analysis of picornavirus P1 proteins. A total of 651 sequences representing the P1 region encoding the capsid proteins (5215 nt) of members of all known picornavirus species and types were analysed with MrBayes v3.2 (nucleotide substitution model GTR+G+I). Convergence was reached after 17 million generations. Sequences of clades 1 and 3 to 8 cluster in monophyletic clades (indicated in different colors). Presented are GenBank accession number, species name , virus name/type, common name , and strain designation (in square brackets). Posterior probabilities of major clades are presented. The scale indicates substitutions per nucleotide (PDF 787 KB)Kodimesavirinae) 2B sequences. The alignment comprises 120 sequences of 22 genera, 38 species, 70 types, and 18 unassigned viruses. Tree inference was conducted with MrBayes, using the nucleotide substitution model GTR+G+I. Convergence was reached after 6 million generations. Unassigned viruses are shown in red. Presented are GenBank accession number, species name , virus name/type, common name , and strain designation (in square brackets) at the tips as well as posterior probabilities at the nodes. The scale indicates substitutions per nucleotide (PDF 295 KB)Supplementary file3Supplementary Fig. S3 Phylogenetic analysis of supergroup 2 (Ensavirinae) 2A sequences. The alignment comprises 82 sequences of 8 genera, 30 species. Tree inference was conducted with MrBayes, using the nucleotide substitution model GTR+G+I. Convergence was reached after 7 million generations. Unassigned viruses are shown in red. Presented are GenBank accession number, species name , virus name/type, common name  and strain designation (in square brackets), at the tips as well as posterior probabilities at the nodes. The scale indicates substitutions per nucleotide (PDF 263 KB)Supplementary file4Supplementary Fig. S4 Phylogenetic analysis of supergroup 3 (Ensavirinae) 2B sequences. The alignment comprises 89 sequences of 8 genera, 30 species. Tree inference was conducted with MrBayes, using the nucleotide substitution model GTR+G+I. Convergence was reached after 12 million generations. Unassigned viruses are shown in red. Presented are GenBank accession number, species name , virus name/type, common name  and strain designation (in square brackets), at the tips as well as posterior probabilities at the nodes. The scale indicates substitutions per nucleotide. Blue and green arrows indicate inconsistent clustering of boosepiviruses and paraboviruses, respectively (PDF 259 KB)Supplementary file5Supplementary Fig. S5 Phylogenetic analysis of supergroup 3 (Ensavirinae) 3A sequences. The alignment comprises 90 sequences of 8 genera, 30 species. Tree inference was conducted with MrBayes, using the nucleotide substitution model GTR+G+I. Convergence was reached after 7 million generations. Unassigned viruses are shown in red. Presented are GenBank accession number, species name , virus name/type, common name , and strain designation (in square brackets) at the tips as well as posterior probabilities at the nodes. The scale indicates substitutions per nucleotide. Blue arrows indicate inconsistent clustering of parabo B and C viruses (PDF 266 KB)Supplementary file6Supplementary Fig. S6 Phylogenetic analysis of supergroup 3 (Paavivirinae) 2B sequences. The alignment comprises 61 sequences of 13 accepted and proposed genera, 29 species. Tree inference was conducted with MrBayes, using the nucleotide substitution model GTR+G+I. Convergence was reached after 4 million generations. Unassigned viruses are shown in red. Presented are GenBank accession number, species name , virus name/type, common name , and strain designation (in square brackets) at the tips as well as posterior probabilities at the nodes. The scale indicates substitutions per nucleotide (PDF 245 KB)Supplementary file7Supplementary Fig. S7 Phylogenetic analysis of supergroup 4 Below is the link to the electronic supplementary material.


NAB2/STAT6 gene fusion, tumor protein-53 (TP53) and/or (telomerase reverse transcriptase) TERT promotor mutation were performed. The risk of metastasis was calculated using the Demicco risk stratification system (RSS). The results were combined and examined to assess the accuracy of risk stratification and classification. The most common location was in non-extremities; 66% were located in soft tissue or subcutaneous areas and 92.8% in deep locations. On microscopic analysis, 38.1% of tumors revealed hypercellularity with a predominant patternless and/or hemangiopericytic growth pattern; 13.4% had ≥4 mitoses/10HPF; 16.5% showed necrosis, and almost half the tumors showed at least focal myxoid areas. Dedifferentiation was observed in three tumors. Immunomarker expression in SFTs was as follows: CD34 92.9%, CD99 57.1%, Bcl2 67.9%, neuroendocrine markers (at least 1) 25.7%, Desmin 14.3%, CK(AE1/AE3) 3%, Apoptotic Protease Activating Factor (APAF-1) 87% and finally Ki-67 ≥ 10% in 14.4%. The NAB2/STAT6 gene fusion was detected in 50 tumors. After a median follow-up of 90 months, 9.3% recurred, 11.3% metastasized, 10.3% died of disease and 76.2% were free of disease. TERT mutations were detected in 40.6% of the SFTs; the TP53 mutation was detected in 17%, and only 9.3% showed both mutations. According to the Demicco RSS, 6.1%, 11.3% and 82.4% of the tumors were classified as high, intermediate or low-risk of metastasis, respectively. All high-risk tumors had ≥4 mitoses/10HPF, necrosis, Ki-67 ≥ 10, HTER and/or TP53 mutation and poor evolution. The intermediate risk SFTs with worse evolution displayed the HTER mutation. Almost all low-risk tumors had a favorable evolution, although four showed at least one adverse factor  and had a worse evolution. An integration of clinical, morphologic, immunohistochemical and molecular findings may improve risk stratification and classification and better predict patient outcome. The unfavorable course seems to be more frequent in high-risk SFTs, although it is not exceptional in low-risk SFTs either; hence, a long-term follow-up is required independently of the assigned risk stratification score. The inclusion of molecular findings in risk stratification