POLR3B encoding the RNA polymerase III subunit B, there is no in vivo model of leukodystrophy based on mutation of this Pol III subunit.RNA polymerase III (Pol III)-related hypomyelinating leukodystrophy (POLR3-HLD), also known as 4H leukodystrophy, is a severe neurodegenerative disease characterized by the cardinal features of hypomyelination, hypodontia and hypogonadotropic hypogonadism. POLR3-HLD is caused by biallelic pathogenic variants in genes encoding Pol III subunits. While approximately half of all patients carry mutations in POLR3BΔ10 (Δ10) on Pol III in human cells and developed and characterized an inducible/conditional mouse model of leukodystrophy using the orthologous Δ10 mutation in mice. The molecular mechanism of Pol III dysfunction was determined in human cells by affinity purification-mass spectrometry and western blot. Postnatal induction with tamoxifen induced expression of the orthologous Δ10 hypomorph in triple transgenic fl-EYFP; Polr3bflPdgfrα-Cre/ERT; R26-Stop mice. CNS and non-CNS features were characterized using a variety of techniques including microCT, ex vivo MRI, immunofluorescence, immunohistochemistry, spectral confocal reflectance microscopy and western blot. Lineage tracing and time series analysis of oligodendrocyte subpopulation dynamics based on co-labelling with lineage-specific and/or proliferation markers were performed.Here, we determined the impact of Pdgfrα-dependent expression of the orthologous murine mutant protein resulted in recessive phenotypes including severe hypomyelination leading to ataxia, tremor, seizures and limited survival, as well as hypodontia and craniofacial abnormalities. Hypomyelination was confirmed and characterized using classic methods to quantify myelin components such as myelin basic protein and lipids, results which agreed with those produced using modern methods to quantify myelin based on the physical properties of myelin membranes. Lineage tracing uncovered the underlying mechanism for the hypomyelinating phenotype: defective oligodendrocyte precursor proliferation and differentiation resulted in a failure to produce an adequate number of mature oligodendrocytes during postnatal myelinogenesis.Proteomics suggested that Δ10 causes a Pol III assembly defect, while western blots demonstrated reduced POLR3BΔ10 expression in the cytoplasm and nucleus in human cells. In mice, postnatal Polr3bΔ10 mutation and developed an animal model that recapitulates features of POLR3-HLD caused by POLR3B mutations, shedding light on disease pathogenesis, and opening the door to the development of therapeutic interventions.In summary, we characterized the  et al. present the first leukodystrophy mouse model based on mutation of Polr3b, a gene encoding a subunit of RNA polymerase III (Pol III). The clinical manifestations of disease in the model, including hypomyelination, hypodontia and craniofacial abnormalities, resemble those seen in patients with leukodystrophy caused by POLR3B mutations.Michell-Robinson  POLR3A) and POLR3B, but also RNA polymerase I subunit C (POLR1C) and POLR3K.1-6 POLR3-HLD presents in previously healthy children in the first few years of life, leading to progressive disability and early death.7 The classic presentation includes hypomyelination, hypodontia and hypogonadotropic hypogonadism.7-10 While neurodegeneration is thought to be primarily driven by hypomyelination with secondary axonal loss, the disease pathogenesis is poorly understood.10-14 We hypothesize that Pol III hypofunction during a critical developmental period causes defective myelinogenesis and hypomyelination.15POLR3-related hypomyelinating leukodystrophy (POLR3-HLD) is one of the most common hypomyelinating leukodystrophies. It is caused by biallelic pathogenic variants in genes encoding subunits of the RNA polymerase III (Pol III) complex; most commonly RNA polymerase III subunit A  because of its predicted impact on Pol III biogenesis.24 The Δ10 mutant was discovered in a mutagenesis screen and initially described as the polr3b splice mutant SlimJim (Slj), which produces a gut phenotype in zebrafish.24 To further understand the mutation, a mouse with a floxed exon 10 allele was generated for studying the gut using the Villin-Cre system, but was not evaluated in other tissues.25 Δ10 has not specifically been detected in POLR3-HLD patients but POLR3B exon deletions have been reported,26 as have missense  and nonsense/frameshift mutations in exon 10.27-31 Furthermore, intronic variants that alter splicing leading to exon skipping in the POLR3B transcript have been reported.33 Taken together, there was sufficient literature supporting the notion that both Δ10 and disease-causing POLR3B mutants had pathophysiological similarities at the gene and protein level, which made Δ10 an interesting candidate for further study in the context of POLR3-HLD.Disease-causing mutations in Pol III subunits impact subunit expression/stability, Pol III biogenesis (complex assembly), nuclear import, and transcriptional activity.17 We then developed an inducible/conditional expression model in vivo, whereby the orthologous Δ10 mutation was induced in a temporal and Pdgfrα-dependent manner during early postnatal development in mice. Expressing Δ10 in this model led to recessive phenotypes, which were reminiscent of the cardinal manifestations of POLR3-HLD, including severe hypomyelination, hypodontia and craniofacial abnormalities. In-depth characterization of the CNS phenotype allowed us to answer fundamental questions about the development of severe hypomyelination in our model, such as whether the primary mechanism was reduced numbers of myelinating cells or reduced production of myelin by a normally sized pool of oligodendrocytes. We found that an early proliferation defect prevented the accumulation of adequate numbers of oligodendrocyte precursors, which was followed by a maturation defect in these precursors, suggesting that putative POLR3-HLD therapeutics will need to target oligodendrocyte precursors to correct hypomyelination.We set out to characterize Δ10 at the molecular level in human cells in order to understand its effects on Pol III. Our proteomics studies suggested that Δ10 expression caused defective Pol III complex assembly, which was accompanied by reduced expression of the mutant protein in the cytosol and nucleus of human cells, like other known POLR3-HLD mutations.Detailed materials and methods for all sections are provided in the 7) were transiently transfected for 24 h with 8.51 µg of plasmid containing human wild-type or mutant POLR3B in 15 cm plates using Jet Prime (PolyPlus) according to the manufacturer's recommended protocol. Cells were harvested, washed with 1× PBS, and snap frozen in liquid nitrogen. The FLAG affinity purification protocol was modified from Kean et al.34 to produce samples for LC-MS/MS. High performance liquid chromatography (HPLC) was performed in a 75 μm i.d. × 150 mm Self-Pack C18 column installed in the Easy-nLC II system (Proxeon Biosystems). HPLC was coupled to an Orbitrap Fusion mass spectrometer (Thermo Scientific) through a Nanospray Flex Ion Source . Label-free quantification (LFQ) intensity for each protein was obtained using MaxQuant (version 1.6.0.16) against the characterized human UniProtKB database (released on 3 June 2018).36 Log2 transformation, imputation and further statistical analysis were performed with Perseus (version 1.6.14.0).37 All purifications were done in triplicate and proteins detected in all experiments were kept for further analysis. Missing values were replaced by randomly generated intensities normally distributed with a width of 0.3 times and a downshift of 1.8 times the standard deviation (SD) of non-zero intensities. Significant differences between Log2 protein intensities from bait purifications and the control groups were then determined using a two-tailed t-test subsequently adjusted for multiple hypothesis testing with a permutation-based false discovery rate (FDR) of 0.05 and an s0 factor of 0.1 with 10 000 iterations. For all analyses, q-value ≤ 0.05 was considered statistically enriched.DNA plasmids were constructed and cloned in p3XFLAG-CMV14 expression plasmids (Sigma). All plasmid sequences were verified by sequencing at each construction step. HEK293T cells (1.6 × 10Pdgfrα-Cre/ERT (‘Pdgfrα-CreERT’),38flPolr3b (floxed exon 10)25 and fl-EYFPR26-stop (‘YFP’).39 Unless otherwise indicated, pups used in the study were generated from crosses between a +/flPdgfrα-CreERT; YFP; Polr3b male and a fl/flPolr3b female. Tamoxifen-treatment induced conversion of unrecombined flPolr3b wildtype-equivalent allele into an exon 10 deletion allele, i.e. ‘Δ10’. Therefore, fl/flPdgfrα-CreERT; YFP; Polr3b, +/flPdgfrα-CreERT; YFP; Polr3b, and fl/flYFP; Polr3b or +/flYFP; Polr3b (Cre-negative control) mice were referred to as Δ10/Δ10, +/Δ10 and CTRLs, respectively, when treated with tamoxifen. By convention, we have referred to mouse proteins in this article using title case  to differentiate them from human proteins  without referring to species explicitly (e.g. Mbp versus MBP).Animal husbandry and all studies described in the publication including animal euthanasia adhered to our animal care and use protocol, as approved by the McGill University Health Centre Animal Resource Division (AUP8055). Parental strains used in the study were Mice underwent transcardiac perfusion under isofluorane anaesthesia with PBS followed by a fixative for fresh frozen (FF) or formalin-fixed paraffin embedded (FFPE) block preparation. Tissues were harvested and sections prepared according to standard protocols for each method. Images of haematoxylin and eosin stained FFPE sections were acquired on a Leica Aperio AT Turbo digital whole slide scanning system at 20× resolution. Fluorescence images were acquired using a Zeiss Axioscan Z1 slide scanner employing a plan-apochromat 10× objective and AxioCam MR R3 camera.40 Activated proteins were transferred to PVDF support using the Bio-Rad Transblot Turbo, developed in Clarity Max enhanced chemiluminescence substrate, and imaged according to Bio-Rad manufacturer's protocols.Brain tissue was prepared by dissection with hindbrain  removed. Tissue lysates were prepared according to standard protocols in RIPA buffer with fresh protease inhibitors, normalized for protein concentration, and run on tris-glycine gels freshly prepared according to the Laemmli protocol with 0.5% trichloroethanol modification.2*-weighted image volumes were acquired using a 3D mGRE sequence with bipolar readout. Reconstructed 4D volumes were corrected for bipolar gradient induced mis-registration and Gibbs ringing prior to calculation of myelin water fraction (MWF) images.42 Whole-brain MWF maps were calculated by fitting the three-pool relaxation model of white matter using a recently developed self-labelled encoder decoder (SLED) network implemented in PyTorch.44 Region of interest (ROI) parcellation was achieved by computing affine and symmetric diffeomorphic transformations between the mGRE volumes and an anatomically labelled reference atlas.45Paraformaldehyde (PFA)-fixed brains of 16 juvenile mice (P18) were used in this study. All imaging was performed using the Bruker Pharmascan 7 T pre-clinical MRI system. Multi-echo gradient-recalled-echo (mGRE) T46-50 Images and CellProfiler pipelines were exported to Amazon Web Services and imaging pipelines were run using Distributed CellProfiler 2.0.0_4.1.3. The results were collated in SqLite databases and subsequently analysed in CellProfiler Analyst 3.0.4.Histological images were exported from Zeiss proprietary format to full depth 16-bit tiff images in FIJI (ImageJ). Binary image masks were manually delineated in FIJI and saved as tiff images. Image processing pipelines were developed in CellProfiler 4.2.1 using representative images.51 All scans , reconstruction and rendering settings were kept consistent between specimens.P18 mice were euthanized and stored in 95% ethanol until imaging. Mice were imaged at 15-μm resolution using a Skyscan 1275 micro-computed tomography (microCT) scanner (Bruker). Raw scan data were reconstructed using NRecon and 3D rendered for visual assessment and measurement in Drishti v3.0 as previously described.n, α, and other mathematical details pertaining to analysis and/or comparisons are detailed in the accompanying figure legends.Statistical analyses were performed with GraphPad Prism 9.4.1 or SPSS v28 0.1.1 (IBM). Biological replicates are presented as individual points in all graphs unless otherwise indicated. For brevity, statistical analyses (tests), 52 POLR3B exon 10 binds POLR3K and the jaw of POLR3A, suggesting that Δ10 would impair Pol III complex assembly or stability .tability . To demotability , suggesttability . Using atability , as summPOLR3B mutations resulting in Pol III complex assembly defects are known to be causal in POLR3-HLD.17 Therefore, we elected to study the Δ10 mutation in vivo in the context of oligodendrocyte precursor cell (OPC) development using an inducible/conditional Pdgfrα-CreERT system. To target Δ10 expression to the largest group of OPCs possible without toxicity, we validated a postnatal tamoxifen injection protocol in control mice. Dosing trials on wild-type animals .38By crossing the ntil P21 . This exPdgfrα-CreERT; YFP;Polr3b triple transgenic model to express Δ10 and YFP in Pdgfrα+ cells. Genotyping PCRs and validation sequencing of the founder strains yielded the expected results , whereas +/Δ10 (n = 72) and CTRL (n = 116) mice had adequate and comparable survival . On macroscopic inspection, Δ10/Δ10 brains had less visible ventral myelin, but overall tissue morphology was normal based on haematoxylin and eosin staining . Howeverex vivo MRI techniques on PFA-fixed brains from P18 mice revealed visible differences in MWF maps indicative of significant hypomyelination in Δ10/Δ10s, in addition to a substantial effect of tamoxifen on myelination overall   microscopy , a labelue (LFB) . At P18,ue (LFB) , cerebelue (LFB)  and braiue (LFB)  of Δ10/Δ55 Our assays included Polr3a, Polr3b, Pdgfrα, Ng2, Olig2 and Mbp. Among these, Ng2 was on trend for increased expression in Δ10/Δ10s, while Olig2 and Mbp were negatively correlated with Δ10 allele dose  that might explain the observed hypomyelination, we conducted YFP+ lineage tracing on histological sections using antibodies against lineage marker antigens. We quantified cells based on the expression of markers of OPCs (Pdgfrα+ and/or Ng2+) or mature oligodendrocytes . We investigated hindbrain, midbrain and forebrain structures at P9, P14 and P18 . At P9, Cell counting indicated steadily reduced numbers of OPCs and OLs over the P9–P18 window in Δ10/Δ10s . We anal56–58 EdU injections at P9 using a 6-hour exposure protocol to assess the percentage of dividing cells demonstrated decreased EdU+ labelling in Δ10/Δ10s compared to +/Δ10s, as well as a decrease in the ratio of YFP+EdU+ to total YFP+ cell population. These studies indicated that the loss of proliferative potential occurred both in terms of the absolute number of dividing cells and the relative proportion of dividing cells among the YFP+ population  interactions, proposing that the mutation affected Pol III facilitated recycling activity by reducing re-initiation, but did not publish experimental confirmation of the mechanism. Our in silico modelling of Δ10 on the human Pol III structure also indicated the likely interaction of POLR3B exon 10 with POLR3K in the jaw region of the human polymerase. Our proteomics data suggested that POLR3BΔ10 caused a severe Pol III complex assembly defect, as well as reduced expression of the mutant protein in the cytoplasm and nucleus of human cells.Pol III assembly and nuclear import is a complex process, which is known to be perturbed in POLR3-HLD, including in association with mutations in 60 testing for nuclear expression indirectly supported that Δ10 does not cause total failure of Pol III complex assembly, consistent with its prior characterization as a hypomorph.25 Taken together, we confirmed that Δ10 perturbs the structural integrity of Pol III.Based on our proteomics data, we concluded that the main mechanism of Pol III hypofunction resulting from Δ10 expression is a likely Pol III complex assembly defect, although we could not categorically rule out complex instability. Indeed, we directly demonstrated that Δ10 has reduced association with Pol III subunits as compared to wild-type protein by AP-MS. However, we also observed that mutant protein was less highly expressed than wild-type at the cytoplasmic and nuclear level when expressed transiently under identical conditions in HEK293T cells. Because Pol III is assembled in the cytoplasm and then imported into the nucleus,in vivo. Moreover, we did not detect multiple splice products in transgenic mice, suggesting that the mutant exon 9–11 junction is a stable product, and that nonsense-mediated decay is unlikely. Finally, in silico analysis does not suggest that free (unassembled) human Δ10 has reduced half-life compared to its wild-type protein counterpart.61 In summary, evidence of transcript instability was not observed in vivo using a highly similar orthologous mutant, while the free mutant protein is not expected to have reduced half-life. We therefore propose that reduced expression of the mutant detected in our human in vitro assays is due to turnover of subunits that fail to be assembled into functional enzymes. In summary, we have elucidated that Δ10 impairs Pol III complex assembly or stability, thereby disrupting its physiological functions upstream of transcription. On the basis of these findings, we elected to study Δ10 in the context of oligodendrocyte development and myelinogenesis.Based on several of our findings, we hypothesize that reduced expression of Δ10 is related to a process downstream of Pol III complex assembly. Although transcript instability is possible, because we detected similar expression of the orthologous Δ10 and wild-type mRNA up to 30 days after induction with tamoxifen when validating brain recombination in mouse studies, it is unlikely to be a significant factor POLR3B variant that causes POLR3-HLD and impairs Pol III assembly or stability.17 Indeed, recent attempts to recapitulate POLR3-HLD in vivo have largely been unsuccessful with the exception of a recent study by Merheb et al.,18 where a constitutively lethal W671R/G672E double mutation in Polr3a was used in the context of a Cre/Lox strategy to elicit a myelin phenotype. The orthologous mutation used in Merheb et al.18 has ∼30% of the transcriptional activity of WT Pol III in Saccharomyces cerevisiae, but complex assembly was not assayed directly. Complex assembly is not known to be significantly affected in the single G672E mutant,16 and the orthologous Y685K/G686E yeast mutant described in Moir et al.19 is not expected to affect subunit interactions. Taken together, there are now two mechanistic models of POLR3-HLD: a milder neurological phenotype based on a Polr3a double mutant that causes reduced Pol III transcriptional output,18 and our more severe model based on a Polr3b mutation that perturbs Pol III structural integrity.The mutation employed in our study explores an alternative molecular mechanism in line with prior characterization of disease-causing variants including a Pdgfrα-CreERT-driven approach because it allowed us to study postnatal development, a critical period of myelinogenesis in the brain. Using an inducible/conditional approach to study myelination has several advantages, the most significant being that lethality is avoidable and the severity of the phenotype can be titrated. Because we elected to study the early oligodendrocyte lineage and OPCs are generated throughout development, we were faced with the reality that we would not be able to induce complete tissue recombination.63 Because of this, we elected to use a triple transgenic strategy in order to better understand the contributions of recombined and non-recombined cells to the overall CNS pathology. Using this method, we observed significant hypomyelination associated with YFP+ cell dysfunction, namely proliferation and maturation defects, despite the presence of YFP-, non-recombined cells. The reporter used in the study is known to be highly specific to recombined cells but is less sensitive and may underestimate the true recombination rate.64 This is consistent with our finding that CC1+ cell density decreased in Δ10/Δ10 mice to a slightly greater degree than would be explained by the decrease in YFP+CC1+ cells alone , or differences in tamoxifen pharmacodynamics in the context of myelinogenesis versus remyelination. Our pilot studies that identified an effect of tamoxifen on myelination led us to design our experiments with equal tamoxifen exposure in all comparator groups.Recombination rates were not amenable to augmentation by administering tamoxifen earlier, or for longer periods. In early pilot studies we found that prenatal tamoxifen administration caused difficulties during parturition in line with prior studies. in mice .66,67 ThPdgfrα expression in non-CNS tissues. Indeed, although Pdgfrα is a high-fidelity marker of the early oligodendrocyte lineage in the mouse CNS, it is widely expressed along the developmental continuum in various neural crest derivatives throughout the body including the developing craniofacial bones and teeth.68-70 We observed an absence of incisors in approximately half of Δ10/Δ10s and small incisors among the rest. Given that many Δ10/Δ10s did not have eruption prior to death, we assume that eruption is not merely delayed but rather that incisors are significantly