systems could improve the precision in the classification of SFTs, especially those of intermediate risk. Future studies will be required to determine the most effective way to incorporate molecular analyses into RSS on SFTs. The coexistence of several adverse factors such as ≥4 mitoses/10HPF, necrosis, Ki-67 ≥ 10%, mutations in HTER and/or p53 may suggest a closer clinical follow-up regardless of the histological appearance of the tumor.Although solitary fibrous tumors (SFTs) have an unpredictable evolution, some specific clinicopathologic factors have been associated with the final outcome. We retrieved clinical, pathological and molecular data of 97 patients with a histological diagnosis of SFT and Signal transducer and activator of transcription 6 (STAT6) positivity. We retrospectively studied the pathological factors predictive of recurrence/metastasis and compared them with the clinical outcome. A wide immunohistochemical study and molecular analysis to detect  Solitary fibrous tumors (SFTs) are mesenchymal neoplasms that can occur at any location, especially pleural, meningeal or extrapleural sites. Location in limbs is infrequent ,38,39,40The evolution of SFTs is often uncertain ,7,8,9,10A patternless and hemangiopericytic growth with variably fibrosis and collagenous deposits are characteristic, but SFTs can show many faces, including round cells, giant-cells, myxoid areas, pleomorphic pattern, fat-forming tumors and dedifferentiated forms ,23,24,25NAB2/STAT6 and its variants confirm a diagnosis of SFT in those cases with unconvincing STAT6 immunoreactivity, and specific gene fusions have been related to prognosis and tumor location [GLI1)-amplified tumor, Kaposi sarcoma and Hodgkin as well non-Hodgkin lymphomas [Strong and diffuse nuclear immunoreactivity for Signal transducer and activator of transcription 6 (STAT6) is almost always diagnosed in SFTs, and CD34, Bcl-2 and CD99 immunoexpression is common ,23,24,25location ,38,39,40ymphomas ,38,39,40Several risk-stratification systems (RSS) have been described ,2,3,4,5,TP53 mutational status and TERT promotor mutational status with clinical outcome.We have previously published a small series of SFTs with at least one histological factor associated with aggressive behavior and explored the correlation between the adverse histological findings and molecular profile with tumor behavior . The maiThe clinicopathological and follow-up data are shown in The representative status of the immunohistochemical profile is shown in  and summNAB2-exon 4/STAT6- exon 2 gene fusion was detected in 35 cases, NAB2-exon 6/STAT6-exon 16/17 in 15 cases  were given in 2 high-risk SFTs, 5 intermediate-risk SFTs and 40 low-risk SFTs. All these cases showed strong and diffuse nuclear STAT6 immunoreactivity. Eleven cases were non-informative for HTER and/or p53 molecular status; these cases were all classified as low-risk of metastasis.The risk stratification system classifications are described in The correlation between clinical and histological findings (mitosis and/or necrosis), the Ki-67 index and molecular status with clinical outcome (metastasis and/or died of disease versus free of disease) in high-risk and intermediate-risk tumors are described in GLI1-amplified tumors; hence, in cases with overlapping morphology and STAT6 immunoreactivity, additional molecular studies are needed to establish a definitive diagnosis [NAB2/STAT6 and its variants confirm a diagnosis of SFT, especially in rare clinical settings, unusual histological findings or unexpected immunohistochemical results. Moreover, specific gene fusions have been related to prognosis and tumor location [Strong and diffuse nuclear immunoreactivity for STAT6 is a very useful tool for the diagnosis of SFTs, although unexpected epithelial, muscular or neuroendocrine marker expression has also been described in these tumors and may lead to confusion with other neoplasms with hemangiopericytic growth patterns ,25,33,42iagnosis ,40,41,42location ,40,41,42location .Most low risk SFTs categorized by Demicco et al. and other RSS follow an apparently indolent course. However, some of these cases may have late recurrence/relapse and/or metastases leading to uncertainty and skepticism among oncologists regarding the specificity of RSS in correctly predicting evolution in all cases ,3,4,5,14Regarding histological predictive factors of aggressiveness, high mitotic counts with a general agreement of ≥4/10HPFs represent the strongest predictor of malignant behavior, as confirmed in the present series where all tumors categorized as high-risk and many of the tumors classified as intermediate-risk revealed ≥4/10HPFs ,6,7,8,42Of all the immunohistochemical markers applied in this series, only Ki-67 ≥ 10 was associated with poor evolution. Similar to mitosis and necrosis, this finding was also found in all SFTs categorized as high-risk. Ki-67 has only been included in the Diebold et al. RSS , and altRecently, loss of APAF-1 immunoexpression has been associated with the progression and poor prognosis in SFTs . APAF-1 NAB2 exon 4-STAT6 exon 2 fusion variant are often located in the thorax and are less cellular with abundant fibrosis [NAB2 exon 6-STAT6 exon 16/17 fusion variants typically show a more cellular round to ovoid cell morphology and are often located in the deep soft tissue of the intra-abdominal/retroperitoneal/pelvic region or in central nervous system [NAB2 exon 6-STAT6 exon 16/17 fusion occur in a significantly younger age group, showing higher mitotic activity and a higher recurrence rate [Molecular studies in SFTs have been progressively implemented in many centers for diagnosis and prognosis. SFTs with the most common canonical fibrosis ,38,39,40fibrosis ,38,39,40s system ,38,39,40NAB2 exon 4-STAT6 exon 2 fusion variant show a transcriptional signature enriched for genes involved in DNA binding, gene transcription and nuclear localization, whereas SFTs with the NAB2 exon 6-STAT6 exon 16/17 fusion variants were enriched for genes involved in tyrosine kinase signaling, cell proliferation and cytoplasmic localization [STAT6 domain composition [STAT6-TAD (contained only the transactivation domain of STAT6) or STAT-Full (fusions with most of the STAT6 domain intact). Tumors with STAT6-TAD fusions had a higher mitotic count (p = 0.016) and were associated with poor prognosis [Recently, a new study reported that SFTs with the lization . In addiposition . They carognosis .We did not observe any direct association of gene fusion variants with aggressiveness or location of any histologic or phenotypic profile in the present series.NAB2/STAT6 fusion gene. Nevertheless, all cases in the present series showed strong and diffuse STAT6 nuclear expression by immunohistochemistry. This finding of strong and diffuse nuclear STAT6 immunoreactivity has previously been implemented in many laboratories as a surrogate for the molecular analysis in solitary fibrous tumors considering the good correlation between the nuclear STAT6 overexpression and the presence of the NAB2/STAT6 fusion gene [NAB2/STAT6 fusion gene represents an intrachromosomal inversion, a non-informative or negative result is not exceptional in gene fusion molecular studies.A limitation of the present study was the fact that 48.5% of the samples 47/97) showed non-informative RNA results for the detection of the /97 showeion gene . FurtherReverse transcription-polymerase chain reaction (RT-PCR) may not be the most suitable method to detect fusion genes in SFTs, and more sensitive techniques such as next generation sequencing (NGS) or RNAseq may have improved the quality of the results. However, these very advanced molecular ancillary tests are also more expensive and not widely available in all pathology labs. RNA analysis failure using RT-PCR could be explained by limited material in the case of a core biopsy or by fixation issues or tissue conservation ,6,7,8,42TERT promoter mutations and the TP53 mutation have been associated with malignant behavior in SFTs [TP53 mutation in the current series was low, in contrast to the TERT mutation that was found in almost half the tumors. Many of the high-risk tumors had TP53 mutations, HTER mutations or both [HTER mutation in the present series showed poor evolution, in line with the previous observation of Demicco et al. where the TERT mutation probably provides no additional prognostic information on tumors already classified as low or high risk [HTER promotor and/or TP53 alterations in SFTs are associated with prognosis, no risk stratification system has so far incorporated these molecular factors [HTER and p53 molecular status, all of which were classified as low-risk of metastasis. in SFTs ,40,41,42 or both ,40,41,42igh risk . At pres factors ,7,8. In Four RSS have been proposed in the literature that classify SFTs into three  or two categories (low vs. high-risk) depending on the system ,3,4,5,14In the present series, all SFTs classified as high-risk by Demicco et al. had Ki-67 ≥ 10. We had four cases categorized as low-risk by Demicco et al. but with a worse clinical evolution. Two out these four cases had a Ki-67 index higher than 10 and when applying the Diebold et al. RSS , those cThe identification of dedifferentiation in SFTs is very important because new evidence has revealed that antiangiogenics are effective, and their use as a first line of treatment should be considered in SFTs, but not in dedifferentiated SFTs for which chemotherapy is more effective .It would be interesting in the future to study the tumor/stromal tissue interaction, and whether the amount of collagen and/or the type of collagen fibers could have any prognostic significance in these tumors. At present, hypercellularity in solitary fibrous tumors, which usually correlates with a lower amount of collagen, has been correlated in some studies with a somewhat more aggressive evolution in SFTs, although hypercellularity is not necessarily always associated with aggressive evolution ,3,4,5,14HTER and/or p53 may suggest the need for a closer clinical follow-up regardless of the histological appearance of the tumor.In conclusion, risk assessment still remains a challenging issue in SFT classification and the final outcome. However, the integration of clinical, morphologic, immunohistochemical and molecular findings may improve risk stratification and classification of SFTs and could guide the clinician when designing risk-adjusted treatment and follow-up. Regardless of the assigned risk stratification score, SFTs may require long-term follow-up considering that low-risk tumors may occasionally show a non-indolent evolution. The inclusion of molecular findings in RSS could improve precision in the classification of SFTs, especially those of intermediate risk. Nevertheless, future studies are required to determine the most effective way to incorporate molecular analyses into RSS on SFTs. The coexistence of several adverse factors such as ≥4 mitoses/10HPF, necrosis, Ki-67 ≥ 10%, mutations in We collected 97 cases of histologically proven SFTs having strong and diffuse nuclear STAT6 positivity. Formalin-fixed, paraffin-embedded tissue (FFPET) was retrieved from the archives at the Pathology Department, Clinical Hospital, University of Valencia; Hospital Universitari i Politécnic La Fe and Instituto Valenciano de Oncología (IVO) Valencia. This study was conducted in accordance with the principles of the Declaration of Helsinki and approved by the local Ethics Committee (IVO 2018-28). Clinical data  and follow-up data  were also retrieved.All the available H&E slides were examined by three pathologists  all blinded to the clinical data. In cases with discordant results, a consensus was reached on a multi-head microscope. The following data were retrieved: histological grade: conventional SFT vs. dedifferentiated SFT; predominant tumor cell morphology: round cells, spindle cells, round and spindle cells, others ; mitotic rate: ≥4/10HPF vs. <4/10HPF; necrosis: yes vs. no; cellularity: high vs. moderate vs. low; nuclear pleomorphism: yes vs. no; fibrosis: yes vs. no; myxoid pattern: yes vs. no.IHC staining was carried out on 3–4 micron-thick FFPET from a single representative block for each primary tumor section. The primary antibodies, source, dilution and staining pattern criteria used are listed in Genomic DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) samples using a QIAamp DNA FFPE Tissue Kit  according to the manufacturer’s instructions. DNA quantification was performed using the Nanodrop One . PCRs for the TERT promoter and exons 5, 6, 7 and 8 for TP53 were performed in the Proflex PCR System . Sequencing reactions were carried out using BigDye v1.1 (Applied Biosystems) and analyzed on a SeqStudio Genetic Analyzer (Applied Biosystems). All sequencing analyses were read on both strands in order to exclude pre-analytical and analytical errors.Total RNA was extracted from formalin-fixed, paraffin-embedded (FFPE) samples using a RNeasy FFPE Kit . We performed PCRs to detect NAB2/STAT6 gene fusions. Primers used are listed in The risk of recurrence/metastasis was calculated using the Demicco scoring system ,2. The c