underdeveloped, most likely due to an underlying hypoplasia. The manifestation of hypomyelination, hypodontia and craniofacial abnormalities is interesting and suggests that further study of the functional similarities between neural crest-derived mesenchymal derivatives and oligodendrocytes may yield greater understanding of the mechanistic link between the disparate phenotypes observed in POLR3-HLD.Emergence of non-CNS anomalies such as hypodontia and craniofacial abnormalities are expected to be caused by Polr3a model, because Merheb et al.18 limited their study to comparisons with wild-type mice. We found that +/Δ10 and CTRL mice have minor differences in oligodendrocyte physiology based on tissue-level measurements. Their similarity in terms of the observed myelination outcomes emphasizes that not all cell-level changes will cause defective myelination outcomes in vivo due to reserve capacity in the myelinating population. Moreover, this highlights the importance of in vivo studies for evaluating whether a particular mutation will produce hypomyelination.Altogether, we conducted an array of studies to confirm and determine the extent of hypomyelination in our transgenic mice. Several histological and biochemical measurements we performed suggested that +/Δ10s were not physiologically equivalent to CTRLs. POLR3-HLD is recessive, and heterozygotes/carriers are not known to have hypomyelination or associated clinical deficits. We attributed differences in results between CTRLs and +/Δ10s to the increased sensitivity afforded by studying the Δ10 mutation at different allelic doses on a genetically identical background. Moreover, the histological and biochemical studies performed here are also incredibly sensitive to changes in myelination relative to clinical MRI used in patient studies. Importantly, we did not observe behavioural or other obvious phenotypic differences between +/Δ10s and CTRLs. Therefore, we determined that characterizing the phenotype in Δ10/Δ10s by comparing to +/Δ10s was most apt, given that the only genetic difference between the two groups is the additional Δ10 allele. Unfortunately, we could not compare our findings in +/Δ10 mice to the prior published Ex vivo MRI and other histological studies  focusing on quantification of myelin lipids agreed with the immunofluorescence results. Depending on the modality of inquiry and brain region, we detected anywhere from ∼50% to >80% loss of myelin at P18.Studies of myelination confirmed severe hypomyelination in Δ10/Δ10s. Notably, marked decreases in Mbp immunofluorescence were identified in sagittal and hemi-coronal sections . Hemi-coin vivo western blots due to using samples in which mutant cells make up a small percentage of the dissected tissue. Ex vivo MRI and histological studies agreed with our studies of OLC protein expression, however our western blot data suggested a more severe decrease in Mbp as compared to the imaging studies focusing on regions of interest that are heavily myelinated. While it is expected to see more severe decreases on western blot since aggregate Mbp across both grey and white matter regions is measured, it is possible that myelin components may be lost to a more severe degree than the cells themselves due to dysfunction among remaining OLCs. In a recent study published by our lab, we observed that Polr3b knockdown was associated with reduced expression of markers of myelination in differentiating oligodendrocytes, including substantially reduced Mbp transcription.71 Because we also observed evidence of differentiation defects, it is likely that remaining CC1+ cells are dysfunctional, explaining the discrepancy in the scale of decreases in Mbp expression and loss of CC1+ cells. Taken together, the model recapitulated severe hypomyelination and produced effect sizes that will be useful for testing therapeutic interventions in the future.Histological results were validated by western blot using midbrain/forebrain tissue lysates at the peak of myelination (P21). Midbrain and forebrain Mbp expression was reduced by >97%, but the exact percentage cannot be ascertained due to a floor effect. Our panel of immunoblotting antibodies also revealed reduced Olig2 expression, which likely reflects reduced numbers of OLCs. Among the other targets tested, none suggested significant changes at the tissue level. As expected, we did not detect significantly reduced Polr3b expression in our 56-58 Indeed, we observed that the Δ10/Δ10 OPC population did not undergo a rapid increase in size from P9 to P14, and mature OLCs did not accumulate in the brain from P9 onward. Direct testing of proliferation with EdU labelling confirmed this hypothesis and demonstrated that a significant proliferation defect was the most upstream factor leading to hypomyelination in our model. Because we did not detect activated Caspase 3 at P9 (or afterward), we concluded that the abrogated proliferation and maturation of the OLC lineage in the early stages produced inadequate numbers of myelinating cells and was directly responsible for the observed hypomyelination.Using lineage tracing methods, we tracked the fate of subpopulations of oligodendrocytes to ascertain the mechanism of hypomyelination. From the outset, we were specifically interested in the question of whether hypomyelination in POLR3-HLD is caused by a failure to obtain adequate numbers of myelinating cells or defective cell function leading to improper or reduced production of myelin and/or myelin components such as Mbp. Our finding that YFP+ cell numbers were already reduced by 50% relative to heterozygotes at P9 suggested a proliferation defect that preceded our observation window, consistent with maximal CNS Pdgfrα+ cell proliferation from late gestation through the first postnatal week.et al.18 in that we developed it based on mutation of Polr3b, and moreover using a mutation that causes defective Pol III biogenesis. Indeed, our model is more severe and uniquely produces CNS-dependent phenotypes common among hypo/dysmyelination mouse models, including ataxia, tremor, seizures and early death.72-76 We expect this to be the case because Pol III complex assembly is upstream from transcription, and disruption of upstream processes typically lead to more wide-ranging effects on cell physiology. Moreover, we detected and quantified a specific defect in proliferation, which occurred at a critical developmental time point, immediately prior to the peak of myelination. This is likely to play a major role in the development of hypomyelination in our model and is also expected to be an underlying factor in the difference in severity between our observations and those of Merheb et al.18Our model differs from that described by Merheb et al.18 Therefore, two animal models making use of mutations in distinct genes, causing Pol III dysfunction by different mechanisms, suggest that reduced myelinogenesis is related to reduced numbers of myelinating cells. Although we cannot yet say with certainty that this is true in POLR3-HLD, in the only existing study quantifying oligodendrocyte-specific immunohistochemistry in a patient, OLIG2+ cells were reduced in number by 70–99% when compared to a control, depending on the brain region.11 Therefore, our current understanding of POLR3-HLD pathophysiology indicates that putative interventions should be targeted to early stages of the oligodendrocyte lineage in order to produce therapeutic benefit, in line with recently published hypotheses by Perrier and colleagues.77We detected steadily decreasing numbers of YFP+ cells in homozygous mice across all endpoints, which decreased in number throughout the observation window. The dwindling numbers of OLCs demonstrated in our model were also suggested by the findings of Merheb POLR3BΔ10 mutation in human cells and developed an animal model of POLR3-HLD using the orthologous mutation in mice. Our proteomics studies demonstrated that Δ10 impacts Pol III structural integrity, likely by impairing assembly of the complex. Postnatal expression of the Δ10 mutant early in the oligodendrocyte lineage using an inducible/conditional strategy in mice recapitulated recessive disease features such as hypomyelination, hypodontia and craniofacial abnormalities. In the CNS, homozygous Δ10 expression was associated with severe hypomyelination, which was caused by significant proliferation and maturation defects in OPCs. Taken together, we produced a unique and novel model of POLR3-HLD: the first of its kind to be based on Polr3b mutation and to recapitulate patients’ neurological and craniofacial/dental abnormalities, opening the door to further study disease mechanisms and test potential therapeutics.In this study, we characterized the awad249_Supplementary_DataClick here for additional data file.


Cylindrical specimens were prepared from a rolled plate that had a (0001) basal texture and was compressed parallel to the c-axis of the grains. Consequently, work softening and fiber texture formation were observed in all the specimens. During the deformation, the development of grain boundaries, which is a typical characteristic of continuous dynamic recrystallization (CDRX), was observed, and the (0001) texture was highly developed with increasing Al content. Although each alloy was associated with the same deformation conditions and mechanisms, the AZ31 alloy exhibited a non-basal texture component. The stacking fault energy contributed to the generation of slip systems and gliding, and it was seen as the main reason for texture variation.Magnesium and its alloys have been restricted in their industrial applications due to problems related to their formability. To overcome this issue, controlling the crystallographic texture is important, and the texture formation mechanism should be investigated in relation to factors including deformation conditions and solute atoms. In particular, the effects of solute atoms on the texture formation behavior should be further analyzed because they can considerably affect the deformation behavior. Thus, in this study, to clarify the effect of aluminum concentration on the texture formation behavior and microstructure, high-temperature uniaxial compression tests were conducted on three types of AZ-series magnesium alloys . Compression was conducted at 673 K and 723 K, with strain rates of 0.05 s Owing to their excellent properties, such as low density and high specific strength, magnesium and its alloys are potential alternatives to conventional structural materials. However, they have low formability at room temperature due to the lack of active slip systems associated with the hexagonal close-packed crystal structure ,2,3. To Fukutomi et al. investigated the texture formation behavior of Al–Mg system alloys that have an FCC structure and different magnesium concentrations during high-temperature uniaxial compression. From the texture measurements, including X-ray diffraction (XRD) and electron backscattered diffraction (EBSD) results ,8,9, theMoreover, one of authors has investigated the effect of Ca precipitation on the texture development of AZ magnesium alloys.  