Ligands at H4R are still being tested pre-clinically and in clinical trials of inflammatory diseases, including rheumatoid arthritis, asthma, dermatitis, and psoriasis. These trials, however, documented only moderate beneficial effects of H4R ligands so far. Nevertheless, pre-clinically, H4R still is subject of ongoing research, analyzing various inflammatory, allergic, and autoimmune diseases. During inflammatory reactions in gut tissues, histamine concentrations rise in affected areas, indicating its possible biological effect. Indeed, in histamine-deficient mice experimentally induced inflammation of the gut is reduced in comparison to that in histamine-competent mice. However, antagonists at H1R, H2R, and H3R do not provide an effect on inflammation, supporting the idea that H4R is responsible for the histamine effects. In the present review, we discuss the involvement of histamine and H4R in inflammatory and inflammation-associated diseases of the gut.Histamine is a pleiotropic mediator involved in a broad spectrum of (patho)-physiological processes, one of which is the regulation of inflammation. Compounds acting on three out of the four known histamine receptors are approved for clinical use. These approved compounds comprise histamine H1-receptor (H The biogenic amine histamine (2-(4-imidazolyl)-ethylamine) is a short-acting local mediator present in virtually all mammalian tissues. It is generated by the enzyme L-histidine decarboxylase (HDC) , and deg1R), H2R, H3R, and H4R in order of their discovery. While H1R and H2R are ubiquitously expressed and bind histamine with relatively low potency (pEC50 ~6), the expression of high potency (pEC50 ~8) histamine receptor subtypes H3R and H4R is rather restricted i) is mobilized from intracellular stores and the concentration of cytosolic cAMP is diminished. Independent of G protein-signaling, β-arrestin 2 is recruited to agonist-bound H4R, initiating mitogen-activated protein kinase (MAPK) cascade activation as well as receptor desensitization and internalization -4-methylpiperazine) was the first selective H4R antagonist developed and it has been used in a large number of laboratories in in vivo and in vitro analyses [4R, inversely agonizing G protein-dependent signaling, while agonizing G protein-independent signaling. Thus, the results gained so far must be re-evaluated accordingly [4R, is an agonist at the mouse and rat H4R. These results, however, were obtained using membrane preparations of insect cells expressing recombinant human H4R together with selected G proteins and have never been reproduced in a native setting [4R are available, providing agonistic and inverse agonistic/antagonistic activities. However, their use may be afflicted with problems concerning specificity, effectivity, and potency, too. Thus, in a complex biological system, studies on receptor function should not be based on a single selected ligand. At least chemical different ligands have to be employed or complementary experimental approaches must be applied. For example, genetic manipulation of the receptor to be studied can be used in addition to ligand-driven experimental systems. Of course, also vice versa, data generated by a genetic-based approach alone should be very carefully validated unless proved by an independent experimental system.Pharmacological ligands, either agonists or antagonists/inverse agonists, or in vivo models using genetically modified animals, are the main tools to identify Hanalyses . JNJ7777ordingly ,93. More setting . Unfortu4R. These mice do not express a functional H4R and without any manipulation they do not demonstrate any biological abnormalities as compared to their wild type (WT) counterparts [4R leads to reduced symptoms [4R, and thus, the involvement of H4R in experimental asthma and dermatitis in mice is widely accepted. Unfortunately, in humans H4R antagonists did not gain the anticipated results regarding effectiveness and safety [4R expression pattern, which is similar, but not necessarily identical in mice and man, as exemplarily demonstrated in 4R pathophysiology, has to be evaluated in a context -dependent way. Using different approaches to manipulate H4R function, it became evident that of all different immune cells, H4R mainly regulates human and mouse mast cells and eosinophils, e.g., their migration and degranulation [4R, it is intriguing to speculate about the biological meaning of this auto-regulatory cycle. In eosinophils, the H4R seems to be only a minor player in comparison to classical activators and may be responsible for the cellular activities’ fine tuning [4R. The observed effects elicited by histamine in cells, tissues, or animals are always the sum of effects elicited by all histamine receptor subtypes present, which are H1R, H2R, and H4R in the case of eosinophils and mast cells. This scenario becomes even more complicated, since, as described above, the histamine receptor subtypes possess different affinities for histamine and effective interstitial concentrations of histamine cannot be reliably measured so far.Mice are widely used to model human diseases since their handling is relatively easy and inexpensive. Furthermore, a huge array of different strains with defined genotypes is available. Among these are mice with a targeted mutation in the Hhr4 locus, which encodes the mouse Hterparts . Howeversymptoms ,40,95. Td safety   are idiopathic, chronic-recurring diseases of the gut. Their two main manifestations, ulcerative colitis (UC) and Crohn’s disease (CD), differ in their clinical, endoscopic, and histologic appearance. In CD, the inflammation appears in diffuse lesions that can be found all over the digestive tract and deeply penetrates the intestinal wall, possibly affecting all layers. In contrast, inflammatory lesions in UC start in the rectum, proceed upwards but do not exceed the colon, and remain superficial at the mucosa ,101,102.E. coli, M. morganii, or L. vaginalis [It has been known for quite a long time that in affected colorectal mucosal samples obtained from CD and UC patients histamine concentrations are elevated in comparison to samples from unaffected sections or healthy control persons ,109. A faginalis .4R as well as of other GPCR is a still ongoing discussion that currently lacks impetus. Since in 2012 the specificity of H4R-selective antibodies was questioned [4R that have been rigorously evaluated according to some general rules [4R proteins by selective antibodies. Other methods such as mRNA detection by RT-(q)PCR or functional assessment using agonistic or antagonistic ligands also bear uncertainties. RT-PCR is a very sensitive detection method, but, besides the risk that trace amounts of contaminating genomic DNA may pose the presence of specific mRNA [4R expression. Alternatively, genetic approaches  are to be used to support expression data gained by other methods. In particular, targeted knock-out approaches would be able to foster our knowledge on the cell type-specific expression of H4R, being applicable not only to the intestine but to all other tissues as well.The detection of functional expression of Hestioned ,115, a cal rules  are stilfic mRNA , it is nfic mRNA . The usefic mRNA ,119. Thu3R are expressed throughout the intestinal tract from mice, rats, monkeys, and humans -induced colitis, the binding of H4R by JNJ7777120 resulted in reduced inflammatory symptoms [4R in the colon. Following studies using mice either or not deficient in H4R expression and using JNJ7777120 in models of chemically-induced colitis confirmed this indication [Since histamine seemed to be functionally involved in gut (patho)physiology, studies aimed at comprehensively identifying the histamine receptor subtypes expressed in the intestinal tract using tissues derived from different species ,122,123.d humans , while tNA level ,121. Subsymptoms , indicatdication ,90. The 4R. Besides the contribution of mast cells to allergic inflammation [+Ly6G+ immature myeloid cells have been described, since they are involved in inflammation-associated carcinogenesis in the gut [As discussed above, mast cells, allocated to the innate immune system, do not only produce histamine, but also respond to stimulation of the Hammation , they alammation ,134. Morammation ,136. In ammation ,138. Thuammation . Expressammation ,141. As  the gut . Whether the gut ,98,143.  the gut , remains4R on eosinophils [4R activity, which diminishes experimental colitis, would also reduce number and, thus, activity of eosinophils in the affected intestinal areas. [Besides mast cells, enhanced numbers of eosinophils have been identified in the inflamed gut mucosa of IBD patients, too ,145. Theinophils ,72,73,80l areas. ,147,148.4R knockout mice as compared to WT mice, reduced numbers of colon-infiltrating neutrophils as well as lower colon tissue concentrations of IL-6, CXCL1, and CXCL2 have been detected upon induction of experimental colitis [4R increases neutrophil infiltration during colitis, presumably in an indirect manner [4R-regulated production of IL-6, CXCL1, and CXCL2 remain ill defined. The related chemokines CXCL1 and CXCL2, which both bind to the receptor CXCR2, are synthesized by variety of immune cells including macrophages and neutrophils themselves, and by epithelial cells. Macrophages have been shown to respond to histamine via H4R [4R is still matter of debate. Eventually, epithelial cells are the most probable source of CXCL1 and CXCL2 in the context of colonic inflammation.Neutrophils belong to those cells first recruited to the site of inflammation, where they combat the potentially harmful intruders applying rather unspecific mechanisms, condoning collateral damage at healthy cells and tissues. Consequently, neutrophils can be detected in high numbers in the colitic mucosa, probably dependent on the enhanced expression of IL-6 and downstream CXCL-chemokines , mediato colitis ,89. Thust manner ,152. How via H4R ,65, howe4R [4R-deficient colon epithelial cells, both pointing to a direct effect of histamine at H4R expressed on mouse colon epithelial cells [Colonic epithelial cells comprise a group of cells forming a tightly linked monolayer epithelium lining the inner surface of the intestine and providing, by several means, a barrier between the gut lumen and the host tissue. Impairment of this barrier would result in the luminal content gaining access to intra- or sub-epithelial areas, followed by an inflammatory reaction ,154. New4R , althoug4R . Neverthal cells . Moreove4R contributes to colonic inflammation, mostly to the innate arm by regulating production and release of the pro-inflammatory mediators TNF, IL-6, CXCL1, and CXCL2 promoting neutrophil recruitment. In addition, in vitro the epithelial cell barrier is battered by histamine/H4R as well, possibly contributing to inflammation. Whether or not colon epithelial cells also are responsible for the histamine/H4R-triggered enhanced release of the mediators mentioned above is not definitively settled so far.In summary, it seems probable that histamine via H2R antagonist cimetidine in colorectal cancer patients [Cancer, in general, is a major public health problem and does not only account for a high physical strain of the patients but also for intensive emotional pressure in both patients and their relatives and, finally, for a large number of deaths. Although anti-cancer therapies, including new strategies and compounds, have advanced strongly in the past decades, they are