Through Although there are many studies about the affected properties improved by the Al content ,16 or thTherefore, in this study, to clarify the effect of Al concentration on the texture formation behavior, AZ-series magnesium alloys with different Al concentrations  were investigated by conducting high-temperature uniaxial compression tests.Commercial cast AZ-series magnesium alloys with different Al concentrations were used as the materials. They were obtained from the Suzhou Chuanmao Metal Material.co. The specimens were cut from the cast ingots in to 10 mm (width) × 10 mm (length) × 4 mm (thickness) sizes, and their chemical compositions were investigated using X-ray fluorescence ((XRF) XRF-1800, SHIMADZU, Tokyo, Japan). Rectangular plates with the dimensions of 60 mm (width) × 60 mm (length) × 40 mm (thickness) were machined from ingots. These plates were hot rolled at a temperature of 673 K, with a rolling reduction of approximately 30%. For uniaxial compression tests, cylindrical specimens with a diameter of 12 mm and a height of 18 mm were cut from the rolled plates using a precision low-rate cutter  and a milling machine . The compression direction of the specimen was set as the normal direction (ND) of the rolled plate, while the elongation directions were parallel to the transverse (TD) and rolling (RD) directions. All the specimens were annealed at 723 K for 3600 s to homogenize the microstructure and for stress relaxation. Thereafter, they were oil quenched to prevent the post-annealing effect on the microstructure.−2 s−1 and 5.0 × 10−3 s−1, up to a true strain of –1.0. To prevent microstructural changes after high-temperature deformation, the specimens were immediately quenched in oil after deformation.Uniaxial compression tests were performed at two different temperatures, namely, 673 K and 723 K, under strain rates of 5.0 × 102 by controlling the voltage at 243 K, in a solution containing 3% perchloric acid and high-purity ethanol. The microstructure was observed using a field-emission scanning electron microscope  and EBSD . The texture was measured using XRD . The five imperfect pole figures of {0001}, {10To analyze the microstructure and texture, pre-treatments were conducted. The deformed specimens were cut parallel to the compression direction, grinded with sand paper, and polished with OP-S silica colloids with 1 µm Si–C particles. Electropolishing was conducted under a current density of 0.20 A/cme method . Thereaf−2 s−1, up to a strain of −1.0. The true strain in the figure is given in terms of the absolute value. All the true stress–strain curves show maximum stresses in the initial stage of deformation that decrease thereafter. Namely, the curves characteristic of work softening can be seen. Furthermore, this phenomenon was observed under all the conditions tested in this study, though there are some differences in the magnitude. There was a difference in the peak stresses of AZ31 and AZ61 in the initial stages; nevertheless, the flow stresses were largely similar during the deformation process. On the contrary, the stress of AZ91 was more dynamic. AZ91 exhibited the most significant work softening at low temperatures and the lowest flow stress at high temperatures.−2 s−1, up to a strain of −1.0. The microstructures are expressed in the same method that of −2 s−1, up to a true strain of −1.0. The pole densities are projected using the form shown in −2 s−1 with the increase in the strain. The (0001) pole densities gradually increased with an increase in the true strain of the three types of magnesium alloy. Because the intensity of the AZ91 alloy increased slightly, it was lower than those of the others during the middle stage of the deformation. However, the intensity of the AZ91 alloy was higher than those of the AZ61 and AZ31 alloys after the final deformation. Therefore, it was implied that the Al content affected the development of the (0001) pole density, and its increment resulted in a higher intensity of the (0001) poles.From the results shown in According to the von Mises theory, magnesium alloys require the activation of at least five or more independent slip systems to accompany the deformation . HoweverThe strain rate and temperature, as deformation parameters, also seemed to contribute to the deformation behavior. In the case of the AZ80 alloy, these parameters affected the flow stress during compression . In part17Al12 precipitation mainly occurs in the Mg–Al binary phase, and it contributes to the strength of magnesium alloys. However, according to its phase diagram, the Mg17Al12 phase in the AZ91 alloy is decomposed at a temperature of 643 K, and its decomposition temperature decreases as the composition reaches that of pure Mg [17Al12 have been already investigated in our previous study [17Al12 phase in the cast, ingot was observed by transmission electron microanalysis (TEM). They were discomposed or precipitated around 688 K (415 °C). Similarly, AZX611 alloy showed two kinds of different precipitation, which are Mg17Al12 and Al2Ca. Because the Ca component was not soluted into the matrix, the matrix of AZX611 alloy showed a more lowered Al content than the AZ61 alloy. However, even in AZX611, the peak temperature of the Mg17Al12 phase occurred in same point. Therefore, only the effect of the Al content was considered as the main reason for the variation in the texture intensity. Because the Al content soluted into the matrix at temperatures of 673 K and 723 K, they had to be conjectured as solute atoms, which could affect the lattice distortion on stacking fault energy and axis ratio. Although Zinc can still affect the formation of different phases, it is difficult to conceive that it significantly affected the overall deformation behavior under low concentrations compared to Al.Mg pure Mg . Moreoveus study ,26. In pTherefore, in this study, the gliding of the pyramidal II <c+a> slip systems and the DRX process were considered the major deformation modes, and the Al content could affect the deformation behavior.The following observations can be made from the results of the high-temperature uniaxial compression tests conducted on AZ-series magnesium alloys: (1) The major deformation mechanism is the glide of pyramidal II <c+a> and the occurrence of DRX; (2) Increasing Al concentration contributed to the strengthening of the basal texture; (3) A tilted texture from the basal pole in AZ31 may be developed due to the other reasons, rather than the variation in deformation mechanism. Based on these results, the mechanism of basal texture formation is discussed.−2 s−1 up to strains of (a) −0.4, (b) −0.7, and (c) −1.0. The red color grains are within 15° from the (0001)-oriented grain, according to the standard triangle. Moreover, the misorientation angle, which ranges from 15° to 180°, is indicated by a black line: high-angle grain boundary (HAGB). The fraction of the (0001)-oriented grain gradually increases when the true strain increases up to −1.0, while the average grain size decreases gradually.Fukutomi et al. suggested that the texture orientation in the Al–Mg alloy formed after deformation is associated with a low Taylor factor . The TayAs mentioned in From the discussion in Many researchers tried to investigate the stacking fault energy of magnesium alloys by calculations or experiments ,29,30. IPure magnesium and aluminum are known as high-SFE alloys . A high In this study, the magnesium alloy containing a higher Al content showed a greater increase in the (0001) intensities, whereas AZ31 showed the development of a tilted texture from the basal axis. These results well support the assumption regarding the relationship between the SFE and the Al content in the Mg alloy. Consequently, the Al content in the AZ-series magnesium alloys contributed to lower SFE and made the perfect slip predominant, inhibiting cross slip. This effect made the magnesium alloy have a stronger (0001) texture intensity.These results showed that the basal texture will be formed or developed when the (0001) orientation had the lowest Taylor factor during the deformation. In addition, this orientation was set in the most of cases. Considering that the value of Taylor factor was determined by the combination of slip systems contributing to the deformation, non-basal texture orientations could be developed by the variation in the combination. In this viewpoint, additive elements to magnesium alloy, which could enhance the SFE, seem to be the key to improve formability. However, further studies about this assumption are required.Continuous types of dynamic recrystallization were observed in all deformation conditions. The microstructures varied through the formation of LAGBs and the development of HAGBs.A fiber texture was formed by recrystallization. The grains which would be grown up or consumed were determined according to the orientation they had before the deformation.The (0001) orientation had the lowest Taylor factor than the other orientations because the slip systems contributing to deformation on this orientation were the combination of the pyramidal <c+a> slip systems. This made the basal texture to be strengthened.The Al concentration in the magnesium alloy contributed to a decrease in the stacking fault energy and inhibited cross slip, leading to the strengthening of the basal texture. On the contrary, the variation in SFE, which can affect the Taylor factor, has possibilities to inhibit the development of basal textures in magnesium alloys.To clarify the effect of aluminum concentration on texture formation behavior and microstructure, three types of AZ-series magnesium alloys with different aluminum concentrations  were experimentally studied by conducting high-temperature uniaxial compression tests. The main results of the study are as follows:


TheNAD+-I (class I) riboswitch stands out because it comprisestwo homologous, tandemly arranged domains. However, previous studiesexamined the isolated domains rather than the full-length riboswitch.Crystallography and ligand binding analyses led to the hypothesisthat each domain senses NAD+ but with disparate equilibriumbinding constants (KD) of 127 μM(domain I) and 3.4 mM (domain II). Here, we analyzed individual domainsand the full-length riboswitch by isothermal titration calorimetryto quantify the cofactor affinity and specificity. Domain I sensesNAD+ with a KD of 24.6 ±8.4 μM but with a reduced ligand-to-receptor stoichiometry,consistent with nonproductive domain self-association observed bygel-filtration chromatography; domain II revealed no detectable binding.By contrast, the full-length riboswitch binds a single NAD+ with a KD of 31.5 ± 1.5 μM;dinucleotides NADH and AP2-ribavirin also bind with one-to-onestoichiometry. Unexpectedly, the full-length riboswitch also bindsa single ATP equivalent (KD = 11.0 ±3.5 μM). The affinity trend of the full-length riboswitch isADP = ATP > NAD+ = AP2-ribavirin > NADH.Althoughour results support riboswitch sensing of a single NAD+ at concentrations significantly below the intracellular levels ofthis cofactor, our findings do not support the level of specificityexpected for a riboswitch that exclusively senses NAD+.Gene regulatory implications and future challenges are discussed.Bacterial riboswitchesare structured RNAs that bind small metabolitesto control downstream gene expression. Two riboswitch classes havebeen reported to sense nicotinamide adenine dinucleotide (NAD The latter enzyme is conservedin all prokaryotes,10 where it convertsiminoaspartate and dihydroxyacetone phosphate into quinolinic acid,which is an essential step in de novo NAD+ production  yielding a gene-off state.22 Thelatter gene regulatory model, however, established a dichotomy inthe field. Specifically, the need to distinguish between one-NAD+-per-riboswitch9 versus two-NAD+-per-riboswitch binding modes.22 A second prediction is that the full-length riboswitch should discriminateagainst the γ-phosphate of ATP but favor NAD+ recognitionbecause these nucleotides are present at concentrations of 0.12–9.6mM and 1.6–2.6 mM in growing bacteria.24To elucidatethe mode of NADe moiety 1d.11,22 nadA motif with the goal of quantifying cofactorbinding stoichiometry, affinity, and specificity for the full-lengthNAD+-I riboswitch, which has not been studied appreciablyin previous investigations.22 Accordingly, we measured riboswitch interactions with NAD+, NADH, ADP, ATP, and the dinucleotide analogue AP2-ribavirin.This work was conducted using individual domain I and II constructsas well as the full-length riboswitch  template sequence was used: 5′-TAATACGACTCACTATAGGCTGCAACATCCCCGTGGGTTGGACGGCGAAAGCCGTAGCGAATCCACTGCAGCCTGTATGGCTTTCAACATCCCCGTCCGCGAAAGCGGACTGAAGGC, where italics indicate the T7 transcriptionstart site and tetraloops are underlined. The sequence GGC was added in P1 to promote a strong transcription start,27 and tandem 2′-O-methylgroups were used at the 3′-end to reduce untemplated transcription.28 The lyophilized desalted template strand wasdissolved in water at a concentration of 100 μM. A 17-mer PAGE-purifiedT7 DNA primer27 at 100 μM was annealedto the template in 1 mL to give 2 μM primer, 2 μM template,10 μM MgCl2, and 0.20 M Na-HEPES at pH 7.0. The reactiontube was mixed gently by inversion, followed by heating at 95 °Cfor 3 min prior to slow cooling to room temperature. A 10 mL transcriptionwas performed in a reaction comprising 0.075 M Na-HEPES at pH 7.5,0.067 M DTT, 0.03 M MgCl2, 21% (w/v) PEG 3350, 0.002 Mspermidine-HCl, 0.0063 M ATP, 0.0063 M UTP, 0.0063 M CTP, 0.008 MGTP, 1 mL of annealed template, 0.26 mg mL–1 BSA,and 0.027 mg mL–1 T7 polymerase, as described.25 The reaction tube was inverted gently againto achieve mixing and incubated for 5 h at 37 °C. The reactionwas spun at 10 000 RCF for 10 min at room temperature. Thesupernatant was ethanol-precipitated and stored at −20 °C.The RNA was purified by batch chromatography using Toyoperl DEAE-650M resin  followed by separation using denaturing6% PAGE. RNA bands were visualized by UV with exposure precautions,29 excised, and eluted.25 A second DEAE-650 M column was used to remove contaminants followedby desalting on a PD-10 column . Purity was assessedby analytical PAGE stained by SYBR Gold (Thermo-Fisher) and visualizedon a Gel Doc XR+ instrument (BioRad). The estimated purity was >99%.The yield per 10 mL of synthesis was 3 mg of RNA based on spectrophotometryat 260 nm. The RNA was stored as a lyophilized powder at −20°C.A DNA template was designed as describedsequence 1b. The nAca sequence; the constructcomprises a 28-mer strand with the sequence 5′-GGGCGCAACAUCCCCGCCGGUUGGGCGCand a 22-mer with the sequence 5′- GCGCAGUGAACCGGCUGCGUCC.The sequence of P1 was modified in a nonconserved segment to promoteduplex formation. A single-strand domain II 50-mer hairpin was synthesizedsimilarly and had the sequence 5′-GCUUUCAACAUCCCCGUCCCGGGCGGCCUUGACCGCAGUGGACUGAAGGC. Each RNA strand was stored as a lyophilizedpowder at −20 °C.Each class I domain was generated by chemical synthesis . We designed two constructs for domainI. A single-stranded domain I construct was synthesized as a 54-mer.The RNA sequence was 5′-GGCGGCAACAUCCCCGCCGGUUGGGCGCGAAAGCGCAGUGAACCGGCUGCAGCC.A 50-mer split domain I construct was also produced from two RNA strandscomprising the wildtype CUGAAGGC1b. A spl31 For the bimolecular structures, we used two folding algorithms.The Duplex fold option was chosen because it predicts the lowest energystructure for two interacting sequences while not allowing intramolecularWC base pairing. The Bifold algorithm predicts the lowest energy structurefor two interacting sequences, allowing for intramolecular WC basepairing. In each case, the default options were selected for folding.The secondary structures are depicted in Figure S1. The propensity of individual strands to adopt hairpinswas comparatively weak relative to the bimolecular split constructsthat form from the 28-mer and 22-mer strands that compose domain Ior the 28-mer and 20-mer strands that compose domain II. The correspondingfree energies are provided in the legend of Figure S1.The free energyassociated with domain I and domain II folding as monomers and intermoleculardimers was evaluated using the “predict secondary structure”and “bimolecular” options of RNAstructure (v6.0.1).m/z value of426.01 . The spectrum indicatedseveral non-nucleotide, singly charged negative ion contaminants.32 NAD+ (MedChem Express LLC) was analyzedin positive ion mode.32 The exact massof the protonated species is 664.12 Da, in agreement with the observedvalue at an m/z value of 664.11. The exact mass of NADH (MedChemExpress LLC) is 665.13 Da for the negatively charged species. Thesample was analyzed in negative ion mode. The observed major specieswas doubly charged at an m/z valueof 663/2 = 331.5 . The exactmass of AP2-ribavirin (Jena Bioscience) is 653.10 Da forthe negatively charged species. The sample was analyzed in negativeion mode. The observed major species was doubly charged at an m/z value of 651/2 = 325.5 . The ATP sample (MedChem ExpressLLC) was analyzed in positive ion mode. The exact mass is 507.00 forthe neutral species, in agreement with the major species observedat an m/z of 508.00 .To confirm thepresence of each ligandfrom commercial sources, each effector was subjected to mass spectrometryusing a TSQ Quantis triple quadrupole system (Thermo Scientific) atthe SUNY Upstate Medical Center  or a Q Exactive PlusHybrid Quadrupole-Orbitrap instrument (Thermo Scientific) at the Universityof Rochester . Exact masses were from the manufacturers.The ADP sample  was analyzed in negative ionmode. The exact mass is 427.03 Da for the negative ion. The specieswas observed at an Aca domainI construct were each dissolved in 75 μL of 0.01 M Na-cacodylateat pH 7.0. The concentration of each dissolved domain I or domainII sample was determined spectrophotometrically at 260 nm based onthe extinction coefficients supplied by Horizon Discovery . An ε260 of 1129.4 mM–1 cm–1 was used for the full-length 100-mer based on the OligoSpec Calculator, whichcorrects the ε260 for hypochromicity. For the splitconstructs of domains I and II, the more concentrated strand was dilutedusing 0.01 M Na-cacodylate at pH 7.0 to match the concentration ofthe more dilute strand. A volume of 75 μL of each strand wascombined to produce an equimolar solution that was heated to 65 °Cfor 3 min. Lyophilized strands of the Aca domainII and the full-length Aai NAD+-I riboswitchwere dissolved similarly in 75 μL of 0.01 M Na-cacodylate atpH 7.0 and heated to 65 °C for 3 min. An equal volume of preheatedfolding buffer  was added slowly to the heated RNA, and the combinedsolution was heated for an additional 3 min at 65 °C followedby slow cooling to 24 °C. Each equilibrated sample was subjectedto overnight dialysis at 4 °C against 2 L of ITC buffer comprising0.10 M Na-HEPES at pH 7.0, 0.10 M NaCl, and 0.02 M MgCl2 using a 3500 MWCO Slide-A-Lyzer Cassette G2 (Thermo-Fisher Scientific).Each effector was weighed on an analytical balance and dissolved inthe used ITC buffer. The final concentration was verified spectrophotometricallyby using known ligand extinction coefficients at 260 nm. The ligandin the syringe was kept in a concentration range of 0.47–1.05mM. The domain I sample in the cell was kept in a concentration rangeof 0.030–0.060 mM. For the Aca domain II and Aai full-length samples, the ligand in the syringe was keptin a concentration range of 0.40–0.63 mM and the RNA in thesample cell was kept at a concentration of 0.025–0.040 mM.All thermograms were analyzed with MicroCal PEAQ-ITC analysis software using a 1:1 binding model. This model was chosenbecause it produced the best thermogram fits in terms of the visualmatch of the data to the isotherm and the χ2 valuesindicating goodness of fit, as described.33 Binding simulations were performed using MicroCal PEAQITC analysissoftware or SEDPHAT34 . Values of ΔH were obtaineddirectly from thermograms. A fit of each thermogram through the inflectionpoint yielded the apparent equilibrium association constant, KA (where KA = KD–1). Using the equation ΔG = ΔH – TΔS, the Gibbs free energy was calculated alongwith the entropy term, −TΔS, where T is the temperature.35 The sample concentration and n value  were allowed to vary during the final fittingprocess. Given the restraints available from the known concentrationof ligand and receptor, the use of the full thermogram range for fitting,an adequate signal-to-noise ratio from heats of titration, and theapplication of specific binding models, the thermograms could be reliablyfit despite low c values36 that were in a range from 0.6–3.1. Duplicate or greater replicatesof each ITC run were performed for each effector. Titrations of effectorinto buffer were performed as controls for heats of dilution . These control titrations were subtractedas the mean or point-by-point corrections from the heats of injectionfor each thermogram, although this did not yield appreciably differentbackground corrections compared with the fitted offset correctionscalculated by MicroCal PEAQITC analysis software, which was the methodchosen for analysis herein. Representative thermograms are providedin Titrations were conductedusing a PEAQ-ITC instrument  with samplesheld at 10 °C and an injection spacing of 180 s. The technicalinjection was 0.4 μL, and the ensuing injections were 2 