still loaded with detrimental effects, such as toxicity or poor response . These epatients ,160. In patients , in whicpatients . Thus, hpatients ,164.4R that has already been discussed above, the absence of H4R expression also leads to a reduction of chemically-induced carcinogenesis in mice [4R decreases in gastric carcinoma during progression, accompanied by the attenuated histamine-induced suppression of proliferation [4R. It is worth noting that others have found H4R expression in such cell lines [4R in detail is far from being finally settled.CRC is the third most common type of cancer in humans and accounts for high mortality. UC patients are at a 2- to 3-fold higher risk to develop CRC in comparison to individuals not suffering from IBD and severity, extent, and persistence of inflammation positively correlates to carcinogenesis. In addition to colitis-associated CRC, CRC appears also sporadic and hereditary ,166. Alt in mice . Whetherferation ,176. Thill lines , which m4R in IBD, especially in the colon, has been demonstrated in different model systems by several laboratories, who mainly conclude a pro-inflammatory function  have not been discussed in this review. Even less differentiated is the picture on the effect of H4R in colon carcinogenesis. It is likely that H4R due to its pro-inflammatory function promotes the pathogenicity of colitis-associated CRC, but an additional effect of H4R on tumor or accessory cells currently can neither be excluded nor confirmed. Therefore, still a lot of work on histamine function on colon carcinogenesis has to be envisaged.Histamine is a mediator that is mainly recognized due to its function in allergy, but takes part also in non-allergic inflammatory reactions. In the gut, the involvement of histamine and Hfunction . Cellula


Results showed that 810-nm PBM affected oxidative phosphorylation in OHSU-971 FAcorr, causing a metabolic switch to anaerobic glycolysis. In addition, PBM reduced the catalase activity, determining an unbalance between oxidative stress production and the antioxidant defenses, which could stimulate the pro-apoptotic cellular pathways. Our data, at the parameters investigated, suggest the safeness of PBM as a supportive cancer therapy. Pre-clinical and clinical studies are necessary to confirm the in vitro evidence.Photobiomodulation (PBM) is a form of low-dose light therapy that acts through energy delivery from non-ionizing sources. During the recent two decades, there has been tremendous progress with PBM acceptance in medicine. However, PBM effects on potential stimulation of existing malignant or pre-malignant cells remain unknown. Thus, the primary endpoint was to assess the safety of PBM treatment parameters on head and neck squamous cell carcinoma (HNSCC) proliferation or survival. The secondary endpoint was to assess any putative anti-cancer effects of PBM treatments. Cell viability, energy metabolism, oxidative stress, and pro- and anti-apoptotic markers expression were investigated on a Human Head and Neck Squamous Cell Carcinoma cellular model (OHSU-974 FAcorr cell line). PBM therapy was administered through the 810 nm diode laser (GaAlAs) device  at the powers of 0, 0.25, 0.50, 0.75, 1.00, or 1.25 W in continuous wave (CW) mode for an exposure time of 60 s with a spot-size of 1 cm Photobiomodulation (PBM) is a low-dose light therapy form, which affects cell metabolism through energy delivery from non-ionizing sources . PhotonsDuring the recent two decades, there has been tremendous progress with PBM acceptance in medicine. Its potent efficacy has been noted in supportive cancer therapy for cancer treatment complications, such as oral mucositis (OM), radiodermatitis, lymphedema, and peripheral neuropathy ,26,27,28Energy metabolism differs in cancer cells compared to healthy cells. Specifically, no-cancer cells are characterized by a high rate of aerobic metabolism. Conversely, cancer cells increase glucose consumption and lactic acid release, despite normoxic conditions, modifying the microenvironment . This coJanus Bifrons relation between the clinical application of PBM and its effects on cancer cells has yet to be elucidated. Specifically, the PBM effect on mitochondria energy metabolism in carcinoma cells remain to be investigated [2 area on an HNSCC cancer cell line, OHSU-974 FAcorr. The first endpoint here was to assess the safety of PBM treatment parameters on HNSCC proliferation or survival. A second endpoint was to investigate any putative anti-cancer effects of PBM treatments. Cell viability, metabolism, oxidative stress, and pro- and anti-apoptotic markers expression were investigated.Thus, the potential stigated . Therefo2 in vitro on endothelial [We performed PBM with an 810 nm diode laser (GaAlAs) device . The laser was equipped with the AB-2799 hand-piece irradiating through a flat-top beam profile . The flat-top hand-piece was chosen because, as we previously showed, it can generate a homogeneous power density on the beam spot-area, which is not affected by the distance from the target . Therefoothelial  and stemothelial ,43, as wothelial  and Bellothelial . To supp2. 0 W denotes the untreated controls. To perform blinded treatments, a 635 nm red-light guide  with the laser device switched to silent mode was used . To avoid bias, the cell growth, PBM treatments, and cell analyses were performed by different operators to ensure blinding. After treatments, cells samples were then processed to evaluate the variation of the energetic metabolism , oxiAs a Human Head and Neck Squamous Cell Carcinoma (HNSCC) model, the OHSU-974 cell line was employed. Since this cell line was isolated from Fanconi Anemia Type A Patients, to avoid any bias associated with the mutation of the FANC-A gene, the cell line has been corrected with S11FAIN retrovirus (OHSU-974 FAcorr). The cell line was obtained from the “Cell Line and DNA Biobank from patients affected by Genetic Diseases” G. Gaslini Institute–Telethon Genetic Biobank Network, Genoa, Italy.2. PBM treatments were performed as described (2.01 above), media was replenished, and cells were maintained in culture for 24, 48, and 72 h. Cells were harvested