μLeach. Lyophilized strands of the split 37 the Kpn preQ1-IIII riboswitch46-mer (MW 14017 Da), the Lrh preQ1-IIriboswitch 77-mer (MW 23442 Da),38 andthe dimeric Fpr preQ1-III riboswitch 101-mer(MW 61693.0 Da).39 Each NAD+-I RNA sample was dissolved in 0.05 M Na-HEPES at pH 7.0 and heatedat 65 °C, followed by the addition of an equal volume of preheatedfolding buffer comprising 0.05 M Na-HEPES at pH 7.0, 0.1 M NaCl, 0.04M MgCl2, and 0.24 M NAD+. Each sample was warmedfor an additional 3 min at 65 °C and then slow cooled in a heatingblock to 24 °C. For size exclusion, the column was equilibratedwith a running buffer of 0.05 M Na-HEPES at pH 7.0, 0.1 M NaCl, 0.02MgCl2, and 0.24 M NAD+. A sample volume of 100μL was injected manually into the column. SEC was conductedon hairpin sequences of domains I and II . Due to the propensity of each hairpin domain sequenceto fold as a monomer–dimer equilibrium , these samples wereexcluded from the ITC analysis. A control sample of the full-length Aai riboswitch without a ligand was run in the unbound stateunder identical buffer conditions . Each sample was eluted in running buffer as 1 mL fractions andmonitored by in-line absorption at 254 and 280 nm.RNA constructs were evaluatedfor multimerization by size-exclusion chromatography. A Superose 610/300GL column (Cytiva Life Sciences) was standardized by using in-houseRNAs with known molecular weights and oligomeric states. The standardsincluded HIV-1 TAR 27-mer (MW 8208 Da),40 to identify interfaces and contacts in domainsI and II based on existing crystal structures retrieved from the PDB. The coordinates analyzed were6tf1 (domain I with ADP bound), 7d81 (domain II with NAD+), 7d7v (U1A in complex with domain I with NAD+), and7d7w (domain I with NAD+). Although multiple interfaceswere identified, we focused on those involving junctions because theseregions are conserved, are important for ligand binding, are functionallyrelevant, and have the potential to provide insight into interdomainpairing. Nucleotides and ions were included in the calculations, butcofactors were not included due to their negligible contribution inthe cases described.We used the program PISAnadA motif, which suggested that the riboswitch senses NAD+ inside cells to regulate the nadA gene encodingquinolinate synthase A , we choseto use a “split construct” of domain I that comprisesa 28-mer paired through WC interactions with a 22-mer, giving riseto a molecule devoid of a joining hairpin loop . Importantly, split constructs avoid the well-knownproblem of forming self-complementary duplexes from hairpin sequencesdue to intermolecular WC pairing.43 The split domain I constructis also more stable energetically than the intramolecular hairpinsformed by individual 28-mer and 22-mer strands . Importantly, the hairpin loop that was removedfrom the split domain I construct is not conserved in the consensusmodel9 and does not participate in cofactorbinding in known crystal structures.22Previouswork described the discovery and validation of the nthase A 1c.9 To i+ interacts with domain I at the P1/J1-1atransition point22  and Acidobacterium ailaaui (Aai), which were analyzed for ligand binding in previousindependent investigations.22 The Aca and Aai sequences exhibit all of the conservednucleotides and secondary structure features present in the consensusmodel.9 First, we tested the binding of Aca domain I to three related ligands: ADP, NAD+, and NADH  but an unfavorable entropic component . Interestingly, NAD+ and NADHshowed average KD values of 24.6 ±8.4 and 55.5 ± 3.6 μM  and entropy  contributions were each favorable  compared to that for related ligands. An independent analysisof the Aca nadA domain I by ITC revealed a KD of 305 μM.22 Similarly, in-line probing of domain I from Aai and Eag yielded KD valuesof ∼300 and ∼200 μM.9 Although our analysis corroborates the observation that NADH bindsmost poorly to domain I among the nucleotides analyzed herein, ourresults showed ∼3.6–5.5-fold better binding based onour average KD of 55.5 ± 3.6 μM, monovalent salts ,and different Mg2+ concentrations (between 10–20mM). Nonetheless, our results support a binding model wherein domainI recognizes a single ADP moiety in the absence of domain II. Thisresult agrees with published cocrystal structures .We next asked whether domain II of the sequence 1b. Like + . Accordingly, ADP bindingaffinity for domain II must be poorer than 1.0 mM, as reported,22 and is likely worse for NAD+ if domainII follows the trend observed for domain I wherein ADP binds moretightly than NAD+ or NADH  butunfavorable entropic  contributions  with an unfavorable entropy. Binding of the full-length Aai riboswitch to NAD+ was comparable to thatof Aca domain I when the associated error was consideredprompted us to test the importance of K+ ions in our bindingbuffer, which yielded an ∼10-fold increase in effector affinityby the lysine riboswitch.48 However, we saw nosignificant change in NAD+ affinity when Na+ was replaced by K+, and the ligand-to-receptor ratioremained between 1.3 and 1.5 . The results suggest that the full-length Aai riboswitchdoes not discriminate between NAD+ and ATP under the conditionstested here. Moreover, monovalent ions such as Na+ andK+ do not affect the binding affinity for NAD+ through site-bound contacts mediated by the RNA and ligand. At present,it is unknown whether monovalent ions exclude ATP from binding.We next tested the binding of ATP to the full-length over ATP 1a. Surpro of 1.2 1. The thAai riboswitch, we conducted ITC simulations to ascertain the feasibilityof detecting two-site binding based on the experimental setup usedherein. In one scenario, we assumed two high-affinity effector-bindingsites with a K1D of 30 μMand a K2D of 50 μM, asreported,22 which produced a sigmoidalisotherm that does not saturate . In a second scenario, we simulated one high-affinity site (K1D of 20 μM) and one low-affinitysite, in agreement with both our data and previous low-affinity recognition(K2D of 3.4 mM) by domain II.22 This simulation shows early binding and producesa longer hyperbolic phase that does not saturate . Based on visual inspection and the much poorerχ2 values of 275 (two high-affinity sites model)and 0.024 (one high-affinity and one low-affinity site) relative tothe one-site model used in our analysis (χ2 of 0.0009),neither two-site model adequately described the observed experimentaldata. Importantly, both simulations produced isotherms that are distinctfrom those observed experimentally here , consistent with a model inwhich one effector binds per full-length riboswitch.Given that our results overwhelmingly supporta ligand-to-receptor ratio of unity for the full-length saturate S3c. In a+ mimics were analyzed for theirbinding affinity to the nadA riboswitch to identifyfunctional groups important for cofactor recognition.9 To complement existing studies, we identified AP2-ribavirin as a ligand of interest to probe cofactor specificitybecause it was not explored previously. This bipartite molecule preservesthe ADP moiety but replaces the nicotinamide group with a planar triazolering attached to a pendant amide . Binding wasdriven by a favorable enthalpy  that offsets an unfavorableentropy . A similar trend was observedfor the binding of ADP and NAD+ to domain I (n = 0.6), as observed forother domain I interactions  are tolerated by domain I.9Titration of AP± 7.1 μM 1, which domain I 1. The liractions 1. The bi byNAD+ 1. TheAPfor NAD+ 1. The renadA riboswitchdomains has the potential to influence binding analysisby ITC. Accordingly, we analyzed each individual split construct domainand the full-length riboswitch by size-exclusion chromatography (SEC)in the presence of NAD+. As a control experiment, we calibratedour column by use of domains from well-folded RNAs characterized inour laboratory by SEC, ITC, and X-ray crystallography. These RNAswere chosen for calibration because they do not self-interact appreciably38 or they form predictable dimers at the concentrations used in chromatography.39 The associated elution profiles and resultingstandard curve are provided (Kav versus log(MW) obeys a linear relationship, as expected for compactbiomacromolecules.50Oligomerization of individual provided 6a. ImporAca domain I split construct50-mer revealed that the sample migrates mostly as a monomer . Indeed, the major peak of the domain I split constructmigrates as a monomer that obeys the linear trend set by the RNA standards. This result was likely causedby a mixture of dimers formed by complementary intermolecular WC pairing as well as the correctlyfolded intramolecular hairpin monomer . This finding justifies our use of split domain constructsinstead of a hairpin sequence for ITC analysis.SEC analysis of the  monomer 6b. This  monomer S1d. Inde monomer S1d. Inde monomer S1d. IndeAca domain II split construct,which produced an asymmetric peak with a prominent prepeak shoulder. Again, these findings illustrate why the domainII hairpin sequence was unsuitable for our ITC analysis.We next analyzedthe shoulder6c. The eAai nadA riboswitchrevealed that the riboswitch migrates as a compact structurein the presence or absence of NAD+, resulting in a singleeluted peak (S6); this suggests that the riboswitchis prefolded prior to effector binding. The Kav versus log(MW) analysis of the full-length riboswitch indicatesthat it obeys the linear trend established by the calibration curve produceda major loss in gene-regulatory function.9 Surprisingly, the crystal structure of isolated domain II suggeststhat C13 bulges into solvent22 22 revealed a notable intermolecularinterface involving J1-1aand J1b-1a. This interface contains eight hydrogen bonds derived fromthe bulged cytosines C11 and C12 in J1-1a and the bulged guaninesG36′ and G38′ in J1b-1a′, which were donatedfrom a symmetry-related molecule (Aai packing interface occurs betweenJ1-1a and J1b-1a in a different domain I structure (PDB 7d7w),22 which reveals eight interdomain contacts . Here, A11 of J1-1a uses its N6 exocyclic amineto interact with the sugar edge of C15′ of J1-1a′; theA11 base also stacks on the G36′ of J1b-1a′. A Mg2+ bridges the phosphate backbones of A11′ in J1-1a′and U17 in J1-1a. A third unique intermolecular J1-1a and J1b-1a′interface occurs in the Ckv nadA riboswitch (PDB 6tf1)11 . Here, the bulgedbase A11 of J1-1a stacks with the G36′ of J1b-1a′ whilesimultaneously coordinating to Na+ and Mg2+ ,which bridges the intermolecular interface. These observations implythat interactions between J1-1a and J1b-1a are influenced by mono-and divalent ions.Analysis of molecule 7c. Speci9 Although onlya single crystal structure existsof domain II, the corresponding interface analysis (PDB 7d81)22 showed that J1-1a and J1b-1a engage in 12 intermolecularcontacts, comparable to those observed for domain I. Notably, WC hydrogenbonds occur between C12/G36′, and the sugar edge of G23 inJ1b-1a interacts with that of C12′  and worse χ2 values. Notably, the average overall n value for the binding of a one-site ligandto the full-length riboswitch was 1.24 ± 0.21 . Indeed, we observed no evidence of two-site bindingusing sequential or interdependent-sites binding models . Notably, each binding modelis expected to produce distinctive thermogram features, such as twoinflections or parabolic curves characteristic of two-site binding,51 which were not observed in our experimental data. Interestingly,we observed that the entropic and enthalpic binding contributionsfor a particular ligand were similar for both domain I and the full-lengthriboswitch .The goal of this workwas to probe the ligand specificity and ligand-to-receptorratios of the full-length boswitch 4 and 5b.4 ± 0.21 1. This sboswitch 1. The sinadA riboswitch is necessary and sufficient for ADP recognition.22 Our work corroborates these observationsand reveals that ADP, NAD+, NADH, and AP2-ribavirineach bind to Aca domain I with KD values between 11.1 ± 0.6 and 55.5 ± 3.6 μM, cannot confirm this proposition.In contrast to domain I, domain II ofthe analysis 1. Our finadA gene regulatory mechanism,we note that an NAD+KD of3.4 mM for domain II (assuming ADP binds with the same affinity asNAD+) would necessitate an effector concentration ∼10-foldhigher than its KD to produce saturationof the domain II binding pocket. Accordingly, cells would need toreach an astonishingly high concentration of ∼34 mM NAD+ to achieve full binding and gene regulation, i.e., a level13-fold higher than the 2.6 mM concentration of NAD+ measuredin aerobically growing, glucose-fed E. coli.23 Remarkably, E. coli cells thatwere fed NAD+ for 26 h reached cofactor levels of only6.2 mM.54 Accordingly, we posit that thebinding of NAD+ to domain II in the crystal structure isan idiosyncrasy of its propensity to self-associate, which may haveresulted in the weak apparent KD of 3.4mM measured outside the context of the full-length riboswitch.In terms of the nadA riboswitch domains include theirhigh degree of sequence homology9 and theirability to self-interact . As such, the “sticky”properties of isolated domains are likely an underlying factor leadingto the reduced ligand-to-receptor ratios observed by ITC for domain I .22 Hence, only the full-length riboswitch appearsto be capable of producing a competent ligand binding pocket, as indicatedby an average ligand-to-receptor ratio of 1.2 ± 0.2 based on The main difficulties in dissecting the functional relevance ofindividual interact 6a–c. Indboswitch 6a–c. Altboswitch 7 and S7.domain I 1. Specif56 Independently folded aptamers of the tandem glycine riboswitch interactedin a trans, glycine-dependent manner.57 At present, it is unclear why domains I andII of the nadA riboswitch did not form a complex,although removal of the interdomain linker could have eliminated acritical structural element required for complex formation. Interdomaincontacts between aptamer 1 and aptamer 2 of the glycine riboswitchutilize multiple patches of A-minor contacts between P1 helices andjunction or loop regions that form reciprocal contacts .58 Thehairpin ribozyme uses interdomain WC pairing at a sharp S-turn bendand a nearby ribose zipper motif to support the docking of loop Aand loop B .55 Although the nadA sequencedoes not exhibit extended adenine sequences suggestive of A-minorinteractions, single conserved adenines are present in the J1-1a consensusmodel that could be employed for tertiary interactions. It is alsoplausible that one or more WC or ribose zipper interactions occurbetween domains, originating from J1-1a and J1b-1a.Our inabilityto reconstitute an intact riboswitch from individualdomain I and domain II constructs 6d was sonadA riboswitch and the tandem glycine riboswitchwherein the latter also comprises two homologous but nonidenticalaptamers connected by a linker.60 Although each glycineriboswitch aptamer binds a single ligand, glycine binding to aptamer1 was more sensitive to intramolecular dimerization than aptamer 2.57 Moreover, aptamer 2 showed much weaker affinityfor glycine. The posited gene regulatory model suggests that ligandbinding to aptamer 1 is linked to dimerization and stabilization ofP1 within aptamer 2, which controls the expression platform.58 Accordingly, domain I of the nadA riboswitch is primarily responsible for NAD+ recognition,which ostensibly provides a scaffold that promotes domain II folding.Like the glycine riboswitch, domain II contains the expression platform,which is likely buried upon formation of the P1′ helix, leadingto a gene-off state  riboswitch also binds NAD+, it does notrecognize the cofactor’s adenosyl moiety, in contrast to the nadA (NAD+-I) riboswitch herein. This is becausethe NAD+-II aptamer senses the nicotinamide riboside moietyof the cofactor,66 which also results in affinity for NMN and NR becausethey are substructures of NAD+. Thus, despite the hypothesisthat nadA domain II would bind the nicotinamide moietyof NAD+ in the context of the full-length riboswitch,9 we did not observe this, and the γ-phosphateof ATP is accommodated readily , other unidentifiedfactors could be required to impart the level of specificity neededfor the nadA riboswitch to exclusively sense NAD+ (Little is known at present about the regulationof bacterial NADprotein 1cis knoity here S8, other


Flaviviridae family, is a novel segmented RNA virus identified in 2014 in the Jingmen region of Hubei Province, China. Up to now, JMTV has been detected in a variety of countries or regions in Asia, Europe, Africa, and the Americas, involving a wide range of arthropods and mammals, and even humans. The JMTV genome is composed of four linear RNA segments, two of which are derived from flaviviruses, while the other two segments are unique to JMTV and has no matching virus. Currently, JMTV has been shown to have a pathogenic effect on humans. Humans who had been infected would develop viremia and variable degrees of clinical symptoms. However, the pathogenic mechanism of JMTV has not been elucidated yet. Therefore, it is crucial to strengthen the epidemiological surveillance and laboratory studies of JMTV.Jingmen tick virus (JMTV), belonging to the  Rhipicephalus microplus from the Jingmen region of Hubei Province, China  genes of the genus Flavivirus, whereas the remaining two segments are specific to JMTV , a novel tick-borne RNA virus, was first identified in 2014 in for JMTV 6. In adfor JMTV . In the dization , 8. ThesJMTV has been reported in many regions of northeastern, northwestern, central, and southern China, as well as in many countries in continental Europe and the Americas since JMTV was first described in Hubei Province, China –11. InJMTV had an extremely wide geographical distribution, covering a number of countries and regions in Asia, Europe, Africa, and the Americas. The earliest detailed description of JMTV appeared in 2014 when it was detected in the Jingmen region of Hubei Province, China. Since then, JMTV has been reported to be endemic and spreading in many areas of China, including Zhejiang, Yunnan, Fujian, Xinjiang, Guangxi, and Heilongjiang provinces , 14–16Haemaphysalis longicornis and Amblyomma testudinarium via the transovarial route , Brazil (25%–67%), Trinidad and Tobago (6%–46%), and the French Antilles (24%–77%)  that encodes a protein, while segment 4 is bicistronic, containing two ORFs and encoding two proteins. Fluorescence particle . This separticle . Proteinparticle . This maparticle . Currentparticle . Segmentparticle , 29.Pairwise distance analysis of all available JMTV genome sequences showed that, with the exception of the JMTV 018 strain from Germany, the nucleic acid identity of segment 1, segment 2, segment 3, and segment 4 of all JMTV strains was 69.4%–100%, 57.4%–100%, 68%–99.9%, and 59.5%–100%, respectively. There were significant differences between JMTV strain 018 from Germany and the currently published sequences of other JMTV strains. Specifically, segments 1, 2, 3, and 4 of JMTV strain 018 shared only 46%–48.2%, 32.1%–38%, 41.1%–42.7%, and 38%–40.3% nucleotide identity with the sequences of other JMTVs, respectively . JMTV stThe phylogenetic tree was constructed by neighbor-joining method to better analyze the phylogenetic relationships of JMTV, and the reliability of the tree was determined by the bootstrap method . The bootstrap values >70% are considered to be significant. All available JMTV and JMTV-related virus sequences could be divided into two major phylogenetic groups, with the first group containing JMTV identified from China, Japan, France, Turkey, Kenya, Tobago together with Amblyomma virus, Rhipicephalus-associated flavi-like virus, Heilongjiang tick virus, and Guangxi tick virus from China, while the second group was composed of JMTV and typical flaviviruses from Asia, Europe, Africa, and South America . Within Ixodidae-derived BME/CTVM23 cell line may be more suitable for the growth of JMTV isolated from Ixodidae (A variety of arthropod and mammalian cells including Vero, Vero E6, BHK-21, C6/36, BME26, DH82, and BME/CTVM23 have been used for viral isolation of JMTV , 17. DesIxodidae . FinallyIxodidae . DH82 anIxodidae . SimilarIxodidae . TransmiIxodidae .JMTV is a novel tick-borne virus, for which studies on human and animal pathogenicity are extremely important. In 2014, an immunofluorescence assay was performed on 145 cattle sera from Hubei and Zhejiang provinces, 54 samples were positive for JMTV, indicating the presence of antibodies against JMTV in cattle blood . These rJMTV is the first segmented RNA virus to be identified and confirmed, and is closely associated with the typical unsegmented flavivirus. A global survey on the epidemiology and pathogenic effects of JMTV has been conducted since its discovery in Hubei Province. It was reported that RNA of JMTV was found in four continental regions worldwide and involved multiple hosts including humans. Despite the fact that no human or animal mortality has been reported from JMTV, it has been shown that JMTV may cause viremia in humans or animals and that infected individuals may show variable degrees of clinical symptoms. More intensive laboratory studies and long-term epidemiological surveillance to elucidate the pathogenicity of JMTV in humans and animals and the global distribution of JMTV would be necessary to avoid possible local or large-scale epidemics caused by the virus, especially in regions where JMTV and JMTV-associated viruses have been detected.In conclusion, we reviewed the recent advances of JMTV, outlined the geographic distribution and transmission vectors of JMTV, and analyzed the JMTV genome and evolutionary relationships to improve our understanding of the virus and develop effective control measures.