and used for protein or biochemical assays. OHSU-974 FAcorr was grown in RPMI supplemented with 10% fetal calf serum and antibiotics at 37 °C with 5% COo-F1 ATP synthase (ATP Synthase) activity was evaluated on 20,000 cells permeabilized with 0.03 mg/mL digitonin for 1 min. Cells were incubated for 10 min in a medium containing: 50 mM Tris-HCl pH 7.4, 50 mM KCl, 1 mM EGTA, 2 mM MgCl2, 0.6 mM ouabain, 0.25 mM di(adenosine)-5-penta-phosphate , and 25 μg/mL ampicillin . ATP synthesis was induced by the addition of 10 mM pyruvate plus 5 mM malate, and 0.1 mM ADP to stimulate the pathway composed by Complexes I, III, and IV. The reaction was monitored for 2 min, every 30 s, with a luminometer , by the luciferin/luciferase chemiluminescent method, with ATP standard solutions between 10−8 and 10−5 M . Data were expressed as nmol ATP/min/106 cells [The F6 cells. Oxygen consumption was measured at 25 °C in a closed chamber using an amperometric electrode . Cells were permeabilized with 0.03 mg/mL digitonin for 1 min, centrifuged for 9 min at 1000 rpm, and resuspended in phosphate buffer saline (PBS). The same solution was used in the oximetry measurements. A total of 20,000 cells were used for each experiment. Moreover, 10 mM pyruvate plus 5 mM malate plus 0.1 mM ADP were added to stimulate the pathway composed by Complexes I, III, and IV. Data were expressed as nmol O/min/10The oxidative phosphorylation (OxPhos) efficiency (P/O ratio) was calculated as the ratio between the concentration of the produced ATP and the amount of consumed oxygen in the presence of respiring substrates and ADP . In effi+ at 340 nm. The assay medium contained 100 mM Tris-HCl, pH 8, and 5 mM NAD+. Samples were spectrophotometrically analyzed before and after the addition of 4 μg of purified lactate dehydrogenase. Data were normalized based on cell numbers [Lactate concentration was spectrophotometrically assayed in the growth medium, following the reduction of NAD numbers .Cultured cells were centrifuged at 1000 rpm for 5 min to remove the growth medium. The pellet was washed in PBS, resuspended in Milli-Q water, and sonicated twice for 10 s in ice, with a 30 s break to prevent mixture warming, using the Microson XL Model DU-2000 . Total protein content was estimated using the Bradford method .To assess lipid peroxidation, malondialdehyde (MDA) concentration was evaluated using the thiobarbituric acid reactive substances (TBARS) assay. This test is based on the reaction of MDA, a breakdown product of lipid peroxides, with thiobarbituric acid (TBA). The TBARS solution contained 15% trichloroacetic acid (TCA) in 0.25 N HCl and 26 mM thiobarbituric acid. To evaluate the basal concentration of MDA, 600 μL of TBARS solution were added to 50 μg of total protein dissolved in 300 μL of Milli-Q water. The mix was incubated for 60 min at 95 °C. Afterwards, the sample was centrifuged at 14,000 rpm for 2 min and the supernatant was spectrophotometrically analyzed at 532 nm [2O2 at 240 nm. The assay mix contained 50 mM phosphate buffer, pH 7.0, and 5 mM H2O2 [Glutathione reductase activity was spectrophotometrically assayed, following the oxidation of NADPH at 340 nm. The assay mix contained 100 mM Tris HCl, pH 7.4, 1 mM EDTA, 5 mM GSSH, and 0.2 mM NADPH . Catalas mM H2O2 . For botDenaturing electrophoresis (SDS-PAGE) was performed on 4–20% gradient gels (BioRad). In total, 30 μg of proteins were loaded for each sample. The following antibodies were employed: anti-Bcl2 , anti-BAX , anti-phospho-p53 (Ser 15) , anti-p53 , and anti-Actin . All primary antibodies were diluted 1:1000 in PBS plus 0.15% tween (PBST). Specific secondary antibodies were employed (Sigma-Aldrich), diluted 1:10,000 in PBST. Bands were detected and analyzed for optical density using an enhanced chemiluminescence substrate , a chemiluminescence system , and UV1D software (UVITEC). Bands of interest were normalized for actin levels in the same membrane.Flow-cytometric analysis of DNA content was performed on cells permeabilized with 0.05% Triton X-100 and stained with 30 mg/mL PI plus 0.5 mg/mL RNase for 30 min. Samples were measured on a FACS-Calibur flow cytometer (Becton Dickinson) and cell cycle phase distributions were achieved from the DNA content histograms of at least 10,000 cells . To evalp < 0.05 was considered statistically significant.Data were analyzed using a one-way or two-way ANOVA, as appropriate, followed by Tukey’s or Sidak’s multiple comparison test, respectively, using Prism 8 Software. Data are expressed as mean ± standard deviation (SD) and are representative of at least three independent experiments. An error with probability The effects of an 808 nm laser on OHSU-974 mitochondrial energy metabolism were evaluated in terms of ATP synthesis and oxygen consumption rate (OCR) after 24 and 48 h from irradiation at different powers (0.25–1.25 W). The results, reported in The mitochondrial metabolism modulation observed after the laser irradiation determined a reorganization of the glucose metabolism. For example, after the first 24 h, the cells treated with 1 W showed a decrement of lactate release, a sign of the anaerobic glycolysis reduction, probably due to the enhancement of OxPhos activity. Conversely, the treatment at 0.25 W induced the opposite effect, incrementing the lactate release D. This pSince altered mitochondrial metabolism is associated with increased oxidative stress, the concentration of malondialdehyde (MDA) has been evaluated as an end-point of oxidative damages. After the first 24 h elapsed from irradiation, only 0.25 W determined an increment in MDA level with respect to the control (0 W), according to the reduced mitochondria activity and P/O ratio. Conversely, the MDA level was higher in all treated samples compared to the untreated sample, after 48 h from the laser treatment, reaching the maximum level in the 1 W sample. A. Since In addition to altering glucose metabolism, PBM treatments affected the OHSU-974 FAcorr viability. In detail, 24 h after the irradiation, 0.25 and 1 W determined a decrement and an increment in the cell number, respectively, compared to the control and the other treated samples A,B. HoweTo