Acheta domesticus, CP), buffalo worm , and mealworm . This goal has been achieved by means of an (HS)-SPME/GC-MS strategy. 67 compounds have been tentatively identified; of these, 27 are present only in the CP and BW flours, while 10 are common in all three flours. The compound with the highest peak’s relative area in gas chromatograms of CP and BW flours is hexadecanoic acid, while in MW it is 1-heptylpyrrolidin-2-one. In general, we have observed that CP and BW flours have 37 compounds in common, and their volatile compositions along with their profiles are more similar to each other than to MW profile.The growing world population, combined with scarcities of agricultural land, water, forest, fisheries, and biodiversity resources, makes it necessary to search for alternative sources of nutrients. For this reason, in recent years, edible insects have been introduced into the diet, even in areas where entomophagy is not traditional. In light of this, the present study aims at characterizing the aromatic profile of three edible insects flours: cricket ( The ever-growing world population inevitably forces an increased food (and even feed) production from available agro-ecosystems, resulting in dramatic pressure on the environment. Scarcities of agricultural land, water, forest, fisheries, and biodiversity resources, as well as nutrients and non-renewable energy, are foreseen and need to be faced.One possible answer for nutrient scarcity could be the introduction of insect-based food in the daily diet of Western populations, since edible insects are a potential reliable source of high quality protein , fats, carbohydrates, and various vitamins and minerals with a very low environmental impact .Regarding the environmental impact, insects are extremely sustainable, as their breeding is not connected to the availability of land. Moreover, insects are cold-blooded animals  and their carbon dioxide and other greenhouse gas emissions are much lower than those from warm blooded animals such as farm animals. In this context, the requirements for water and nutrients are also reduced. Furthermore insects’ protein bioconversion rate is also considerably higher than that of farm animals .Additionally, many insects also feed on organic waste, thus solving another very topical problem, namely that of food waste: the United Nations estimates that, around the world, about one third of food destined for human consumption is thrown away .There are nearly 1900 insect species reported for human consumption and more than 2 billion people use insects as food. Edible insects are mostly consumed in Asia and Central America, while in the Western world, entomophagy remains rare or even a taboo .Although health and environmental issues are powerful motivations to entomophagy, it has been suggested that people are more likely to be persuaded by a positive eating experience . In thisRegarding the appearance, since in Western countries the acceptance of insect consumption is very low, the main form of insect consumption is as an ingredient; typically, a powder obtained from grinding dehydrated insect or protein extracts is added to food products such as cookies, snack, bars and protein shakes. In fact, incorporating insects in a non-recognizable form, such as a powder or a flour, reduces the negative perception of eating insects. In fact, it has been reported that the low acceptability of insects as food is mostly associated with emotional factors such as disgust, as well as unusual tastes and textures, and thus the use of edible insects in a unrecognizable form as an ingredient could be a potential means to increase edible insect acceptance . For insThe flavor of edible insects depends on the insect species, their stage of development, and for insect-based food also on the method of drying and preparation. Even though the edible insect flavor is generally described as tasty , it is sMost of the studies on edible insects are focused on their nutritional composition, while there is still a lack of knowledge on organoleptic properties of these novel foods. This gap needs to be undertaken, since it is known that the analysis of the volatilome of a food product provides an important indication of the safety and the authenticity of a food product .One of the reasons is definitely the extreme variability of the volatile compounds among species, stage of development and, above all, edible insect processing methods.A very recent comprehensive review on volatile compounds found in edible insect  reports Acheta domesticus), buffalo worm larvae  and mealworm larvae (Tenebrio molitor).In the present study, we describe the volatile profile obtained by (HS)-SPME/GC-MS of flours of three (out of the only four) edible insects approved as novel foods in the European Union -SPME/GC-MS.Acheta domesticus—CP), buffalo worm larvae (Alphitobius diaperinus—BW) and meal worm larvae (Tenebrio molitor—MW) have been analysed by HS-SPME/GC-MS under the same experimental conditions , which account for around 15% in CP flour and around 12% in the BW one. Conversely, this compound has not been detected at all in the MW flour. Remarkably, only two (#11 and #56) out of the six identified fatty acids were detected in MW flour; moreover, hexanoic acid (#11), the fatty acid with the shortest chain detected here, is present only in the MW flour . Together, the six fatty acids detected account for around 22% and 18% in CP and BW flours, respectively, and less than 2% in MW flour.The compound with the highest peak’s relative area in the MW flour volatile profile is 1-heptylpyrrolidin-2-one #45, which accounts for about 18% in MW flour and less than 1% in both CP and BW flours. Noticeably, the five 1-alkylpyrrolidin-2-ones represent by far the compounds with the highest peak’s relative areas present in the MW flour’s volatile profile, where their overall amount is about 36%. On the contrary, the 1-alkylpyrrolidin-2-ones accounts for only a total relative area of 0.4% and 3% in CP and BW flours, respectively.27 out of the 67 detected compounds listed in A. domesticus sample. Moreover, several different pyrazines were found in the HS-SPME GC MS profile of T. molitor larvae affected by roasting processes [Among the MRPs, we found 5 alkyl pyrazines  which account for around 24% of the total area in CP flour and around 14% in BW flour, while they are completely absent in MW flour. Pyrazines, which possess roasted, toasted, nutty, coffee-like, or cocoa-like odor notes, are the most abundant heterocyclic compounds among the volatile MRPs . Interesrocesses .On the other hand, the five 1-alkylpyrrolidin-2-ones , as mentioned above, reach overall around 36% of the total area in MW flour, while contributing to about 0.4% and 3% in the chromatograms of CP and BW flour, respectively. Pyrrolidin-2-ones , besides being Maillard products deriving from γ-aminobutyric acid ,26, haveN-heterocyclic compounds’ flavor deriving from Maillard reactions [Also, the seven compounds with a piperidine moiety  could be considered as MRPs, since it was demonstrated that lysine can generate piperidine in the presence of sugars , and pipeactions . Furthereactions . The cumN-valeryl-l-proline-alkyl esters (#60 and #63) were detected only in CP and BW samples where they account for an overall relative area of around 2% in CP flour and around 15% in BW. N-valeryl-l-proline-alkyl esters have been identified in waste chicken feathers [Two feathers  but alsofeathers  and in afeathers .Besides, we identified five methlylketones which are reasonably produced by lipid oxidation . The fivγ-Heptalactone (#26) and γ-nonalactone (#37) were detected only in MW flour. Both of these γ-lactones have been described as important flavor compounds; in particular, the former possess a fatty milky coconut odor while the latter gives a fruity aroma . We alsoThe comparison of the three flours’ volatile profiles shows a great similarity, in terms of compounds detected, between those of CP and BW flours compared to the volatile profile of BW. In fact, for CP and BW flours, we detected the same 37 compounds; consequently, the differences between the CP and BW flours’ volatile profiles depend only on the different relative abundances of these compounds. Conversely, in the case of MW flour, only 10 compounds out of the 38 volatiles identified for this flour are in common with the other two flours; thus, in this case, the volatile profile of MW flour appears completely different, mainly in qualitative terms.What mostly affects the volatile profile seems not to be the insect phylogenetic classification  nor its development stadium , but probably the flour production process. In fact, CP and BW were purchased as flours, while MW has been bought as whole larvae and hand grinded.The presence of a high number and relative amount of MRPs in the three flour samples (17 MRPs in CP and BW flours and 12 in MW flour with a cumulative area of about 43% in both CP and BW flours and around 49% in MW flour) could, at first sight, be considered an experimental artifact due to the relatively high temperature (130 °C) and long time (1 h 45 min) at which the insect flours were exposed during the SPME analysis, which, in principle, could simulate a sort of cooking process. However, this hypothesis must be discarded since Maillard reaction products are also reported in volatile profiles of proteinaceous flours from legumes obtained by (HS)-SPME/GC-MS at 65 °C  or by soTenebrio molitor larvae (the same larvae from which we obtained MW flour), the authors found some pyrazines [Tenebrio molitor larvae.Interestingly, in a (HS)-SPME/GC-MS study on the characterization of volatiles deriving from the roasting process on yrazines . The facyrazines  than thoAcheta domesticus “Cricket Powder”, purchased from GRIG Distribuce s.r.o. Czech Republic;CP: flour from Alphitobius diaperinus, purchased from isaac nutrition Gesellschaft mit beschränkter Haftung. Germany;BW: flour from Tenebrio molitor, purchased from Sahawa, Germany. The larvae were hand grinded in a mortar to obtain a powder similar to the CP and BW flours.MW: dried larvae from Tenebrio molitor—also known as the (yellow) mealworm—was authorized as novel food on 3 May 2021 (reg. EU 2021/882 and reg. EU 2022/169), and was the first insect-based novel food authorized in the EU.The larval form of the insect species Acheta domesticus) was authorized as novel food on 3 March 2022 (reg. EU 2022/188).The adult form of house cricket  was authorized by the EFSA on 4 July 2022 (EFSA-Q-2018-00282 ON-7325) and the EU executive regulation has not yet been issued. The larval form of The SPME fiber used in this work was coated with divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS), 50/30 μm thickness .About 100 mg of flour was placed in a 4 mL vial equipped with the Mininert cap and kept at 130 °C in an oil bath for 1 h without stirring. The fiber was introduced into the vial and exposed for 45 min to the head space of the sample. After insertion of the fiber into the injection port of the GC apparatus, desorption of the extracted volatiles took place at 250 °C for 2 min. After each analysis, the fiber was kept in the GC injection port at 270 °C for 5 min, and after that a blank run was recorded to check the cleaning of the sorbent.For each sample, the analysis was done in triplicate.All the analyses were carried out on a Saturn 2000 GC-MS instrument . The GC apparatus was equipped with a 1078 split/splitless injector with a SPME liner inside. All injections were performed in split mode with a 10:1 split ratio.m/z range; 1 s scan cycle time; transfer line temperature 170 °C; manifold temperature 110 °C; ion trap temperature 150 °C. The compounds were identified by comparison of their linear retention indices calculated using a linear alkane mixture  and their mass spectra with those reported in standard libraries [A Varian FactorFour™ VF5-ms capillary column  was used and the carrier gas was helium 5.5 IP, supplied at a flow rate of 1.1 mL/min. GC separation was achieved using the following column oven temperature program: initial temperature 50 °C for 2 min, then increasing to to 100 °C at 30 °C/min, then to 250 °C at a rate of 6 °C/min, at the end the temperature of 280 °C was reached at 65 °C/min and held for 2 min. The EI-ion trap mass spectrometer operated at the following conditions: ionization energy 70 eV; 40–600 ibraries .Acheta domesticus), buffalo worm larvae (Alphitobius diaperinus) and meal worm larvae (Tenebrio molitor—MW), all approved as novel food by the European Union.In this preliminary study, we described the volatile profiles of three edible insect flours, namely flours from adult crickets (The volatile profiles obtained by (HS)-SPME/GC-MS from each of the three flours under investigation appear quite peculiar and seem to depend both on insect species and flour processing.On the basis of this preliminary evidence, SPME can be regarded as an effective and promising tool for future applications aimed at describing and discriminating flour of different insects and/or obtained by different technological processes.The potential effectiveness of insect flours’ volatile profiles as a fingerprint for insect species supports the upcoming application of (HS)-SPME/GC-MS to assess the authenticity of these products and detect possible food frauds deriving from their adulteration with lower value flours (either from other insect species or deriving from legumes).