confirm the activation of the apoptotic process in the lasered OHSU-974 FAcorr cells, the expression of phospho-p53, p53, Bcl2, and Bax has been evaluated, by western blot. Data show that in the first 24 h after the laser irradiation, the p53 phosphorylation appeared impaired only in 0.25, 0.5, and 0.75 W cells, while other samples displayed an expression level similar to that of the control. Conversely, after 48 h of laser irradiation, all samples except the one treated at 1.25 W showed the impairment in the activation of p53, suggesting a minor ability to check the DNA damage . The evain vitro, and in vivo studies showed conflicting results concerning the effects of PBM on the proliferation of cancer [The effect of PBM on cellular energetic metabolism was described ,8. Mitocf cancer ,57,58,59f cancer ,54. Therf cancer ,60,61. Tf cancer .2), which, 24 h after treatment, drastically increased the mitochondria activity. However, 48 h after the irradiation the sample experienced a drastic decrement in oxygen consumption and ATP synthesis, in line with the effect of the other parameters. Basically, 48 h after the irradiation, the 808-nm laser light led to mitochondria uncoupling with the exclusion of 0.75 and 1.25 W . This means that the treatment with an 808-nm laser not only determined a decrease of the mitochondrial energy function but also a partial OxPhos inefficiency, causing a further reduction of the energy production and an increment of oxidative stress production. Interestingly, the effect seems to follow the window effect previously described for the interaction of 980-nm wavelength with isolated-mitochondria of mammals [Our data showed that 808-nm wavelength PBM at different powers and fluences interact with mitochondrial energetic metabolism of OHSU-974 FAcorr, as previously observed in normal cells ,12,26,41in primis, to maintain active energy metabolism. [More in general, since the cancer cells are characterized by the Warburg effect, which consists of a metabolic switch from aerobic metabolism to anaerobic glycolysis, resulting in a sharp increase in glucose consumption , the impabolism. ,62.2O2, can oxidize the cysteine residues of proteins and support cancer cell development, proliferation, and adaptation [The increase of mitochondria respiratory chain activity and even more mitochondria unbalance show the unphysiological implementation of ROS production . Note thaptation ,64,65. Captation .OHSU-974 FAcorr cells irradiated through 808-nm experienced oxidative stress, as pointed out by the increment of glutathione reductase activity. This enzyme plays a role in the pathway involved to maintain the redox balance of cancer cells under their pro-oxidative environmental conditions and provides survival support for tumors against chemotherapeutic drugs and radiotherapy ,68,69. H2). Therefore, regardless of the effect of laser light therapy on mitochondria activity , the OHSU-974 FAcorr cells seem not able to counteract the oxidative stress generated, despite the enhancement of GRx activity, probably because of a severe inhibition of catalase activity by the 808-nm PBM. Since, mitochondrial ROS modulation control pro- or anti-tumorigenic cell signaling , the effThe 808-nm laser treatment on OHSU-974 FAcorr also determines the alterations in the vitality rate. Our data, in fact, show that 24 h after the irradiation, the cell number decreased in the sample treated with 0.25 W and increases with 1 W treatment, following the modulation of the energy metabolism. However, 48 h and 72 h after the irradiation, a decrement in the cellular viability and an increment of apoptotic bodies were observed. These data are confirmed by the expression of two apoptosis markers, Bcl2 and Bax, anti- and pro-apoptotic proteins, respectively. As reported by the WB signals, the ratio between Bcl2 and Bax drastically decreased after 48 from the laser treatment, suggesting an imbalance towards the activation of apoptosis, which could be responsible for the cell number reduction. Moreover, the p53 pathway activation also appeared inhibited, indicating that the treated cells were less able to verify the DNA quality and to manage possible errors.2, observed that “PBM decreases the redox state of oral cancer by possibly increasing glycolysis and affects normal and tumor cells through distinct pathways”. Additionally, human cervical cancer cells (HeLa cell line) experienced an 808-nm PBM induced pro-apoptotic effect dependent on irradiation dose [2 and 3 J/cm2, than the highest, 30 J/cm2. Given the heterogeneity of the experimental setup, laser parameters, and results, a comparison between our data and literature has no consistent conclusions. However, our data seem to follow the preliminary results on oral squamous cell carcinoma (SCC) cell line of Gonçalves De Faria and collaborators , which tion dose . The mor2) [2 [2 [2) [2) [2) [2) [The 800-nm range of wavelengths showed also a significant inhibitory effect on the proliferation of SCC cells originated from the tongue   and SCC 2) [2  and 805-2) [2 [2 ). Conver2 [2 [2) , human o [2) [2) , SCC of In conclusion, our data met the primary and secondary endpoints. Indeed, despite the limitations of the in vitro experimental setup, our results consistently showed the harmful effect of 808-nm PBM on the OHSU-974 FAcorr. The ability of 808-nm wavelength laser light to interact with cell photo-acceptors localized into the mitochondria respiratory chain and the catalase enzyme negatively impacts carcinoma cell homeostasis. This action-reaction effect would affect the energetic metabolism equilibrium between the mitochondrial oxidative phosphorylation and glycolysis followed by lactic acid fermentation, as well as the antioxidant enzymes chain of cancer cells. Therefore, the OHSU-974 FAcorr cells would fail to counteract the increasing ROS production, which would stimulate the pro-apoptotic cellular pathways. Despite the promising results, pre-clinical and clinical studies are necessary to confirm the PBM safeness as supportive therapy for toxicities related to anti-cancer treatment and better investigate its possible anti-cancer coadjuvant therapy use.